The Arab League for
Palm & Date Research
National Centre for
Agricultural Technologies
papers
Volume I (English)
ARAB PALM
CONFERENCE 2011
Proceedings of the First International Scientific Conference for the
Development of Date Palm and Dates sector in the Arab World.
4 - 7 December 2011, corresponding to
9 - 12 Muharram 1433 H - Conference Hall, Building 36
KACST Head Quarters - King Abdullah Road - Riyadh, KSA
4
Proceedings of the
First International Scientific Conference for the
Development of Date Palm and Dates sector in the
Arab World.
Volume I (English)
Riyadh, Saudi Arabia
4-7 December 2011
National Centre for Agricultural Technologies
King Abdulaziz City for Science and Technology, Post
Box No.6086, Riyadh 11442
i
ii
Kingdom of Saudi Arabia
First International Scientific Conference for the
Development of Date Palm and Dates sector in the
Arab World.
Scientific Committee
Dr.Nasser S. Al-Khalifah
(Convener)
Dr. Ali Al -Jaloud
Dr. Ibrahim Al-Ruqaie
Dr. Mahar Shahata
Dr. Hassan Al- Ayedh
Dr. Ahmad Al-Abdulkader
Co-Organizer
The Date Palm and Dates League of the Arab World
iii
iv
Proceedings of the
First International Scientific Conference for the
Development of Date Palm and Dates sector in the
Arab World.
Volume I (English)
Compiled by
Dr.Nasser S.Al-Khalifah
Dr. Shanavaskhan A.E.
Mr. Amer Yasif Al-Khaybari
Riyadh, Saudi Arabia
4-7 December 2011
King Abdulaziz City for Science and Technology, Post
Box No.6086, Riyadh 11442
Kingdom of Saudi Arabia
v
vi
CONTENTS
Foreword
…………………………………………………….. ix
Preface
……………………………..…………………….. xi
List of papers ………………………….……………………….. xiii
List of authors ……………………..…………………………….. xxi
vii
viii
Foreword
More than hundred and fifty scientists, academicians, government officials,
business persons, students and farmers representing eighteen countries attended
the first scientific conference for the development of date palm and dates in the
Arab world held at the King Abdulaziz City for Science and Technology, Riyadh,
Saudi Arabia. The conference was jointly organized by King Abdulaziz City for
Science and Technology (KACST) and the Date Palm and Dates League of the
Arab world (DPLAW).
The conference was formally inaugurated by the Honourable Minister of
Agriculture Dr.Fahad Balghunaim and his excellency president of KACST Dr.
Mohammed Al Suwaiyel. The four day deliberations covering seven themes were
presented into nine oral sessions and one poster session. Date palm and dates
production was the opening theme in which 14 papers were presented in two halls as
parallel sessions.
In the sessions of agriculture biotechnology fifteen presentations were made by
the scientists, out of which seven were on the micropropagation and related aspects
of date palm. One of the lead papers overviewed the current status and prospective
of date palm biotechnology and the other one highlighted the potential application of
gene silencing or RNA interference to the control of disease and insect pests of date
palm. Novel techniques for distinguishing male and
female plants using leaf
scanning technique as a biophysical and engineering contribution to plant research
were also discussed.
Disease and pests of date palms and their management was one of the
important themes selected for this conference. Twenty papers were presented on these
subjects. Various aspects related to red palm weevil infestation, nematode attack,
bacterial, fungal and viral diseases were discussed. There were four presentations on
post-harvest and storage of date palms. There were discussions on the prospects of
dates industry and palm tree by-products. For the effective storage of dates, innovative
methods using modern technology tools have been discussed.
ix
The highlight of the Date palm and Dates processing session was the presentations
on non-conventional usages of date palms. Speakers explained various usages of other
palms and recommended the same possible usages to the date palms. Other speakers
explained the usages of palm wood, leaf and seeds for making useful products. The
last session was on the economic aspects of date palm industry. Date palm production
economics and market distribution systems of Algeria, Sudan, Yemen and Saudi
Arabia were discussed in this session. Two open forum discussions on developing
investment in date palm and date sector and dates loss and opportunities of utilization
were held as evening sessions. These sessions were attended by many people and
deliberations on the subject matters by the participants have been recorded.
My sincere thanks go to all the participants for making this conference a successful
event. The unconditional support received from the president and vice presidents of
KACST and all the executive committee members of RABITAH is greatly
acknowledged. The Scientific committee members and the researchers namely
Dr.Shanavaskhan, A.E., Messrs Ejaz Askari, Amar Al-Khaybari, Abdulaziz Al-Oufi,
Meshal Al-Mutairi,
Fahad Al-Otaibi, Mohammed Al-Dukhail, Abdulmannan Al-
Hindi, Majed Al-Orf and Nasser Al-Hamid extended their full support to organize this
conference in a successful manner. Dunia Intermedia deserves a word of thank for
their excellent reprographic works. The public relation department of KACST has
done excellent work in providing necessary helps in organizing the events.
Convener
Dr.Nasser Saleh Al-Khalifah.
x
Preface
This is the proceedings of the first international scientific conference for the
development of date palms and dates sector in the Arab world held at the King
Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia from 4th
– 7th December 2011. The papers contained in this volume are non-peer reviewed
articles presented during the conference and collected for documentation . Authors are
solely responsible for the content of their articles. However the compilers have made
efforts to make it uniform to some extent. Due to the large sizes and severe
incompatibility problems a few tables and figures were omitted from some articles.
Some articles published in this volume were submitted to the editorial board of
Emirates Journal for Food and Agriculture(EJFA) for publication, with author’s
consents. The other articles that were not published in EJFA, the authors are free to
publish their article in the journals of their choice.
Compilers
xi
xii
List of papers
Envision of an International Consortium for Palm Research
OP01
Jameel M. Al-Khayri and Charles L. Niblett
OP02
OP03
Development of Tall X Dwarf Hybrid Date Palm
C. Sudhersan and Y. Al-Shayji
Assessing Fruit Characteristics to Standardize Quality
Norms in Date Cultivars of Saudi Arabia
I.A.AL-Abdulhadi, S. Al-Ali, K. Khurshid, F. Al-Shryda,
A.M.Al-Jabr and A. Ben Abdallah
Effect of different levels of elemental sulfur fertilizer on the Growth,
fruit quality, yield, and nutrient levels in the leaves of Mishrig Wad
OP04
laggai date palm cultivar under Al Mukabrab Conditions.
Dawoud Hussien Dawoud and Fatima A. Ahmed.
3
21
33
47
OP05
Improving Nutritional Status, Yield and Fruit Quality of Date
Palm by Nitrogen Forms, Potassium and Sulfur fertilization
Hassan A. Kassem
57
OP06
Effect of Gibberellic acid on fruit quality of Samani and
Zaghloul date palm cultivars
Ghazawy, H.S; Bakr, E.I , S.EL-Kosary and A.EL-Bana
75
Effects of water stress and arbuscular mycorrhizal fungi on reactive
oxygen metabolism and antioxidant production by date palm seedlings.
OP07
Kasrati A., Oussouf F.M. and Qaddoury A.
OP08
OP09
PP01
Relationship between yield and fruit quality and leaf and
fruit mineral content of Zaghloul dates in the Egyptian
calcareous soil as result of flower boron spray and soil
application with some micronutirents.
A.M. Attalla, A.M. El-Kobbia and S.M. El-Nawam
83
95
Physical characteristics of fruit of different date palm
cultivars during various maturity stages.
Imran Ullah
107
Effect of amount of pollen on anatomy and quality of
Zagloul date palm fruit (Phoenix dactylifera, L.)
Alaa El-Din Kh. Omar
121
xiii
Evaluation of pollination ability and metazenic effects of seedlings
date palm males, on two female cultivars Mishrig Wad Laggai and
Mishrig Wad Khatiab under Khartoum state condition
PP02
Dawoud H.D., and Fatima A.El-Rauof
133
Effect of some Artificial Ripening Treatments used as Environmentally
Safe on fruit quality and storage ability of "Sewy" Dates.
PP03
El-Kafrawy, T.M. and N. Abdel- Hamid
147
PP04
Effect of time and patterns of fruit thinning on yield and
fruit quality of Saidy date palm cultivar
El-Salhy A.M., A.A. El-Bana, H.A. Abdel-Galil and E.F.S. Ahmed
163
PP05
Effect of some activators and wounding on rooting and
growth of Phoenix dactylifera L. cv. Haiany aerial offshoots
Lobna M. Abdel-Galeil; Tahani Y. Saber; M.A. Abd El-Baky
and A.A. El-Bana
177
PP06
Role of yeast as a new biofertilizer for improving yield and
fruit quality of both Hayani and Sewi date palm cultivars.
Gadalla, E. G, Abo Rekab, Zeinab A. M and Nady, N .H
199
PP07
Impact of reducing soil in the area of date palm dike (Jura)
and the annual amount of irrigation water and fruits cover
at date palm with the conditions of organic farming
Rashed M. F.
225
PP08
Influence of nutrients spray application on the yield traits of
"Zaghloul Cv." date palm
El Assar A. M. and O. A. M. El Sehrawy
241
Date palm biotechnology: current status and prospective-an overview
S. Mohan Jain
255
Influence of shoot-tip callus induction medium on in vitro
morphogenesis of date palm inflorescence explants
Adel Ahmed Abul-Soad
283
Effect of number of subcultures and cultivar on in vitro
multiplication through direct organogenesis of date palm
(Phoenix dactylifera L.)
Abd EL-Baky M. A., A. Seaf El-Dean and H. A. A. Metwally.
297
Protoplast culture before and after fusion between two different
cultivars for improvement of date palm ( Phoenix dactylifera L. ).
311
OP10
OP11
OP12
OP13
xiv
Chabane D, Assani A and Bouguedoura N.
Effect of somaclonal variation , subculture number and
explant type on in vitro micropropagation of date palm cvs .
Sewi, Zaghloul and Samani.
OP14
Essam Abdel-Rahman Madboly, Amina Hamed Gomaa and
Mohamed Ahmed Eisaa
327
Application of in vitro culture technique for propagation of
two cultivars of Egyptian date palm
M.K. El-Bahr and Taha. H. S
355
Importance of Inflorescence Tissues in Micropropagation of
Select Date Palm (Phoenix dactylifera L.) Genotypes
OP16
Larbi ABAHMANE
371
OP15
OP17
OP18
OP19
OP20
OP21
OP22
OP23
OP24
Growth Promotion of date palm plantlets ex vitro by
inoculation of rhizosphere bacteria
Hala M. A. Farrag, Abeer.H.E. Abd-El Kareim and Rasmia S.S.Darwesh
385
Studies on storage under minimal growth condition of date
palm callus explants
Zeinab E. Zaid.,Maiada M. El-Dawayati., EL-Sayed I. Baker,
and Amina H. Gomaa
401
A new concept for production and scaling up of bioactive compounds
from Egyptian date palm (Zaghlool) cultivar using bioreactor.
Taha H. S., S.A. Bekheet and M. K. El-Bahr
421
Potential Applications of Gene Silencing or RNA Interference
(RNAi) to the Control of Disease and Insect Pests of Date Palm
Charles L. Niblett and Ana M. Bailey
Biophysical and Engineering Contributions to Plant Research
Artmann GM. , I. Digel, P. Linder, and Mrs. Aysegül Temiz Artmann
Identification of sex-specific DNA markers for Date Palm
(Phoenix dactylifera L.) using RAPD and ISSR techniques
Rania A. A. Younis, Omayma M. Ismail and S. S. Soliman
441
459
467
Genetic analysis of abnormalities in tissue culture-derived
date palm (Phoenix dacylifers L.), Barhi cultivars
Kh. A. Soliman, R. M. Rizk, and S. S. H. El-Sharabasy
481
Physiological Modifications in Date Palm Seedlings in
Response to X-Ray
497
xv
Norah A. Al-Enezi1 and Jameel M. Al-Khayri
First report on finger printing of famous date palm cultivars of Pakistan
using SSR markers for identification of their true to type suckers.
OP25
Muhammad Mansoor, Michael Baum2 and Aladdin Hamwieh
OP26
PP09
PP10
OP27
Genetic diversity analysis of the date palm (Phoenix
dactylifera L.) cultivars grown in Tunisian oasis :
morphological and molecular traits and their reports
with agronomic performances
Ali FERCHICHI and Hammadi HAMZA
Protocol for in vitro regeneration of plantlets from root tip
explants differentiated from shoot tip explants and genetic
stability of date palm cultivars.
Essam Abdel-Rahman Madboly and Amina Hamed Gomaa
OP32
535
561
Bt: A new strategy towards date palm orchards resistant to
Red Palm Weevil
S.A. Bekheet
575
Acremonium as an endophytic bioagent against date palm Fusarium wilt
Hatem M. El-Deeb and Youssef A. Arab
Effect of Some Streptomyces Isolated from Date Palm
Rhizosphere on Some Toxigenic Fungi in Saudi Arabia in Vitro
OP30 M.A.Naggar, Muneera, D.F. AlKahtani, T.M.Tabt, E.A.Sarhan
and K.Mostafa
OP31
523
Molecular and Morphological Identification of Some Elite
Varieties of Date Palms in Saudi Arabia.
Nasser S. Al-Khalifah, Ejaz Askari and Shanavas Khan A.E.
Dubas Bug (Ommatissus lybicus Aschc & Wilson), A New Pest
of Date Palm and Canary Palm in Helwan Governorate – Egypt
OP28
Abd Rabou Eid Hussain
OP29
509
595
607
621
Some Inorganic Salts for Production of Sterile Adults of the Red Palm
Weevil Rhynchophorus ferruginous (Coleoptera: Curculionidae)
Aziza Sharaby, F.M. EL-Hawari, S.A. Ibrahim
641
Observations on the biology and ecology of Oryctes
agamemnon arabicus Fairmaire, 1896 (Coleoptera:
Scarabaeidae): pest of date palm tree in Tunisia
Rasmi Soltani
649
xvi
OP33
OP34
A trap for autodissemination of the endomopathogenic fungus
Beauveria bassiana by red palm weevil adults in date palm plantations
R. El-Sufty, S. Al Bgham, S. Al-Awash, A. Shahdad and A. Al Bathra
Eectrophoretic mobilities of haemolymph protein in date
palm pest Rhynchophorus ferrugineus.
Annie Thomas and C.R.M. Nair
Activity of insect fauna during night in date palm orchards of Central Iraq.
OP35 Mohammed Zaidan Khalaf; Ali Khaywon Shbar; Falah Hansh Naher
OP36
Effect of Fusariosis on the metabolism of phenolics compounds of
date palm leaflets, resistant and sensitive to the disease.
Saida OUAFI, Nicole BOUNAGA.
Population dynamics of the Red Palm Weevil
(Rhynchophorus ferrugineus (Oliv.) on date palm plantations
in 6th October Governorate.
OP37
El-Lakwah, F. A. M. ; EL-Banna, A. A; El-Hosary, Rasha A
and El-Shafei, W.K.M.
667
681
693
705
715
OP38
The study of fungal infections of dates during storage
Oustani Mabrouka
PP11
Acaricidal , Ovicial and Repellent Activities of Some Plant
Extracts on the Date Palm Dust Mite, Oligonychus
afrasiaticus Meg. (Acari: Tetranychidae)
Badr El-Sabah A. Fetoh and Kholoud A. Al-Shammery
PP12
Occurrence of some diseases in date palm trees in Upper
Egypt and its control
Eman S. H. Farrag and Sabry Y. M. Mahmoud
763
High-Rise Fully Automated Refrigerated Warehouses, New
Trend for Date Storage in KSA
Atef M.Elansari & Sultan Al-Thagafi
779
Exploiting the potential of freeze technology to improve quality
and yield, and streamline the product supply of Dhakki dates
OP40 Shahzada A. Saleem, Ahmad K. Baloch, Ambreen A. Saddozai,
and Waqar A. Khan
793
OP39
OP41
Quality of steamed and microwaved dates
Isameldin B. Hashim, Alia Akram and Hanan S. Afifi
OP42
Effect of X-Ray Irradiation on food borne pathogens and
xvii
731
749
809
OP43
OP44
Sensory Properties of Dates
Aleid Salah, Kirk Dolan, Sanghyup Jeong , Muhammad
Siddiq , Bradley Marks, Karim Maredia
823
Enhancement of Date Palm as a Source of Multiple
Products: Examples from Other Industrialized Palms
Dennis V. Johnson
833
Effect of biological treatments of date palm on feeding lambs.
A.A. Mahrous M. A. El-Manylawi
845
Study on the effect of the Use of Diets Containing Different
Levels of Crushed Date Seeds on Growing Assaf Lambs
OP45 H.A. Al - Shanti , K.J. Al -Shakhrit ,M.F. Al - Banna , I.E .Abu
Showayb and A.M. Kholif
857
Composition and functional properties of the date fruit residue
(DFR) a by-product of date syrup (Debis) production
Isameldin B. Hashim and Ali H. Khalil
875
OP47
Treated effect of palm pollen grains extract (Phoenix dactylifera
L.) on the sterility induced by acrylamide in male rabbits.
A.A.Sawad
889
OP48
Response of growing New Zealand white rabbit to dietary date
stone meal with or without commercial enzyme supplementation.
EL-Manylawi, M.A.F.
899
PP13
Analyzing the Role of Agricultural Extension services in
Date Palm Orchards in Khairpur, Pakistan.
Shakeel Ahmed Ibupoto, Wazir Ali Maitlo and Mushtaque Ahmed Jatoi
911
OP46
PP14
PP15
Development of New Innovative Procedure for Accelerated
Ripening of Dhakki Dates.
Shahzada A. Saleem, Ambreen A. Saddozai, Muhammad Asif,
Muhammad Mansoor and Ahmad K. Baloch
Model Study of Application Total Quality Management
(TQM) for Safe and food Quality Foods Products by ISO
9000 Quality Standards and HACCP System
Implementation at Date Palm Packaging house
E.EL-Tanboly, M. A. EL-Hofi, and Azza Ismail
A Study on the Economic Feasibility of Date Palm
Cultivation in the Al-Hassa Oasis of Saudi Arabia
OP49
Al-Abbad, M. Al-Jamal, Z .Al-Elaiw, F. Al-Shreed, and H. Belaifa
xviii
921
937
955
Improving the Market profit distribution effected date palm
production in Yemen
OP50
Mohamed Abdulrahman Hashem Al-Hebshi
969
A pilot study to evaluate the use of date palm residues (leaves,
Leaf, Kornav) such as organic fertilizer in the in the desert areas
OP51
Oustani Mabrouka
979
OP52
Preparation of novel product by using mixture of meat and dates
Hanaa A. Abdel Aziz and El-Kafrawy, T.M.H.
xix
999
xx
List of Authors
Abahmane, L.
OP 16
Al-Jamal, M.
OP 49
Abd-El Kareim,
OP 17
Al-Khalifah, N.S
PP 10
Abdelbaky,M.A.
PP 05
Al-Khayri, J.M.
OP 01
Abdel-Galil, H.A
PP 04
Al-Khayri, J.M.
OP 24
Abdel-Hameed, N
PP 03
Al-Shakhrit, K.J.
OP 45
Abdoulhadi, I.A.
OP 03
Al-Shammery, K.A.
PP 11
Abo Rekab
PP 06
Al-Shayji
OP 02
Abul-Soad, A.A.
OP 11
Al-Shreed, F.
OP 49
Afifi, H.S.
OP 41
Al-Shryda, F.
OP 03
Ahmed, E.F.S.
PP 04
Annie Thomas
OP 34
Ahmed, F.A.
OP 04
Arab, Y.A.
OP 29
Akram, A.
OP 41
Artmann, A.T.
OP 21
Al-Shanti, H.A.
OP 45
Artmann, G.M.
OP 21
Al- Hebshi, M.A.
OP 50
Asif,M.
PP 14
Al-Abbad, A.
OP 49
Askari, E.
PP 10
Al-Ali, S.
OP 03
Assani, A.
OP 13
Al-Awash, S.
OP 33
Attalla, A.M.
OP 08
Al-Banna, M.F
OP 45
Bailey, A.M.
OP 20
Al-Bathra, A.
OP 33
Baker, E.I.
OP 18
Albgham, S.
OP 33
Bakheet, S.A.
OP 27
Al-Elaiw, Z.
OP 49
Bakr, E.I.
OP 06
Al-Enezi, N.A.
OP 24
Baloch, A.K.
OP 40
Al-Jabr, A.M.
OP 03
Baloch,A.K.
PP 14
xxi
Baum, M.
OP 25
El-Deeb, H.M.
OP 29
Bekheet, S.A.
OP 19
El-Hawari, F.M.
OP 31
Bekheet, S.A.
OP 27
El-Hofi, M.A.
PP 15
Belaifa, H.
OP 49
El-Hosary, R.A.
OP 37
Ben Abdallah, A.
OP 03
El-Kafrawy, T.M
PP 03
Bouguedoura, N.
OP 13
El-Kafrawy, T.M.
OP 52
Bounaga, N.
OP 36
El-Kobbia, A.M.
OP 08
Chabane, D.
OP13
El-Kozary, S.
OP 06
Chellan, S.
OP 02
El-Lakwah, F.A.M.
OP 37
Darwesh, R.S.
OP 17
El-Manylawi, M.A
OP 44
Dawoud, D.H.
OP 04
EL-Manylawi, M.A.F. OP 48
Dawoud, H.D
PP 02
El-Naggar, M.A.
OP 30
Digel, I.
OP 21
El-Nawam, S.M.
OP 08
Eisaa, M.A.
OP 14
El-Rauof, F.A.
PP 02
El Assar, A.M.
PP 08
El-Salhy, A.M.
PP 04
Elansari, A.M.
OP 40
El-Sehrawy,O.A.M
PP 08
El-Bahr, M.K.
OP 19
El-Shafei, W.K.M.
OP 37
El-Bahr, M.K.
OP 15
El-Sharbasy, S.S.H.
OP 23
EL-Baky, M.A.A
OP 12
El-Sufty, R.
OP 33
El-Bana, A.
OP 06
El-Tanboly, E.
PP 15
El-Bana,A.A.
PP 04
Farrag , E.S.H
PP 12
El-Bana,A.A.
PP 05
Farrag, H.M.A.
OP 17
El-Banna, A.A.
OP 37
Ferchichi, A.
OP 26
El-Dawayati, M.M.
OP 18
Fetoh, B.E.A.
PP 11
El-Dean, A.S.
OP 12
Gadallah, E.G
PP 06
xxii
Ghazawy, H.S.
OP 06
Khurshid, K.
OP 03
Gomaa, A.H.
OP 14
Kirk, D.
OP 42
Gomaa, A.H.
OP 18
Linder, P.
OP 21
Gomaa, A.H.
PP 09
Lobna , M.A.
PP 05
Hamwieh, A.
OP 25
Mabrouka, O.
OP 38
Haana, A.A.
OP 52
Mabrouka, O.
OP 51
Hamza, H.
OP 26
Madboly, E.A
PP 09
Hashim, I.B.
OP 41
Madboly, E.A.
OP 14
Hashim, I.B.
OP 46
Mahmoud, S.Y.M.
PP 12
Hussain, A.R.E.
OP 28
Mahrous, A.A.
OP 44
Ibrahim, S.A.
OP 31
Mailto, W.A.
PP 13
Ibupoto, S.M
PP 13
Mansoor, M.
OP 25
Imranullah
OP 09
Maredia, K.
OP 42
Ismail, A.
PP 15
Marks, B.
OP 42
Ismail, O.M.
OP 22
Matwally, H.A.A.
OP 12
Jain, S.M.
OP 10
Mostafa, K.
OP 30
Jatoi, M.A.
PP 13
Nady, N.H
PP 06
Johnson, D.V.
OP 43
Naher, F.H.
OP 35
Kahtani, M.D.F
OP 30
Nair, C.R.M.
OP 34
Kasrati , A.
OP 07
Niblett, C.L
OP 01
Kassem, H.A
OP 05
Niblett, C.L.
OP 20
Khalaf , M.Z.
OP 35
Omar, A.E.
PP 01
Khalil, A.H.
OP 46
Ouafi, S.
OP 36
Khan, W.A.
OP 40
Oussouf, F.M.
OP 07
Kholif, A.M.
OP 45
Qaddoury, A.
OP 07
xxiii
Rasheed, M.F.
PP 07
Shbar, A.H.
OP 35
Rizk, R.M.
OP 23
Showayb, I.E.A.
OP 45
Saber, T.Y.
PP 05
Siddiq, M.
OP 42
Saddozai, A.A.
PP 14
Soliman, K.A
OP 23
Saddozai, A.A.
OP 40
Soliman, S.S.
OP 22
Salah, A.
OP 42
Soltani, R.
OP 32
Saleem, S.A
PP 14
Taha, H.S.
OP 15
Sallem, S.A.
OP 40
Taha, H.S.
OP 19
Sanghyup, J.
OP 42
Thabt, T. M.
OP 30
Sarhan, E.A.
OP 30
Younis. R.A.A.
OP 22
Sawad. A.A.
OP 47
Zaid, Z.E.
OP 18
Shahdad, A.
OP 33
Zeinab, A.M
PP 06
Shanavaskhan, A.E.
PP 10
Sharaby, A.
OP 31
xxiv
Date Palm and Dates Production
1
2
OP 01
Envision of an International Consortium for Palm Research
Jameel M. Al-Khayri1 and Charles L. Niblett2
1
Department of Agricultural Biotechnology, College of Agricultural and Food
Sciences, King Faisal University, Saudi Arabia, jkhayri@kfu.edu.sa
2
Venganza, Inc., 840 Main Campus Drive, Raleigh, NC 27606, USA,
niblett@venganzainc.com
Abstract
An increasing number of insects and diseases are destroying palm trees of high
economic and aesthetic value throughout the world. They include the extremely
serious red palm weevil (Rhynchophorus ferrugineus) occurring from the Middle East
to Asia and California, USA; R. palmarum vectoring the very serious red ring
nematode (Bursaphelenchus cocophilus baujard) in the Caribbean and Central and
South America; at least three species of Fusarium, one possibly airborne, occurring
from Morocco to Florida, USA; Phytophthora palmivora destroying the oil palm
industry in Colombia; Ganoderma causing serious losses from Malaysia to Florida,
USA; and phytoplasmas including lethal yellowing and Al-Wijam. These pests affect
numerous palm species and many municipalities and industries. Efforts to reduce
damage caused by these pests are being made by scientists worldwide. Individual
efforts may be duplicative and sometimes unsuccessful. Interdisciplinary approaches
combining expertise of pathologists, entomologists, biotechnologists, and breeders
should be more effective. We propose the formation of an International Consortium
for Palm Research (ICPR) to devise control methods for palm pests and palm
improvement in general. Funding of this nonprofit Consortium is envisioned to include
donations from potential beneficiaries in proportion to the potential benefits received.
Potential donors might include agricultural ministries and public and private
organizations in various affected countries as well as international organizations
interested in palm development. The host of ICPR is envisioned to be the Kingdom of
Saudi Arabia with an international board directing activities and awarding meritorious
research proposals. Tangible evidence of scientific accomplishment, publications,
ancillary funding, and international patent applications would be key criteria for
receiving research awards.
Key words: Agriculture, Arecaceae, Biotechnology, Collaboration, Consortium,
Development, Research, Palm, Pests
3
Introduction
Many species of palm trees provide food, shelter, fiber, income and aesthetic value
to millions of the world’s citizens. Despite these major economic and social
contributions, palm production throughout the world is threatened by an increasing
number of insect and disease pests, and overall production inefficiency [55]. In this
communication we propose the establishment of an International Consortium for Palm
Research (ICPR) to foster research collaboration with the objective of supporting palm
improvement on a global basis. Despite notable research progress, numerous problems
still persist and threaten the existence of many palm species.
Consortium is a Latin word meaning partnership, association or society. It can be
further defined as “an agreement, combination, or group (as of companies) formed to
undertake an enterprise beyond the resources of any one member” [1]. This
organizational and funding approach is a common practice to support various aspects
of agriculture worldwide, where the research needs for a commodity are greater than
the resources of a single member. These groups may be formed under several different
names including associations, foundations and growers groups for commodities as
diverse as potatoes, grapes, wheat, hops, soybeans, corn, cotton, horseradish, citrus,
etc. Each consortium collectively funds research specific to their commodity needs
through a self-tax or check-off system. The growers contribute a certain amount or
percentage per unit of production (pound, box, bushel, barrel, acre, hectare, etc.). The
funds are collected and pooled, and then allocated to support meritorious competitive
research projects deemed necessary to improve production practices and sustain yields
for that particular commodity. We propose this consortium approach for solving
problems in palm production on a global basis.
Background
The order Arecales contains only one family, the Arecaceae (syn. Palmae), which
comprises 2,400 species in 190 genera. Recently a new phylogenetic classification of
Arecaceae was published [2], and modern molecular techniques have contributed
significantly to establishing genetic relationships among the palms [3, 4, and 5].
Moore and Uhl [6,7] stated that the palms with the greatest importance in world
commerce are the coconut (Cocos nucifera) and the African oil palm (Elaeis
4
guineensis); both are prime sources of vegetable oil and fat. The top 5 coconut
producing countries are Indonesia, Philippines, India, Brazil and Sri Lanka,
respectively. Collectively, they produce 50.7 million tons annually, or 93% of the
world’s production [8] Coconut has culinary, domestic and industrial uses, and nearly
12 million hectares are planted in 86 countries. About 96% of the crop is grown by 10
million resource-poor families, on holdings under 4 ha. and more than 80 million
people depend directly on coconut and its processing for their livelihoods [9].
Oil palm provides a major source of oil for cooking, biodiesel fuel production and
many industrial uses. It is cultivated in tropical areas of Asia, Africa and South
America. With the global demand for edible vegetable oils increasing strongly in
recent decades, palm oil production has expanded rapidly to meet that demand. Since
the 1990s the area under cultivation increased about 43%. Seventeen countries produce
palm oil, and about 4.5 million people earn a living from it. The five major producing
countries are Indonesia, Malaysia, Thailand, Colombia and Nigeria, respectively.
Collectively, they produce 47 million MT annually, or 94% of the 50.2 MMT
produced, with Indonesia alone producing 51% of the total [10, 11].
The date palm (Phoenix dactylifera) also is of major importance. It has been
cultivated as a tree crop for at least 5,000 years [12]. A very important plant
throughout the world, it is perhaps the most important plant in Saudi Arabia and
throughout the Middle East. It has high socioeconomic importance, due not only to its
food value, but also its capacity to provide many other products such as shelter, fiber,
clothing, aesthetic beauty and furniture [13]. It has high natural tolerance to very
adverse growing conditions, including drought, salinity and high temperatures [14]. In
2007 nearly 1.1 million ha of date palm were harvested, yielding 6.91 million tones.
The major producers were Egypt (19%), Iran (15%) and Saudi Arabia (14%) [8].
In addition, many species of palms are used extensively as ornamentals in warm
regions throughout the world, or indoors when a tropical effect is desired. The word palm
is even used to indicate the tropical or verdant nature of municipalities such as Palm
Beach, Palm Coast and Palm Springs in the United States. Sales of palms for ornamental
purposes provide millions of dollars annually to economies throughout the world. Annual
sales are valued at $70 million in California and $127 million in Florida [15] alone.
5
Breeding palms for pest resistance or for other desirable characteristics by
traditional breeding techniques is very difficult and time consuming. It is hindered by
life cycle longevity, the palm’s highly heterogeneous genetic nature, and the difficulty
to propagate uniform plants in large numbers. However, with recent scientific
advances palm improvement can be accomplished through biotechnological
approaches [16], such as genetic transformation [17, 18, 19, 20] and in vitro selection
[21, 22, 23]. Combining these approaches with other emerging molecular techniques
holds great promise to control insects [24] and disease pests of palm [Niblett and
Bailey 2011, these proceedings].
Palms are affected by large number of pests, including insects, nematodes and
diseases caused by fungi, bacteria and phytoplasma [25]. Four of these major pests
will be discussed as examples:
Red Palm Weevil (RPW): The RPW, Rhynchophorus ferrugineus, a member of
Coleoptera: Curculionidae, is a concealed tissue borer and lethal pest on over 20
species of palm, including date palm (Fig. 1) and coconut palm [26, 27]. Gomez and
Ferry [28] indicate it "has become the most important pest of the date palm in the
world". Its aboriginal home is South and Southeast Asia, where it is a key pest of
coconut [29, 30]. First reported on date palm in Iraq [31], then from Rass-El-Khaima
in the UAE in 1985, it reached the eastern region of the Kingdom of Saudi Arabia in
1985, and afterwards spread to numerous other areas in the Kingdom [32]. It was
recorded in Iran in 1990 [33], then in Egypt in 1992 [34]. By 1994, it had been found
in the south of Spain [35] and in 1999 in Israel, Jordan and the Palestinian Authority
Territories [36].
The RPW has now spread to all the countries of the Gulf region in the Middle-East,
infesting approximately 5% of the palms in the region with an annual infestation rate
of about 1.9% [26, 37]. FAO has now identified it as a category-1 pest of date palm in
the Middle-East [38] Subsequently, the weevil moved from North Africa into Europe,
where it was reported for the first time in Spain, Portugal and the Canary Islands [53,
54] and in 2009 in the Caribbean (Curacao, Dutch Antilles), potentially from date
palms imported from Egypt for landscaping [39]. In August of 2010 the RPW was
reported in Laguna Beach California [15], where it now poses a serious threat to
6
California’s $30 million dollar date crop industry, as well as ornamental palm tree
sales valued at $70 million in California and $127 million in Florida.
The RPW can be controlled by insecticide applications, but it is expensive and
pollutes the environment. Instead, an integrated pest management approach has been
adopted. It includes prohibition of movement of infested plants, extensive monitoring
of insect populations, the use of food-baited pheromone traps to reduce adult weevil
populations and strategic insecticide applications. But this program is labor intensive
and expensive to implement.
Fusarium diseases: Bayoud disease (BD) of date palm is caused by the soil-borne
fungus Fusarium oxysporum f. sp. albedinis (FOA). It is a lethal root rot and vascular
wilt disease of date first reported in 1870 in the Drâa valley of Zagora, Morocco. It has
been spreading continuously eastward, and within one century had killed more than
twelve million palms in Morocco and three million in Algeria [37, 40]. Oases that
formerly had 300-400 palms per hectare were reduced to 40-50 palms per hectare [41],
and BD was destroying the most renowned varieties such as Medjool, Deglet Nour,
and Bou Fegouss). BD also has reduced the production of desirable annual crops
formerly supported under date palm culture, and has accelerated the desertification of
the region, with farmers abandoning their land and moving to large urban centers.
FOA is a soil inhabiting fungus, persisting as hard-walled chlamydospores in dead
tissues of diseased palms. Chlamydospores are released from disintegrating tissues
into the soil, where they remain dormant and survive for more than eight years.
Controlling BD is difficult, if not impossible, by current cultural methods once FOA
becomes establishes in a plantation, because of the persistence of the fungus, the
movement of soil and the flow of irrigation water. Individual infected plants can be
eradicated and the soil fumigated with methyl bromide or chloropicrin, but this is
expensive and time-consuming. Some BD-resistant date palm selections have been
reported [41], but introgression of resistance into desirable varieties by conventional
breeding is difficult and time-consuming.
In addition to FOA there are several additional Fusarium pathogens of palm. Elliott
[42] recently confirmed the spread of new palm diseases in Florida including two
formae speciales of Fusarium oxysporum, with f. sp. canariensis (FOC) causing
7
Fusarium wilt of Canary Island date palm (Phoenix canariensis) and a new forma
specialis causing Fusarium wilt of queen palm (Syagrus romanzoffiana) (Fig. 2) and
Mexican fan palm (Washingtonia robusta). FOC also attacks Canary Island date palm
in Australia, Italy, France, Japan, the Canary Islands and California, where it can kill
40-50 year old trees [43, 44, 45]. The California isolate caused sufficient concern to
warrant a quarantine to protect the California date industry [43]. Fusarium remains a
threat to palm species worldwide.
Phytophthora palmivora: Bud rot disease or “pudricion del cogollo” is a major
disease of African oil palm in Colombia (Fig. 3). It has killed thousands of trees and
been known for more than 40 years in Central and South America. But the causal
agent has only recently been identified as Phytophthora palmivora [46]. It has spread
widely in Colombia and threatens to make oil palm production unprofitable. Bud rot
of coconut also is caused by P. palmivora [47]. It would be economically ruinous to
the Malaysian oil palm industry if P. palmivora was introduced there. Two species of
Ganoderma currently cause butt rot and serious losses in oil palm production in
Malaysia [48].
Phytoplasmas: Phytoplasmas are bacteria without cell walls, and they cannot be
cultured in microbiological media. There are several different phytoplasmas that affect
palms. These diseases usually occur in tropical or subtropical climates and cause
symptoms ranging from mild yellowing to death of the infected plants. Transmission
from plant-to-plant requires an insect vector, usually a leafhopper or plant hopper.
Probably the best known and most destructive phytoplasma disease is lethal
yellowing (LY) of coconut, which has killed millions of coconut palms in Florida and
throughout the Caribbean (Fig. 4). It infects and kills many species of palm including
P. dactylifera, and is vectored by a plant hopper, Haplaxius crudus (previously
Myndus crudus) [49]. Texas Phoenix palm decline (TPPD) occurs on Phoenix
canariensis in Texas and on Phoenix sp. and Sabal palmetto in Florida [50]. Al Wijam
(AW), another phytoplasma disease, was first observed on date palm in Al Hassa,
Saudi Arabia [51], and it was recently characterized by Alhudaib et al. [52].
Phytoplasma are currently classified by comparing restriction fragment length
polymorphism (RFLP) patterns for polymerase chain reaction (PCR) amplicons of
8
their 16S rDNA and naming them after the major phytoplasma whose RFLP pattern
they most closely resemble. Therefore, LY is Candidatus Phytoplasma palmae
subgroup 16SrIV-A; TPPD is Candidatus Phytoplasma palmae subgroup 16SrIV-D;
and AW is Candidatus Phytoplasma asteris group 16SrI [49, 50, 52].
Phytoplasma diseases cannot be controlled, but symptoms are diminished and tree
life extended by injections of tetracycline. This is practical only for "specimen trees"
in an expensive landscape [49]
Genetic resistance is available in palm species to some or all of the serious pests of
palm, but it has not been introgressed into those which are susceptible because of the
difficulties in palm breeding. Current and emerging molecular techniques show
promise for overcoming many of these impediments.
3. Structure and function of the international consortium for palm research
Justification: Numerous researchers are working on palm improvement worldwide.
Unfortunately, many are working in isolation, their research may be poorly funded,
and their research may inadvertently duplicate, or repeat that of others. To minimize
these issues, to maximize research output and to give palm research new visibility we
envision the creation of the International Consortium for Palm Research (ICPR).
Objective: To foster innovative research collaborations for palm improvement,
productivity and utilization.
General Priorities:
Improve resistance to palm pests and other biotic stresses.
Enhance palm tolerance to abiotic stresses and global climate change.
Promote conservation and utilization of genetic diversity of palm.
Develop novel industrial applications for palm products (e.g. Bio-energy,
Pharmaceuticals, etc.)
Multidisciplinary: The ICPR advocates interdisciplinary research approach involving
different science disciplines including biotechnology (e.g. bioinformatics, genetic
engineering genomics, molecular biology, plant tissue culture), entomology, plant
breeding and genetics, plant pathology, and plant physiology.
Membership: ICPR membership is free and open to any person having an interest in
palms and palm improvement. Membership may be revoked if privileges are abused.
9
Location: We propose that the ICPR headquarters be located at the Date Palm
Research Center, King Faisal University, Al-Hassa Saudi Arabia because of their
proven expertise in palm technology, the interest expressed by the institution, and its
proximity to many of the important research problems.
Governance: The ICPR will be managed by a 5 member, unsalaried Board of
Directors (BoD) representing different geographical regions, commodities and
expertise. They will normally serve three-year terms, except for the initial BoD, where
to ensure continuity and orderly succession one member will serve one year, two will
serve two years, and two will serve three years. The BoD will make the daily
management decisions, and their decisions will be made by majority vote and
announced to the Membership. The BoD shall present proposed bylaws to the
membership for ratification and adoption, and they shall solicit the involvement of the
membership by electronic polls and for review of manuscripts and submitted grant
proposals, and for other issues as they may occur.
Bylaws: The ICPR bylaws will be drafted by the BoD and sent out to the membership
for ratification. Those of the West Chester University Research Consortium might be
viewed as an example at: http://www.wcupa.edu/wcurc/bylaws.htm
Funding acquisition: Support funding will be solicited from growers and grower
organizations involved with date, oil, coconut and ornamental palms. Equally
important are the countries, states, municipalities and companies that depend on palm
trees to provide the ambiance in which they do their business. These include such
diverse agencies as the governments of Saudi Arabia and Malaysia, Colombian Oil
Palm Growers, the Walt Disney Company, cities of Palm Springs, Palm Beach,
Miami, Miami Beach, California Date Growers Association, Florida, Arizona, Nevada
and California Departments of Tourism, Florida and California Ornamental Growers
and Nurserymen’s Associations, etc. – all contributing in proportion to the value that
palms bring to their agency or institution.
Services and activities: The ICPR will maintain an active and up-to-date website,
with a bulletin board for notices of general interest, a list of active members, including
names, addresses and email addresses, a periodic electronic newsletter, and a literature
collection of recent major palm publications accessible as PDF files.
10
Funding of research proposals: Research priorities will be established annually by
the BoD, and when funds are available a call for proposals will be sent to the
membership. Proposals will be peer-reviewed by a committee of the membership
and/or outside experts selected by the BoD. Key criteria for receiving research awards
will include quality and appropriateness of the proposal, tangible evidence of scientific
accomplishment, publications in international refereed journals, ancillary funding, and
international patent applications. Within three months following completion of the
research, a detailed report of 5 pages will be submitted to the BoD, along with any
publications or patent applications.
To ensure the success of the ICPR, we are keen to receive comments and
suggestions from all interested parties.
Acknowledgments
The authors thank Dr. J. Romeno Faleiro, Plant Protection Laboratory, ICAR
Research Complex for Goa, India (jrfaleiro@yahoo.co.in) and Dr. Monica L. Elliott,
Fort Lauderdale Research and Education Center, University of Florida, USA
(melliott@ufl.edu) for providing photographs and information about palm insects
and diseases.
11
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[52] Alhudaib K., Y.Arocha, M. Wilson and P. Jones. 2007. “Al-Wijam”, a new
phytoplasma disease of date palm in Saudi Arabia. Bull. Insectol. 60: 285-286.
[53] Faleiro J.R. 2006a. Insight into the management of red palm weevil
Rhynchophorus ferrugineus Olivier based on experiences on coconut in India and
date palm in Saudi Arabia. In Proceedings of the 1st International Workshop on
Red Palm Weevil, 28–29, November 2005, IVIA, Valencia, Spain.
[54] Faleiro J.R. 2006b. A review of the issues and management of the red palm weevil
Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date
palm during the last one hundred years. Int. J. . Tropic. Insect Sci. 26: 135–154.
[55] El-Juhany L.I. 2010. Degradation of Date Palm Trees and Date Production in
Arab Countries: Causes and Potential Rehabilitation. Aust. J. Basic Appl. Sci. 4:
3998-4010.
16
Fig. 1. Red palm weevil infestation. Source: [38]
Fig. 2. Queen palm in Florida dying from Fusarium wilt
17
Fig. 3. Thousands of oil palms dying in Colombia from infection by Phytophthora palmivora
18
Fig. 4. Coconut palms in Jamaica dying from lethal yellowing phytoplasma
19
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OP 02
Development of Tall X Dwarf Hybrid Date Palm
C. Sudhersan and Y. Al-Shayji
Biotechnology Department,
Food Resources and Marine Sciences Division
Kuwait Institute for Scientific Research
P.O.Box 24885, Safat 13109, Kuwait.
Email: schellan@kisr.edu.kw / sudher_san@yahoo.com
Abstract
Tree height in date palm (Phoenix dactylifera L.) is one of the major difficulties
affecting date production. Climbing on tall date palms for various pre-harvest and
post-harvest activities is dangerous, difficult and expensive process. Therefore,
research focussing towards the development of dwarf date palm trees is necessary for
the date crop improvement. Recently, the authors have attempted to develop dwarf or
medium size date palm through hybridization technique. Female date palm cultivars
Barhi, Madjhool and Sultana were cross pollinated with male pollen of Phoenix pusilla
(dwarf date palm) and produced interspecific hybrid seeds. The resultant hybrid seeds
failed to germinate due to embryo abortion at the seed maturity stage. Therefore, the
hybrid embryos were isolated under aseptic conditions during the early stages of fruit
development for in vitro culture. A protocol for the isolation of hybrid embryos and
production of hybrid plantlets via in vitro embryo rescue method was developed.
Isolated young hybrid embryos were cultured in vitro using the standardized embryo
rescue procedure and produced hybrid plantlets. Rooted plantlets were acclimatized to
the greenhouse environment and hardened to the field condition. A hardened Tall x
Dwarf hybrid date palm plantlet was planted in the KISR date palm orchard and
maintained for field evaluation. This is believed to be the first successful trial on Tall x
Dwarf hybrid production in date palm.
21
Introduction
Date palm (Phoenix dactylifera L.) is one of the oldest crops known to man.
However, date crop improvement is lagging behind due to its long life cycle,
heterogeneous nature and slow growth habit. It is necessary to improve this tree crop
for the high quality and economically feasible date fruit production. Date palm crop
improvement through breeding is slow when compared to other crops, due to the time
consuming nature to obtain hybrid palms from the first inter-varietal crosses.
During past centuries, through natural crossing within the species more than 2000
date-palm cultivars were developed and few of them were propagated clonally for
quality fruit production worldwide. In 1948 a date breeding program was started at
USDA, Indio, California by Nixon and Farr and their results showed only 1 % of the
female population had the potential for improvement and inclusion in further advance
breeding trials (Carpenter 1979; Carpenter and Ream 1976; Nixon and Farr 1965;
Ream 1975). This breeding program was terminated in the year 1978 and some of the
plants developed were conserved in the National Date Palm Germplasm Repository in
California (Krueger 1998). The earliest breeding attempts conducted in the University
of Arizona in 1912 with date palm cv. Deglet Noor failed to give similar or better
quality fruit than the parent (Anon 1982). In Algeria, an early flowering male variety
was selected through breeding programs (Monciero 1959). Breeding for bayoud
disease resistance was carried out in Morocco (Saaidi et al. 1981). However, there is
no report on interspecific hybridization on date palm for the development of dwarfs.
Therefore, recently an interspecific hybridization trial using female date palm
(Phoenix dactylifera L.) and male dwarf palm (Phoenix pusilla) has been attempted to
develop short hybrid date palm. The attempt was successful but the resultant hybrid
seeds were not viable. Therefore, Tall x dwarf hybrid plants were produced through
the in vitro embryo culture method. The details are presented herein.
Materials and Methods
Dwarf palm pollen was obtained from Phoenix pusilla introduced to Kuwait in the
year 2000 (Sudhersan 2004). Female date palm cvs. Barhee, Medjhool and Sultana
were selected from the tissue culture date palm orchard established in 2000 at the
Kuwait Institute for Scientific Research (KISR) campus, Kuwait. Male pollen was
22
collected from P. pusilla during the summer months (May-July) when no date palm
pollen was available. Pollen was stored in sterile glass bottles at 4° C temperature for
8-9 months. Late-maturing female date palm inflorescences (inflorescence open in late
April) of the selected date palm cultivars were pollinated with dwarf date palm pollen.
Late maturing date palm inflorescences were selected for this experiment in order to
avoid mixing of date palm pollen which is available during the early periods
(February-March) in the atmosphere. The unopened mature female inflorescences of
the selected date palm cultivars were opened with a surgical knife and the dwarf date
palm pollen was dusted over the flowers and covered immediately with paper to avoid
date palm pollen mixing. The entire interspecific cross-pollination process was
carefully carried out and paper covers were removed only after 15 days. Fruits were
carefully observed during their different stages such as hababouk (7 days after
pollination); kimri (35-120 DAP); khalal (125-175 DAP); rutab (180-200 DAP); tamar
(200 DAP). Seed development was also observed frequently (every 15 days) by
opening the fruit at different stages.
Fruits produced by this interspecific crossing were collected during their different
developmental stages (Zaid and De Wet 2002) to check the viability of the hybrid
embryos. For this purpose the fruits were washed in soap solution for few minutes and
treated with 30 % commercial Chlorox with a drop of Tween 20 for 30 min followed
by three wash in sterile distilled water. The immature seeds were isolated from the
surface sterilized fruits under aseptic conditions under a laminar hood and cultured on
MS basal culture media. After 15 days under dark in vitro culture, the seeds showed a
projection on the dorsal side where the embryo is located. The embryos were isolated
under the aseptic conditions and cultured in different culture media using MS basal
salts, different concentrations of sucrose and growth hormones. Embryo viability
during the stages of fruit development and the stage at which the embryo abortion
occurs were identified.
The immature hybrid embryos rescued in vitro were maintained up to the
maturation stage in cultures and made to germinate through embryo culture procedure.
Germinated hybrid embryos were transferred to the hormone-free MS basal medium
for plantlet growth and elongation. The hybrid plantlets elongated to 10 cm height
23
were transferred to the rooting media containing 0.1 mg/l NAA. Rooted plantlets were
acclimatized using the standard acclimatization procedure.
Results
The interspecific crossing was successfully carried out between female date palm
(Phoenix dactylifera) and male dwarf date palm (P. pusilla). Both the species were
found to be compatible with each other and fertilization occurred. Post-fertilization
changes in the fruits of interspecific cross were similar to normal date palm. Fruit
development and seed development were similar during the early stages.
Seed development occurred in both the treatment and the control at the initial stages
of fruit development. Later, at kimri stage, seed growth and development was arrested.
At the late khalal stage, fruits showed an interior cavity and a small rudimentary seed
at the anterior end of the fruit. The rudimentary hybrid seeds were small and similar to
a grape seed in shape and size (Fig. 1). Rudimentary seeds were 5 mm long, 3 mm
wide and weighed an average of 40 mg, whereas normal seeds were 25 mm long, 12
mm wide and 120 mg in weight. Viable hybrid embryos were observed up to the
Khalal stage of fruit development. At the last stage during the dehydration of the seed
and embryo, abortion of embryo occurred due to the lack of enough endosperm tissue
to hold the embryo inside.
During the different stages of seed in in vitro culture, viable embryos responded to
different types of culture media (Table 1). The embryos isolated from the khalal stage
seeds germinated in MS basal medium without any growth hormones. The embryos
isolated from early stages of its development failed to germinate in the hormone-free
MS medium. Mature hybrid embryos germinated in the hormone-free MS media
(Table 2). Healthy adventitious roots were produced when the hybrid plantlets were
transferred to rooting culture media containing 0.1 mg/l NAA. A total of 5 rooted
plantlets were acclimatized up to the greenhouse stage. One of the hybrid plantlet
planted inside the KISR date palm orchard in the year 2008 showed normal growth
and development in the field (Fig.2).
The hybrid plant has not yet produced flowers. The leaves of the hybrid plant were
smaller than the same aged normal control date palm and larger than the male parent.
The hybrid produced axillary shoots similar to the female parent and unlike the male
24
parent (Figs. 2-3). The stem height was less than the control female parent and more
than the male parent.
Discussion
In date palm cultivation, pruning, pollination, fruit thinning, bunch removal and
fruit harvesting are highly essential for good quality fruit production. The cost of date
production increases when the trees grow taller, due to the high labour cost in many
date producing countries. Mechanization is also expensive and unjustifiable in the case
of small growers. Frequent climbing for fruit harvesting is highly dangerous in the
case of taller old trees. Tree height is one of the major constraints to good quality date
production. Therefore, an attempt was made to cross date palm with a dwarf palm
species related to the date palm.
The pollen of the male dwarf palm was compatible with the date palm flowers.
Fertilization and fruit set were normal. However, seed development was totally arrested
at the fruit ripening stage. Previous reports of such interspecific crosses revealed that
pollen from Phoenix reclinata, P. canariensis, P. roebelenii and P. rupicola crossed
with date palm for fruit-quality improvement, failed to produce better-quality fruit;
whereas the cross between the date palm and P. sylvestris produced slightly larger fruits
than normal (Nixon 1935). In the present study, using P. pusilla pollen, fruits were
equal or larger in size depending on different date palm cultivars but were seedless
(Sudhersan et al., 2009). The seedless nature was due to the poor development of
endosperm during the fruit growth. Boyes and Thompson (1937) found shrivelled small
seeds with floury endosperm in interspecific crosses due to chromosome imbalance in
the endosperm. Similarly, chromosome imbalance may be the reason for the poor
endosperm development in this interspecific cross. Brink and Cooper (1947) suggested
that endosperm breakdown was the main reason for the failure in interspecific and
intraspecific crosses in plants. Johnson et al. (1980) suggested that endosperm
breakdown was due to post-fertilization incompatibility. The endosperm plays a role in
embryo nutrition as it accumulates reserves of starch, proteins and lipids. Genetic
analyses suggest that maternal and endosperm tissues may regulate each other’s
development (Lopes and Larkins 1993). The failure of endosperm development in the
present study causes embryo abortion in the hybrid seed.
25
The embryo abortion at the late khalal stage was confirmed through in vitro seed
culture at different stages of fruit development. The seeds of date palm use to desiccate
during the fruit ripening stage. During the desiccation the embryo also desiccates and
positioned inside the endosperm. In the present hybrid case since there is poor
endosperm development and the endosperm is not enough to hold the embryo during
desiccation at the fruit maturity, the hybrid embryo aborted at the fruit ripening stage.
The hybrid seed endosperm was very soft and shriveled at maturity during seed
desiccation and is palatable.
Isolated hybrid zygotic embryos cultured on the different culture media showed
different types of morphogenesis depending upon the media composition and embryo
stage. Embryo maturation occurred in media with high sucrose and normal embryo
germination occurred in media with 30-40 g sucrose/l. Callusing occurred in media
with PGRs. Seeds collected at the rutab stage failed to respond in all the three type
culture media due to the embryo abortion at this stage. A majority of the embryos
isolated from the early khalal stage seeds germinated into rooted plantlets after 60-90
days of culture in the PGR-free MS culture media with 40 g/l sucrose. Therefore,
embryos of the khalal stage seeds are the ideal choice for the germination and hybrid
plantlet production.
The hybrid plant which panted in the field has well established similar to the control
date palm. The vegetative growth characteristic features were similar to the female
parent (date palm). However, leaf length, number of leaflets, number of spines on the
leaf base and stem height were differed from both the parents. Few more years will take
to get the complete vegetative and reproductive characteristic features of this newly
developed interspecific hybrid date palm. This study is a preliminary study which paves
the way for the production of more interspecific hybrids of date palm. Further research
is necessary for mass production of the dwarf palm males through tissue culture since
the dwarf palms cannot produce any offshoots like the date palm and more such
interspecific hybrids need to be produced for the field experimental study.
Acknowledgement
The support and encouragement given by the KISR management is hereby acknowledged.
26
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
Anon, 1982. Date production and protection. FAO Plant production and
Protection Paper 35.
Boyes, J. W. and Thompson, W. P. 1937. The development of the endosperm and
embryo in reciprocal interspecific crosses in cereals. J. Genet. 24:14-227.
Brink, B. A. and Cooper, D. C. 1947. The endosperm in seed development. Bot.
Rev. 13: 423-541.
Carpenter, J. B. 1979. Breeding date palms in California. Ann. Rep. Date Grow.
Inst. 54:13-14.
Carpenter, J. B. and Ream, C. L. 1976. Date palm breeding a review. Ann. Rep.
Date Grow. Inst. 53:25-29.
Johnson, S. A., den Nijs, T. P. N., Peloquin, S.J. and Hanneman Jr. R.E. 1980.
The significance of genic balance to endosperm development in interspecific
crosses. Theor. Appl. Genet. 57: 5-6.
Krueger, R. R. 1998. Date palm germplasm: overview and utilization in the
USA. In: Proceedings of the 1st International Conference on Date Palms, Al-Ain,
UAE. pp. 2-37.
Lopes, M. A. and Larkins, B. A. 1993. Endosperm origin, development and
function. Plant Cell 5:1383-1399.
Monciero, A. 1959. Le palmier-dattier au Sahara. First FAC International
Technical Meeting on Date Products and Protection, Tripoli, Libya. Paper No. 3.
Nixon, R. W. 1935. Metaxenia and interspecific pollination in Phoenix. Proc. Am.
Soc. Hort. Sci. 33:21-26.
Nixon, R. W. and Farr, J. R. 1965. Problems and progress in date breeding. Ann.
Rep. Date Grow. Inst. 42:2-5.
Ream, C. L. 1975. Date palm breeding - a progress report. Ann. Rep. Date Grow.
Inst. 52:8-9.
Saaidi, M., Toutain, G., Bannerot, H. and Louvet, J. 1981. The selection of date
palm (Phoenix dactylifera L.) for resistance to boyoud disease. Fr d' Outre Mer
35:241-249.
Sudhersan, C. 2004. Introduction of a multipurpose palm Phoenix pusilla in
Kuwait. Palms 48:191-196.
Sudhersan, C., Al-Shayji, Y. and Jibi Manuel, S. 2009. Date palm crop
improvement via T x D hybridization integrated with in vitro culture technique.
Acta Hort. 829:219-224.
Zaid, A. and De Wet PF. 2002. Date palm propagation. In: Zaid A (ed) Date palm
cultivation, FAO Plant Production and Protection Paper No. 156, Rome, Italy.
27
Table 1. Types of culture media used for hybrid seed germination
Media type
Media components
EG
MS basal salt + Vitamins +30 g/l sucrose
EM
MS basal salt + Vitamins +60 g/l sucrose
SE
MS basal salt + Vitamins + 100 mg/l 2,4-D + 6 mg/l 2iP + 40 g/l
sucrose
EG-embryo germination; EM-embryo maturation; SE-somatic embryogenesis
Table 2. In vitro responses of different stages of hybrid seeds to different culture media
Media Type Responses to culture media type
Seeds of 100 DAP
Seeds of 120 DAP
Seeds of140 DAP
EG
No seed germination
30 %embryo germinated
No response
EM
10% embryo enlarged
50% embryo enlarged
No response
SE
50 % embryo callused
80% embryo callused
No response
DAP-days after pollination; EG-embryo germination; EM-embryo maturation; SEsomatic embryogenesis
28
Fig. 1. Phoenix pusilla
Fig. 2. Hybrid sterile seeds
29
Fig. 3. T x D Hybrid date palm
30
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32
OP 03
Assessing Fruit Characteristics to Standardize Quality Norms in
Date Cultivars of Saudi Arabia
I.A.AL-Abdulhadi1, S. Al-Ali1, K. Khurshid1, F. Al-Shryda1,
A.M.Al-Jabr1 and A. Ben Abdallah2
¹ National Date Palm Research Centre, P. O. Box 43, Al Hassa-31982, Saudi Arabia
² FAO Project: UTFN/SAU/015/SAU, NDPRC, P.O.Box 43,
AlHassa-31982, Saudi Arabia.
Email: abdallah.benabdallah@fao.org
Abstract
The date palm (Phoenix dactylifera L) is the most important crop of the Gulf region
in the Middle-East. The Kingdom of Saudi Arabia with an estimated 25 million date
palms produces nearly a million tons of dates annually with a good potential for
exporting the surplus produce. In order to standardize quality norms of major date
cultivars in the Kingdom, studies were taken up at the National Date Palm Research
Centre, Al Hassa, Saudi Arabia to ascertain fruit characteristics of dates (Tamar stage)
in the cultivars Khalas, Sheshi and Reziz with respect to fruit weight (g), size of fruit
(length and breadth), number of fruits /500g, fruit moisture, colour and texture.
Khalas recorded the maximum fruit length in all the three categories of large,
medium and small sized fruits. With regard to the breath of fruits, the cultivar Sheshi
registered the highest values. Further, Sheshi recorded the highest fruit weight values,
which in turn influenced the number of fruits per unit weight, with Sheshi recording
the least number of fruits per 500g. The cultivar Khalas had the least fruit moisture and
the highest mean ∆E for colour indicating light colour (yellow) fruits. We also studied
four texture parameters viz, hardness, springiness, cohesiveness and resilience of
Khalas, Sheshi and Reziz date cultivars.
Findings of this investigation will strengthen the data base of fruit quality norms in
major Saudi Arabian date cultivars and boost export of dates from the Kingdom,
besides protecting the identity of the cultivars studied.
Key words: Phoenix dactylifera L. cultivars, quality norms, Saudi Arabian dates
33
Introduction
The date palm (Phoenix dactylifera L) is one of the oldest fruit trees of the world
and is closely associated with the life of the people in the Middle East including the
Kingdom of Saudi Arabia since ancient times. In the Middle-East date palm has been
cultivated at least since 6000 BC (Al-Qarawi et al. 2003). The Arab countries of the
Middle-East and North Africa account for 60 per cent of the world’s production where
800 different kinds of date cultivars are reported to be grown (Mikki 1998; Al-Afifi &
Al-Badawi, 1998).The with an estimated 25 million date palms the Kingdom produces
nearly a million tons of dates annually accounting for about 15% of the global date
production (Anonymous 2006). The date fruit is a good source of food, providing
fiber, carbohydrates, minerals and vitamins besides having antimutagenic and
anticarcenogenic properties (Baloch et al. 2006; Al-Farsi et al. 2005; Ishurd &
Kennedy 2005; Vayalill 2002; Mohamed 2000).
Worldwide 2000 or more date cultivars are known to exist (Ali- Mohamed &
Khamis 2004) .Saudi Arabia has a rich diversity of about 400 date cultivars of which
10 varieties including Khalas, Sheshi and Reziz are popular and have a high consumer
preference (Anonymous 2006).
With an estimated three million palms the Al Hassa oasis is the largest in the
Kingdom where El-Baker (1952) listed 15 date palm varieties of commercial
importance, while Asif et al. (1982) listed 25 cultivars from the Al-Hassa oasis.
Further, Asif et al .(1986) grouped the Al-Hassa date palm cultivars based on the
season of production and categorized the three cultivars under investigation in this
study as mid-season cultivars.
Khalas is widely cultivated in the Al Hassa oasis and considered by many as the
best date of the world, with mostly medium to big sized fruits that make a delicacy as
both fresh (rutab) and dry (tamar) dates which store well. Sheshi produces mostly
medium to large fruits , plumpy and firm in texture . Skin separation of fruits is
common in this cultivar which often lowers the quality and value of the produce. Reziz
is widely cultivated in the Al- Hassa oasis and is distinguished by small dark coloured
fruits that have a characteristic flavour ( Asif et al .1986).
34
In view of the increasing cultivation and surplus date production in the Kingdom
there is an emphasis on exports where ascertaining the quality norms of specific date
cultivars is crucial as emphasized by the National Committee on date palm in Saudi
Arabia. Determining fruit characteristics of major Saudi Arabian date cultivars will
also ensure that the cultivar is protected.
Ripening of dates is characterized by four stages viz. Kimri , Khalal ,Rutab and
Tamar stage depending on the colour, softness moisture and sugar content (Farahnaky
& Afshari-Jouybari 2010). CODEX norms based on fruit weight are available for
major North African date varieties viz. Deglet Nour and Majhoul . International
standards for export of dates also demands homogeneity of colour , size and texture
(Anonymous 1985) . Jaradat and Zaid (2004) while studying quality traits of date palm
fruits revealed that fruit colour, shape, size, ripening and their interactions
predominantly reflect differences in consumer preferences, with fruit colour, softness
and consumption stage explaining 65.5 per cent of the variability in the economic
value of date palm cultivars. Given the importance of the subject and lack of
information on fruit characteristics of Saudi Arabian dates, studies were taken up at the
National Date Palm Research Centre, Al Hassa, Saudi Arabia to ascertain fruit
characteristics of dates (Tamar stage) in the cultivars Khalas , Sheshi and Reziz with
respect to fruit weight (g) , size of fruit ( length and breadth) , number of fruits /500g ,
fruit moisture, colour and texture.
Materials and Methods
Date samples of major Saudi Arabian date cultivars viz. Khalas , Sheshi and Reziz
were collected at Tamar stage . Based on consumer preference as assessed from
interviews with consumers (50) and key market stake holders (10) fruits in one
kilogram of dates were sorted out as large, medium and small. The weight/ fruit (g) for
the three cultivars as perceived by consumer preferences is given below
Cultivar
Fruit weight (g)
Large
Medium
Small
Sheshi
> 11
8-10
<8
Khalas
>10
7-9
<7
Reziz
>7
5-7
<5
35
The average fruit weight (cultivar wise) for the above three categories was recorded.
Further, the number of fruits per 500g as recommended by CODEX norms was also
recorded for the above cultivars. Fruit size was determined by recording the fruit
dimensions pertaining to length (mm) and breath (mm) of the above mentioned cultivars.
Fruit colour measurements ranging from dark to clear were recorded for 10 date
samples using a spectrophotometer (Hunter ColorFlexTM, USA) through digital imaging
under controlled conditions. Analysis of pictures was performed using easy match QC
software, which is a windows- based computer programme that performs numerical
calculations on data measured by ColorFlexTM , stores sample measurements and /or
printed representation of data. The software also directly controls instrument
standardization, measurement and diagnostics .Colour differences between samples
were computed with the equation given below and represented by mean ∆E.
√∆E= ∆L2 + ∆a2 + ∆b2
Where L, a, and b are the colour values of the samples. The colour value ‘L’
measures relative white(100) to black(0). The value’a’ measures relative green(-) to
red (+) while the’b’ colour values measures relative yellow (+) to blue (-).
Further, fruit texture analysis (mm) with respect to hardness, springiness,
cohesiveness, and resilience of dates, cultivar wise was carried out using a texture
analyzer (TA.XT plus/TA.HD plus/MT.LQ plusTM, England).
Hardness (mm) or firmness, also indicating extent of tissue softness was measured
through a force test. Springiness (mm) is related to tissue elasticity and measured in
the degree to which a product can be extended / stretched before breaking.
Cohesiveness (mm) represents steadiness and measures tissue strength. Resilience
(mm) of dates is the ability of the tissue to return to the original form after being
compressed or stretched.
Fruit moisture was also measured for three cultivars using the protocol given below
by Marzouk and Kassem (2010) . Fruits were washed with tap water, then rinsed
twice with distilled water and cut into small pieces with a knife. Then an amount of
36
fresh weight sample was weighed ( fresh weight) and dried to a constant weight (g) in
an air drying oven at 70o C, then weighed ( dry weight).
Fruit moisture and dry matter contents were calculated as;
Fruit moisture content (%) = Average fresh fruit weight - Average dry fruit /
Average fresh weight X 100
For each of the three cultivars, all observations (except number of fruits/ 500g) were
recorded on a 10 replicate basis with each replicate being the mean of three
observations. Data was subjected to ANOVA using the randomized block design. In
case of number of fruits /500g observations were recorded on a three replicate basis
and data was subjected to ANOVA using the completely randomized design. With
regard to the physical parameters viz. weight / fruit (g), number of fruits /500g and size
of fruit (length and breadth) observations were recorded for each of the three fruit
categories viz. large, medium and small. All data were analyzed through the Web
Based Agricultural Statistics Software Package (WASP 1.0 -http://www.icargoa.res.in)
Results on the above physical characteristics (fruit weight, size, number of
fruits/500g) and other quality parameters viz. fruit moisture, fruit colour and texture
are presented and discussed below.
Results and Discussion
I. Fruit size
Results presented in table 1 reveal that the cultivar Khalas recorded the maximum
fruit length (39.60-31.45mm) in all the three categories of large, medium and small
sized fruits and was statistically superior to Sheshi and Reziz . With regard to the
breath of fruits, the cultivar Sheshi registered the highest values ( 25.70 – 21.80 mm)
and was significantly different from Khalas and Reziz which second and third
,respectively. Sakr et al .(2010) reported fruit length to significantly differed among
the fruits of eight date palm cultivars studied with the cultivar Kuboshy and Zaghloul
registering the maximum fruit length of 6.65 and 6.10 cm, respectively, while the
cultivar Samany registered the maximum breath of 3.31 cm. Asif et al. (1986)
categorized fruits of both Khalas and Sheshi as medium to big and that of Reziz to be
small. In the Western region of Saudi Arabia, large fruit size characteristic of the
37
cultivar Anbarah from Al Medina is reported to be a unique variant found with
relatively low frequency and high polymorphic index (Jaradat & Zaid 2004).
II. Fruit weight related characters
With regard to weight of individual fruits, the cultivar Sheshi recorded the highest
and significantly superior values (11.60 – 7.05 g) in all the three categories of large,
medium and small fruits tested. Consequently, fruit weight influenced the number of
fruits per unit weight, with Sheshi recording the least number of fruits per 500g (39.00
– 72.67) and was significantly different from Khalas and Reziz in the three categories
of large, medium and small fruits (Table 2). Sakr et al. 2010 recorded maximum and
minimum fruit weight of 28.71g and 8.50g in the cultivar Samany and Amhat,
respectively. Fruit size in dates of the Egyptian cultivar Zaghloul were enhanced with
the application of organic manures or its supplementation with mineral NPK compared
to mineral fertilization alone (Marzouk & Kassem 2010).
III. Fruit moisture and colour
Fruit moisture in dates is an important quality parameter that contributes to the
quality of dates. Results presented in figure 1 indicate that the cultivar Khalas had the
least fruit moisture of 13.96 per cent and was significantly different from Sheshi
(15.24 %) and Reziz (15.52%) which were statistically at par.
Hussein et al.1979 classified dates on the basis of moisture content as dry date with
less than 20 per cent moisture that require high temperature and sun intensity for
maturity with about the same level of sucrose and reducing sugars. Semi-dry dates
were found to have moisture levels ranging from 20-30 per cent with low sucrose
content and soft dates with more than 30 per cent moisture with low sucrose content
and must be eaten fresh. According to this classification the dates studied in this
investigation would therefore be classified as dry dates as moisture levels were less
than 20 per cent ranging from 13.96 to 15.24 percent ( Figure 1).
With regard to fruit colour (Figure 2) Khalas recorded the highest mean ∆E of 25.34
indicating light colour (yellow) and was statistically at par with Sheshi (23.44), both of
which were significantly different from Reziz (14.83) where the darkness increased
(Figure 3) . Fruit colour constituted the most important trait in the Gulf Cooperation
Council countries of the Middle-East when quality traits were scored on fruits of 203
38
date palm cultivars. Further, predictors of fruit economic value for date palm cultivars
revealed that yellow colour had 70 per cent economic value in Saudi Arabia like in the
United Arab Emirates as against 64 per cent in Qatar. In Bahrain, Kuwait and Oman
different shades of red colour predominated (Jaradat &Zaid 2004) .
Studies conducted in Iran on Mazafati dates showed that red colour in Khalal stage
turns to black during the Rutab stage and that colour change may not be a suitable
indicator for ripening of Mazafati dates (Farahnaky & Afshari-Jouybari 2010).
Marzouk and Kassem (2010) reported that fruit colour in dates is enhanced with the
application of organic manures or its supplementation with mineral NPK compared to
mineral fertilization alone. Mansour (2005) recorded five colours in date fruits ranging
from bright yellow in the cultivar Aglany, yellow with red spots in Samany, orange in
Amry , and bright red in Zaghloul and scarlet-red in both Hayany and Bent-Aisha.
IV. Texture of fruits
Fruit texture constitutes an important quality parameter in dates. We studied four
texture parameters viz, hardness, springiness, cohesiveness and resilience of Khalas ,
Sheshi and Reziz date cultivars that were measured as a force in g.sec ( Table 3). With
regard to hardness, the cultivar Reziz registered significantly highest value (9166.81)
indicating it is produces the hardest fruits compared to Khalas and Sheshi which were
statistically at par . Jaradat and Zaid (2004) reported that with regard to fruit softness
and semi-dry dates influenced 60 per cent of the predictors for fruit economic value in
Saudi Arabia. Semi-dry and dry dates predominated in Oman and UAE, while in
Bahrain, Kuwait and Qatar soft dates predominated. In Tunisia, date cultivars were
clustered on the basis of fruit consistency as soft fruit, semi-dry fruit and dry fruit
cultivars (Hammadi et al .2009).
With regard to the preferred quality parameters of springiness and resilience the
cultivar Sheshi recorded significantly superior values of 0.77and 0.12, respectively.
However, with regard to fruit cohesiveness, Khalas was the best (0.40) but was
statistically at par with Sheshi (0.46) , both of which were significantly different from
Reziz (0.27). Except for fruit hardness, there are no previous reports pertaining to the
texture parameters of springiness, cohesiveness and resilience in dates. As compared
to the cultivar Reziz, both Khalas and Sheshi have a high consumer preference in the
39
Kingdom which can be attributed to the superior attributes of springiness,
cohesiveness and resilience.
This investigation will strengthen the data base for quality norms in dates of major
Saudi Arabian cultivars and will support export of dates from the Kingdom, besides
protecting the identity of the cultivars studied.
Acknowledgement
The authors thank the Ministry of Agriculture, Kingdom of Saudi Arabia for
providing the necessary support to conduct this trial at the National Date Palm
Research Centre (NDPRC) , Al - Hassa, through the FAO funded project
UTF/SAU/015/SAU.
Thanks are also due to Mr. Adnan Al-Afaliq, General Director of the NDPRC for
his support in carrying out the study.
40
References
[1]
Al-Afifi M and Al-Badawi A (1998) Proceedings of the first international
conference on date palm , Al Ain, United Arab Emirates .643pp.
[2]
Ali- Mohamed AY and Khamis ASH (2004) Mineral ion content of the seeds of
six cultivars of Baharaini date palm (Phoenix dactylifera L). Journal of
Agricultural and Food Chemistry 52, 6522-6525
[3]
Al-Qarawi AA, Ali BH, Al-Mougy SA and Mousa HM (2003) Gastrointestinal
transit in mice treated with various extracts of date (Phoenix dactylifera L). Food
and Chemical Toxicology 41, 37-39.
[4]
Al-Farsi M, Alasalvar C, Morris A, Barron M and Shahidi F (2005)
Compositional and sensory characteristics of three native sun-dried date (Phoenix
dactylifera L) varieties grown in Oman. Journal of Agricultural and Food
Chemistry 53, 7586-7591.
[5]
Anonymous (2006) The famous date varieties in the Kingdom of Saudi Arabia (
Ed. Ministry of Agriculture, Kingdom of Saudi Arabia and Food and Agriculture
Organization of the United Nations). pp 245.
[6]
Anonymous(1985) Codex standards for dates. CODEX STAN. pp6.
[7]
Asif MI , Al-Tahir OA and Al-Kahtani MS (1982) Inter-regional and inter-cultivar
variations in dates grown in the Kingdom of Saudi Arabia. In, proceedings of the
first symposium on date palm. King Faisal University, Al- Hassa.
[8]
Asif MI, Al-Ghamdi AS, Al-Tahir OA and Latif RAA (1986) Studies on the date
palm cultivars of Al-Hassa oasis. In, proceedings of the second symposium on date
palm in Saudi Arabia. King Faisal University, Al Hassa Saudi Arabia.pp 405-413.
[9]
Baloch MK, Saleem SA , Ahmad K , Baloch AK and Baloch WA (2006) Impact
of controlled atmosphere on the stability of Dhakki dates .Swiss Society of Food
Science and Technology 39, 671-676.
[10] El-Baker AJ (1952) Date cultivation in Saudi Arabia. Report number 31. FAO
,Rome, Italy.
[11] Farahnaky A and Afshari-Jouybari, H (2010) Physiochemical changes in
Mazafati date fruits incubated in hot acetic acid for accelerated ripening to
prevent diseases and decay. Scientia Hortculturae 127, 313-317.
41
[12] Hammadi H, Mokhtar R, Mokhtar E and Ali F (2009) New approach for the
morphological identification of date palm (Phoenix dactylifera L.) cultivars from
Tunisia. Pakistan Journal of Botany 41(6) : 2671-2681.
[13] Hussein FM ,El-Khatny S and Wally YA (1979) Date palm growing and date
production in the Arab and Islamic world. Ain Shams Press (Arabic) , Egypt.
[14] Ishurd O and Kennedy JF (2005) The anti-cancer activity of polysaccharide
prepared from Libyan dates (Phoenix dactylifera L). Carbohydrate Polymers
59,531-535.
[15] Jaradat AA and Zaid A (2004)Quality traits of date palm fruits in a centre of
origin and centre of diversity. Food, Agriculture and Environment 2(1) ; 208-217.
[16] Mansour HM (2005) Morphological and genetic chatacterization of some
common Phoenix dactylifera L. cultivars in Islamia region. M. Sc. Thesis.
Botany Department, Faculty of of Science, Suez Canal University, Egypt.
[17] Marzouk HA and Kassem HA (2010) Improving fruit quality, nutritional value
and yield of Zaghloul dates by application of organic and / or mineral fertilizers.
Scientia Hortculturae 127, 249- 254.
[18] Mikki MS (1998) Present status and future prospects of dates and date industry in
Saudi Arabia. Pp 469-507.In M.Al-Afifi,M and A.Al-badawi (eds). In
proceedings of the first international conference on date palm , Al Ain, United
Arab Emirates. March 8-10,1998.
[19] Mohamed AE (2000) Trace element levels in some kinds of dates . Food
Chemistry 49, 107-113.
[20] Sakr MM , Abu Zeid IM , Hassan AE , Baz AGIO and Hassan W M
(2010)Identification of some date palm (Phoenix dactylifera ) cultivars by fruit
characters. Indian Journal of Science and Technology 3(3) : 338- 342. ISSN :
0974-6846.
[21] Vayalill PK (2002) Antioxidant and antimutagenic properties of aqueous extract
of date fruit (Phoenix dactylifera L. Arecaceae ). Journal of Agricultural and
Food Chemistry 50,610-617.
42
Table 1. Fruit size of major Saudi Arabian date cultivars
Fruit Size
Cultivar
Fruit Length(mm)
Fruit Breath(mm)
Large
Medium
Small
Large
Medium
Small
Khalas
39.60a
38.50a
31.45a
23.60b
24.50b
20.16b
Sheshi
35.30b
26.00b
29.50b
25.70a
25.50a
21.80a
Reziz
26.57c
25.26b
24.68c
18.60c
17.69c
17.08c
CD (p=0.05)
0.66
1.19
1.11
0.89
0.77
0.67
Values followed by different letters within the same column are significantly
different at 5% significance level.
CD : Critical Difference
Table 2. Weight per fruit and number of fruits per 500g of major Saudi Arabian date cultivars
Cultivar
Weight per fruit (g)
Number of fruits per 500g
Large
Medium
Small
Large
Medium
Small
Khalas
10.70b
8.65b
6.05b
44.00b
60.67b
83.67b
Sheshi
11.60a
10.30a
7.05a
39.00c
52.33c
72.67c
Reziz
7.55c
6.08c
4.71c
65.67a
78.33a
109.67a
CD (p=0.05)
0.15
0.41
0.27
4.66
2.00
2.31
Values followed by different letters within the same column are significantly
different at 5% significance level.
CD : Critical Difference
43
Table 3 . Texture of major Saudi Arabian dates
Cultivar
Texture Parameters (g.sec)
Hardness
Springiness
Cohesiveness
Resilience
Khalas
3441.72b
0.56b
0.40a
0.10b
Sheshi
3817.34b
0.77a
0.46a
0.12a
Reziz
9166.81a
0.46c
0.27b
0.08c
CD
2102.93
0.81
0.08
0.12
(p=0.05)
Values followed by different letters within the same column are
significantly different at 5% significance level.
CD : Critical Difference
Cultivar
Reziz
15.52
Sheshi
15.24
Khalas
13.96
13
13.5
14
14.5
15
15.5
16
% Moisture
Figure 1. Fruit moisture in Saudi Arabian date
cultivars [CD : 0.89 ; p=0.05 ]
Cultivar
Reziz
14.83
Sheshi
23.44
25.34
Khalas
0
5
10
15
20
25
Fruit Colour (Mean ∆E)
Figure 2. Fruit colour of Saudi Arabian date cultivars
[CD : 2.10 ; p=0.05]
44
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ه f5ا "? I I3 "L oو Ogا_Pت أ LLد !%ه #ا " ر ا $د 2^8ض ا "25
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45
46
OP 04
Effect of different levels of elemental sulfur fertilizer on the Growth,
fruit quality, yield, and nutrient levels in the leaves of Mishrig Wad
laggai date palm cultivar under Al Mukabrab Conditions.
Dawoud Hussien Dawoud and
Fatima A. Ahmed
dawoudhussien@hotmial.com
fatimaraouf.ahmed0@gamail.com
Abstract
This study was carried out for three successive seasons {٢٠٠٧-20١٠}to investigate
the effect of soil application of elemental sulfur on the growth rate , fruit quality , yield
, nutrient levels in the leaves of Mishrig Wad Laggai date palm cultivars
under Al
Mukabrab condition. The results revealed that significantly greater values of growth,
fruit weight, yield, total and reducing sugar percent, protein, leaf N, P, K, Ca, Mg, Cu,
Zn, Mn and Fe.
The experimental design was randomized complete block design, three replicates,
four trees per plot; the significant variation between treatments was determined by
Duncan's multiple range tests at 5%.
Introduction
Date palm one of the most important fruit crops growing in the Sudan , which needs
a lot of research efforts to increase , it's yield quantitatively and qualitatively. In Sudan
date palm is traditionally grown entirely on the limited light soil along the banks of the
rivers and vallies. These soils are light, highly fertile and renewed annually by the
flood, thus fertilization is not common. Recently, however cultivation of date palm
extended to high terrace soils which are characterized by calcareous soil or alkaline
soil reaction with soil pH 8.3-8.8.
The effect of Sulfur fertilization on growth , quality and yield of many crops have
been reported by Dihllon, (1978), Joshi, (1975) and Singh, (1979) .In addition to the
direct nutrition effect of sulfur on plant growth, this element may have a number of
beneficial side effects. The acidifying effect of Sulfur on calcareous or high pH soil
may increase the availability of other essential nutrients. Omer (1975) reported that the
application of Sulfur resulted in an increase in the up-take of Phosphorus, Iron ,and
47
Potassium ,also Khapaji , (1986) stated that the application of Sulfur to Al Hassa with
high pH values
reduced its pH and increase the availability of phosphorus. Several
investigators reported that
Sulfur fertilizer increase the protein content of the crop,
Patil, (1981), Bahl, (1986) and Das , (1975).
Soil pH was significantly decreased by Sulfur application as reported by Al-Ane et
al (1977), Hattar and kochler (1978), Also El karouri (1969) found that soil pH
acidification resulted in a progressive decrease in
soil pH and an increase in
Phosphorus up-take by Fasulia. Fatima and Gafer (1996) found a positive correlation
between Sulfur and the growth rate, quality and macro and micro nutrient levels in the
leaves of Snap bean.
Singh and Singh (1977) reported that Sulfur application resulted
in reducing soil pH and increased the uptake of N, P, K, Ca, Mg, Fe, Mn, Zn and Cu.
So the main objective of this work was to study the effects of different levels of
elemental Sulfur fertilizer on the growth rate, fruit quality, nutrient levels in the leaves
of M. W. laggai and the pH levels of the treated area.
Material and methods
The experiment was conducted during 2007/2008and 2009/2010 on mature M.W.L
date palm cultivar growing in block (4B) Al Mukabrab private date palm project, in
clay calcareous with soil pH 8.8,(Appendix 1) Five treatments of Sulfur application
were used on 72 trees ,the design of experiment were randomized complete block
design ,with three replications, each plot consisted of 4 trees .The study started in
November 1999 .and repeated on the same trees for the following years the Sulfur
fertilizer was applied in the form of elemental Sulfur in a trench one meter far around
the stem,(10 cm depth,10cm width) then irrigated directly-All the cultural practices for
the experimental orchard done as recommended by A.R.C.
The irrigation water from artesian well, bubbler irrigation method was used. The
growth rate of the leaves was obtained by periodic reading of a measuring tape
attached to a young leaf. As the leaf elongated, it was pulled past the head of a nail
driven into the tree trunk . Aldrich (1942) and Aldrich, Crawford, Nixon and Reuther
(1942). The total yield was obtained by weighing, picked fruits, excluding field culls.
Samples of 50 dates were collected from each bunch of each palm at the picking. The
samples were weighed, measured and graded after they have been wiped free of dirt
and calyxes removed, total sugars reducing sugars and protein were determined by the
48
method used by Sinclair et al 1941.Sulfur was determined in pinnae ,samples collected
from each tree in treatment plots. The method used was the same as described by Furr
and Cook (1952). Each leaf sample consisted of twelve pinnae, Four taken from the
mid portion of the blade of each of the three youngest mature leaves. These leaves are
described as ‘Top head’ by Reuther (1948)
The samples were dried in a forced draft oven at 80°c, ground in a Wiley mill and
analyzed for Nitrogen by the standard kjeldahl method
recommended by Chapman
(1961). Phosphorus was determined using Vandate-molybdate-yellow method. Sulfur
was determined using Barium sulphate method described by Chapman and Pratt
(1961), iron was determined using Dry Ashingo-phenanthorline method Atomic
Absorption Spectrophotometer was used to determine the other nutrients as
recommended by (Chapman 1961). Soil samples for pH yearly taken from the root
zone area of the treated trees.
The experiment was laid out in a randomized complete block design, with three replicates.
The treatments means were separated by Duncan's multiple range tests at 5%level.
Results and discussion
The data in table (1a and b) show the effect of various sulfur treatments on the growth
rate, fruit quality, yield of M.W.L. ,the rate of leaf growth was increased as a result of
sulfur application ,this is in line with The findings of Broomfield (1973), Rethee and
Ghahal (1977) and Aulakh (1977), fruit weight (g) also increased with the increase of
sulfur application Singh (1980) reported that the increase in fruit weight as a result of
sulfur fertilizer may be due to the increase of dry matter and this theory may justify the
positive correlation between the yield and sulfur application. All the experimental
findings were in line with Mathur (1976), Pareek (1973) Dihllon, (1978) and Singh
(1979). Sulfur is a vital constituent of all plant proteins and of some plant hormones.
Sulfur deficiencies slow down protein synthesis for two reasons. The S-containing
amino acids are vital constituents of protein. Other amino acids may accumulate if S is
deficient furthermore, S is essential for the action of enzymes involved in nitrate reduction
as reported by Nightingale (1952). sulfur -deficiency slows the formation of all amino
acids. sulfur –deficient plants therefore, tend to accumulate nitrate nitrogen in their tissues.
Several investigators reported that sulfur fertilizer increased the protein content of
many crops Singh, (1970); Bhuity (1974) Laurence (1976) Patil (1986) Bahi (1986)
49
and Singh (1968) Das (1975) and Fatima (1996) found that application of sulfur
increased seed protein content of wheat, maize, rice, snap beans 1974. Dube and
Misra (1970) observed that in black gram sulfur deficiency reduced the yield, quality
and protein content of seeds.
Also the data in table (1a,b)shows effect of sulfur on total, reducing sugar of M.W.L
fruits , data show the positive correlation between the sugar and the sulfur fertilizer.
Also this finding may due to the effect of Sulfur on N level in Mw.laggai leaf, Sulfur
increase the uptake of N in many crops. Also this findings are in agreement with
Dawoud and Salih (1997).
Data in table (2) show the effect of Sulfur on the nutrient levels and show positive
correlation between N, P, K, Mg, Ca, Cu, Mn, Fe and Zn. This may be due to the
acidifying effect of sulfur on calcareous or high pH soil Omer (1970) reported that :the
application of Sulfur resulted in an increase in the uptake of N, P, K, Ca, Mg, Fe, Zn,
Cu and Mn .Some research workers with sulfur reported that the uptake Nitrogen,
Phosphorus and Potassium was increased with sulfur application, Subbrroa and Gosh,
(1981);Singh, (1980).
Data presented in table (2) showed that soil pH was significantly affected by Sulfur
fertilizer application, soil pH was significantly lowered by Sulfur application as
reported by Al-Ane et al. (1977), Hatter kochler (1978). This decrease in soil pH
might possibly due to the formation of sulfuric acid in the soil as a result of Sulfur
oxidation which is an acidifying process .
Aulakh and Dev (1976) reported that sulfuric acid is a good source of Sulfur when
applied in alkaline soil because it reduced soil pH improves the availability of other
nutrients to plants and has an effect on microorganisms’ activities.
Recommendation
The results obtained in the study, clearly indicated that,-with in the tested levels of
elemental Sulfur fertilizer, the highest leaf elongation, fruit quality, yield, macro and
micro nutrients levels in the leaves of Mishrig Wad Laggai date palm cultivar was
produced form 400 g elemental Sulfur per tree to the soil, Accordingly, this practices
is proposed to recommended to date palm growers at Al. Mukabrab area.
50
References
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pod yield and protein content of the groundnut Indian J. Agric. Sci. 56(6)428-433 .
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10. Das, S.K. and Data, N. P. (1973) Fertilization for increased production and grain
quality. Fert. News 18(9)3-10 .
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nutrients to foster grapefruit trees Under New Halfa conditions. A paper submitted
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Agriculture Research Corporation .
13. Dhillon, N. S. and Dev. G. (1978). Effect of elemental Sulfur application on
soybean (Glycine maxmerril ). Indian Soc. Soil Sci. 26(1)55-57.
14. Dikson, T. and Aswir, C.T (1974) .The role of sulfur in maintaining lucre yield in
the lockyery valley. Aust. J. Exp. Agric.Anim. Husb. 14(69) :515-519 .
51
15. Dube, S. D. and Misra, P. H. (1970). Influence of sulfur on growth and content of
the peas. Indian Soc. Soil Sci. 18:375-378.
16. Elgazzar, A. M; Elazzab, S. M. and Elsafy, M. (1979). response of Washington
Naval orange to foliar application of iron chelate. Alex. J. Agric. Res. 27(1); 19-26
17. Elkaroui, M. O. H. (1969). Effect of soil acidification on phosphate availability in
alkaline kuru soil. Annual report of the Hudeiba res. Station, Sudan.
18. Elkarouri, M. O. H. (1981). Effect of elemental sulfur in the availability of
phosphorus in saline sodic soil. South Khartoum Area, soba research station,
Khartoum, Sudan.
19. Elkarouri, S. M. (1970). Investigation of yellowing of fasulia. Annual report of
the Hudeiba Res. Station, Sudan .
20. Hatter, B. I.. and kochler, F. e. (1978) .Effect of levels of caco on oxidation of
elemental sulfur and on plant uptake. Agronomy Abstracts. Ann. Meet. P. 154.
21. Joshi, D. C. and Seth, S. P. (1975). Effect of sulfur and phosphorus application
on characteristics, nutrient uptake and yield of wheat crop. J. Indian soc. soil sci.
23(2); 217-221.
22. Khafaji M. S. Al-Barrak, and y. Abed Elhadi use of sulfur for increasing the
availability of some nutrients in soils under date palm cultivation Al-Hassaoasis
college of agricultural and food sciences ,King Faisal University , Al- Hassa ,
Saudi Arabia second symposium on date palm (page 386-392) .
23. Laurence, R.C.N; Gibbons, R.W. and young, C.T. (19796). Changes in yield,
protein, oil and maturity of groundnut cultivars with application of sulfur fertilizers
and fungicides. J. Agric. Sci. (camb.) 86(2); 245-250.
24. Mathur, and Singh,H.G. (1976). Metabolic changes associated with the
prevention of chlorosis by application of elemental sulfur and foliar sprays of
squestrine (138-Fe) in Pisum sativum Annuals. Botany, 40;833-836.
25. Misra, N.M, and Dupe, S.D. (1966). Effect of sulfur fertilization on yield of
onion, Fert.News.11, (10); 18-19, 33.
27.Nightingale, G.T. (1952). Effect of sulfur deficiency on metabolism in
tomato. Plant physiol.7; 565-595
52
28.Omer, A.M; Eldamaty, A. H; Hamid, H. and Elsherbini, A.E. (1970).Effect of
gypsum on yield , quality and mineral constituent of peanut plant .J. Soil sci. (U.
A. R.) 10;15-121.
29- Patil, J.D. (1981). Effect of B, S, and FYM on the yield and quality of groundnut .
J. Maharashtra Agric. Univ. 6[1] ; 17-18 .
30- Rathee, O.P. and Chahal , R.S. (1977). Effect of phosphorus and sulfur
application on yield and chemical composition of groundnut in Ambala. Soils and
Fert. Abstr. 42 (11) : 7132.
31- Reuther W. and Graword C.L. (1945). The effect of temperature and bagging on
fruit set dates. Date grow's Inst. Rept . 23; 3-7.
32- Singh,B. and Singh , M . (1977). Sulfur status of hissar soil s and availability of
sulfur as affected by application. soil and Fertl. Abstr. 40 (5); 2241.
33-Singh, H.G. (1971). Sulfur application prevents chlorosis and ensures larger crop
yields on alkaline calcareous soil. Indian Farming, 21(1) :212-213.
34- Singh, H.G.. and Singh, M.P. (1980) . Effect of foliar sulfuric acids on dry matter
weight of plants Fert. News , 31(9): 23-30 .
35- Singh, M. and Singh , N. (1977). Effect of sulfur and selenium on oilin raya
containing amino acids and quality of oil in raya (Brassica juncea coss.) in normal
and sodic soils . Indian PL. /physiol. 20(1) :56-62 .
36- Singh , N; Subbian , B.V. and Gupta , Y.P. (1970). Effect of the sulfur fertilization
on chemical composition of groundnut and mustard . Indian J. Agron . 15(1) :24-28.
37- Subbarroa, A. and Gosh , A.B. (1981). Effect of intensive cropping and fertilizer
use on crop removal of S and Zn and Their availability in soil. Fertilizer Research
2:306-308.
53
Table (1-a) Effect of different levels of elemental Sulfur on growth rate, fruit
quality of Mishrig Wad Laggai date palm cultivar under Al Mukabrab conditions.
Treatment
Season
Leaf length /cm/year
Fruit weight(g)
Fruit Volume (cc)
100 g
2007
-2008
21d
2008
-2009
20d
2009
-2010
22d
2007
-2008
6.7d
2008
-2009
6.8d
2009
-2010
6.7d
2007
-2008
5.70d
2008
-2009
5.6c
2009
-2010
5.7d
200 g
300 g
400 g
500 g
28c
33b
36a
37a
28c
34b
37a
36a
29c
32b
36a
36a
7.2c
9.3b
11a
9.4b
7.1c
9.1b
10.9a
9.3b
7.3c
9.2b
11.1a
9.5b
6.12c
7.82b
9.35a
7.99b
6c
7.7b
9.3a
7.9b
6.2c
7.8b
9.3a
8.0b
control
17e
15e
17e
5.3e
5.2e
5.1e
4.42e
4.3d
4.33e
* Means within columns followed by the same letter are not significantly
different at p: 0.05 according to new Duncan's multiple range test.
Table (1-b). Effect of different levels of elemental Sulfur on fruit and yield
of Mishrig Wad Laggai date palm cultivar under Al Mukabrab conditions
Treatment
season
100g
200g
300g
400g
500g
600g
Protein
Sugar%
2007
2008
2009
-2008
-2009
-2010
2.8c
2.9c
3.1c
3.82a
3.5b
2.1d
2.70bc
2.9b
3.2ab
3.91a
3.5ab
2.od
2.90c
2.85c
3.0c
3.85a
3.4b
2.2d
2007
-2008
R
53d
54c
55b
56a
56a
50e
2008
-2009
T
58e
59bc
92b
65a
65a
52b
R
58c
59b
62b
65a
65a
52d
T
59d
60c
63b
66a
66a
53e
Yield /tree/ Kg
2009
-2010
R
52d
53c
54b
55a
55a
49e
T
57d
59c
62b
65a
65a
52e
2007
2008
2009
-2008
-2009
-2010
380d
409c
437b
459a
465a
250e
374d
403c
431b
453a
459a
245e
375d
405c
434b
455a
461a
244e
*Means within columns followed by the same letter are not significantly different at p: 0.05 according
to new Duncan's multiple range test
R : Reducing sugar
T : Total sugar
54
Table (2) Effect of different levels of elemental Sulfur on nutrients levels in the leaves of
M.W.L. date palm growing under Al Mukabrab condition
%
Treat
ment
PPM
Soil pH
N
P
K
Ca
Mg
Fe
Mn
Zn
Cu
1st
year
2nd
year
3nd
year
100
(g)s
1.5 d
0.12 c
0,52 e
1.02 d
o.27 d
1350 e
388 e
43 e
59 e
8.7 e
8.5 e
8.4 e
200
(g)s
1.7 c
0.12 c
0.55 d
1.05 e
0.3 c
2114 d
492 d
51 d
74 d
8.5 d
8.4 d
8.3 d
300
(g) s
1.8 c
0.14 c
0.61 c
1.07 c
0.35 b
2156 e
409 c
87 c
104 c
8.3 c
8.2 c
8.1 c
400
(g)s
2.0 b
0.18 b
0.8 b
1.12 b
0.40 b
4202 b
570 b
11 b
135 b
8.2 b
8.1b
8.0 b
500
(g)s
2.2 a
0.25 a
0.92 a
1.21 a
0.45 a
4488 a
1192a
480a
185 a
8.1 a
8a
7.8 a
Cont
rol
1.0 e
0.06 d
0.31 f
0.6 e
o.17 e
230 f
45 f
20 f
17 f
8.8 f
8.8 f
8.8 f
Means within columns followed by the same letter are not significantly different at p: 0.05 according to
new Duncan's multiple range test
55
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56
BZي .و 2 Lر زة
OP 05
Improving Nutritional Status, Yield and Fruit Quality of Date
Palm by Nitrogen Forms, Potassium and Sulfur fertilization
Hassan A. Kassem
Plant Production Dept., College of Food and Agricultural Sciences, King Saud
University, Saudi Arabia.
hkassem60@yahoo.com
Abstract
The present study was conducted during 2008/2009 and 2009/ 2010 seasons on
Zaghloul date palm grown in calcareous loamy sand soil and irrigated with derange
water. Trees were fertilized with 1000g/palm of actual nitrogen from ammonium
sulphate (N1), ammonium nitrate (N2) or urea (N3) as well as two levels of potassium
(K) and elemental sulfur (S) in order to study their effect on the nutritional status,
yield and fruit quality. Data showed that the N1 increased yield compared to N2 and
N3. Also, the N1 gave better results of fruit weight, length and color than the N3 one.
In general, fruit chemical characteristic were improved by the N1 and N2 compared to
N3. Fruit TSS, sugars, anthocyanin and dry matter content were increased while,
decreased fruit acidity and tannins by the N1. Furthermore, the K2 and S2 rates greatly
enhance the fruit physico-chemical characters . Leaf N, K, Ca, Fe and Zn content were
increased by N1, K2 and S2 applications. Fruit N, P, K, Fe and Zn increased by N1
whereas, fruit Cd, Pb and NO3 was highest with N2 form. The higher rates of both
K2O and S increased fruit N, Fe, Mn, Cd, Pb and NO3.
Keywords: Fertilization; Date Palm; nitrogen form; K2O, productively; derange
water; calcareous soil.
Introduction
Date palm (Phoenix dactylifera) is the most common fruit trees grown under the
hot, semiarid and arid- regions. In Egypt many farmers rely on date palm cultivation
and according to FAO (2010), Egypt is considered the first country of the top ten date
producers (1130000 tones). Zaghloul date is the most economically important soft
cultivars grown in Egypt which is usually harvested and consumed at the Khalal (Bisr)
57
stage. It is mostly cultivated under little rain and high evapotranspiration conditions.
Palms of the present study are growing in loamy calcareous soil and irrigated with
drainage water. These conditions have a great influence on nutrient uptake validity.
Tisdale and Nelson (1978) stated that loamy and sandy loamy soils might with time
become deficient in N, P, K, Mg and B. Therefore the adaptation of a proper
fertilization program, in terms of adequate rates, appropriate sources, efficient methods
of application and application timing are important strategies for better yield and fruit
quality (Fageria and Baligar, 2005).
Nitrogen and potassium are the two most needed nutrients by palms for optimum
growth, yield and fruit quality (Tung et al., 2009). Nitrogen is one of the major
nutrients that have many important roles in plant development and physiological
process. Nitrogen levels and forms, cultivars and soil physical and chemical properties
can be factors related to its use by plants (Abd El-Khalek, 1992; Kage et al., 2003; Li
et al., 2007). The form of the applied nitrogen can have a significant effect on plant
growth and productivity (Owusu, et al., 2000; Houdusse et al., 2007; Sady et al.,
2008). Ammonium (NH4+) and nitrate (NO3-) as well as urea are the major forms of
nitrogen applied and one source or another may be preferred according to plant species
(Marschner, 1995). The response of a large number of fruit trees to the application of
different nitrogen forms has been reported (Kassem et al., 1995; Saleh et al., 2000;
Kassem, 2002).
Potassium is also an important nutrient for date palm growth and productivity (ElHammady et al., 1994; Kassem et al., 1997; Bamiftah, 2000; Al-Kharusi et al., 2009).
Potassium is necessary for basic physiological functions such as formation of sugars
and starch, synthesis of proteins, cell division, growth and fruit formation and it
enhanced fruit size, flavor, and color. (Obreza, 2003; Abbas and Fares, 2008).
Potassium has been shown to promote plant disease reduction, and potassium stress
can increase the degree of crop damage by bacterial and fungal diseases (Kettlewell et
al. 2000; Holzmueller et al. 2007). Furthermore, in the last few years, there has been
an increase concerns about using sulfur application to reduce alkalinity in calcareous
soil (Abbey et al., 2002) and it has become one of the most limiting nutrients in
agricultural production (Eriksen et al., 2004).
58
In the present market economy, product quality has become increasingly important.
Therefore, the present study was carried out in order to investigate the efficiency of
using different nitrogen forms as well as potassium and sulfur fertilization rate on
yield, leaf and fruit mineral content and fruit marketing and edible quality of Zaghloul
dates grown in calcareous soil and irrigated with derange water.
Materials and Methods
Plant materials and experimental design
The present study was conducted during 2008/ 2009 and 2009/2010 seasons on 22
year Zaghloul date palm trees grown in calcareous loamy sand soil at a private orchard
located in Mariut region, near Alexandria, Egypt .
Palms were planted at 10 meters apart and irrigated with derange water. Soil and
irrigation water analysis is presented in Table (1). In December of 2008 and 2009
years, cattle manure (~1.5% N) and calcium superphosphate (15.5% P2O5) were
applied at the rate of 25-30 and 1.5 kg /tree, respectively. All the routine agrotechnical operations were carried out according to the traditional schedule for date
palm plantation. The leaf/bunch ratio was adjusted, in both years, by the end of the
blooming season to meet the value of 10:1 for all experimental palms.
One level of actual nitrogen (1000g/palm) from either ammonium sulphate [(21.5%
N ) N1], ammonium nitrate [(33.5% N) N2] or urea [(46 % N )N3] , two potassium
(K2O) levels (K1= 500 and K2 = 1000 g K2O/palm) from potassium sulphate (48%
K2O) and two elemental sulfur levels (S1 = 750 and S2 = 1500 gm/palm) were
applied either alone or in combinations with each other in order to study their
influence on leaf mineral content , palm yield and fruit quality.
In both experimental years; nitrogen fertilizer was divided into four equal doses and
added in March, April, May and July. Potassium fertilizer was divided into three
equal doses and added in March, May and August. Elemental sulfur was added as a
single dose in December with the manure fertilizer. Twelve fertilization treatments
representing all possible combinations of the three forms of nitrogen fertilizer, two
levels of potassium and two levels of elemental sulfur fertilizer were used (3×2×2=12
treatments). Each treatment was added as a broadcast on the soil surface (~ 1.5 meter
apart from the palm trunk) and palms were irrigated immediately after adding the
59
fertilizer. The experiment was conducted as a split–split plot in randomized complete
design with three replicates (1 replicate = one palm) for each treatment. The soil
moisture content was kept at an appropriate field capacity for sandy soil (50–75%) as
described by Klocke and Fischbach (1984) and Miles and Broner (1998) .
Yield determination
In both years, palms were harvested in mid- October when fruits reached the Khalal
stage (full mature, crunchy and red in color) and the average fruit yield/date and bunch
weight were recorded in kilograms. Additionally, fruit samples were randomly taken
from four different bunches in order to determine fruit physical and chemical quality
characteristics.
Fruit physical characters
In a fruit sample of 30 mature dates for each replicate; fruit weight (g), length (cm),
width (cm) and shape (length/width) were determined. Also fruit color was measured
by using a degree of color intensity as follows: (1) = 100% green, (2) = 25% red, (3) =
50% red, (4) = 75% red and (5) = 100% red.
Fruit chemical characters
In a fruit sample of 40 mature dates for each replicate fruit chemical characteristics
were determined as follows: Fruits were peeled at evenly spaced location on the
equatorial region of the fruits and were cut into small pieces with a clean knife.
Anthocyanin content in 1 g fruit peel tissue was determined according to Fuleki and
Francis (1968). Five grams were taken from the whole fruit fresh (peel + pulp) to
extract the reducing and non-reducing sugars by water at 85°C and the 3-5,
dinitrosallicylic acid according to the method of (Barbin, 2006). The non-reducing
sugars percent were determined by hydrolysis with hydrochloric acid into reducing
sugars. The percentage of reducing and total sugars power was determined according
to AOAC (1995). Non-reducing sugars were calculated by the difference between total
sugars and reducing sugars. Another 5 g was taken to determine the soluble tannins
content (g/100 g fresh weight) as illustrated by Abou Sayed et al. (1997). In the fruit
juice, the percentage of total soluble solids (TSS) was determined using hand
refractometer and acidity as malic acid was determined according to AOAC (1995).
60
Fruit dry matter and nutritional status
A sample of 40 mature fruit was taken to determine fruit dry matter and mineral
content. Fruits were cut into pieces with a clean knife, then an amount of the fresh fruit
was weighed (fresh weight) and dried to a constant weight (g) in air drying oven at 70◦
C, then weighed (dry weight) and fruit dry matter content was calculated as follows:
Fruit dry matter (%) = [average dry weight/average fresh weight] × 100
A leaf sample of three consecutive leaves located just below the fruiting zone (about
two years old) was taken at random from each replicate in mid-October of both years.
Leaf samples were washed with tap water, rinsed twice in distilled water and dried in
air drying oven at 70 ◦C. Dried leaves and fruits were grounded and digested with
H2O2 and H2SO4 according to Evanhuis and De Waard (1980). Suitable aliquots were
taken for the determination of the mineral content. Nitrogen was determined by the
Kjeldahl method (AOAC, 1995). Phosphorus was determined by ascorbic acid method
according to Murphy and Riley (1962). Potassium and sodium were determined by
flame photometer. Ca, Mg, Fe, Zn, Mn, Cu, Pb and Cd contents were measured using
an atomic absorption spectrophotometer (Model 305B). The concentrations of N, P, K,
Ca, Na and Mg were expressed as percentages, while Fe, Mn, Zn, Cu, Pb and Cd as
parts per million (ppm) on dry weight basis. Fruit nitrate content was determined
spectrophotometrically at 540 nm, then calculated as mg/kg dry weight as described by
Singh (1988).
Statistical analysis
The obtained results were evaluated using three ways analysis of variance
(ANOVA). Treatments means and the main effect of nitrogen form, potassium level
and sulfur level was compared using LSD test at 0.05 significance level. Calculations
were carried out using the software package Statistical™ for Windows version 6.1
(Statsoft Inc., 2001, Tulsa, Oklahoma, USA) .
Results
Yield
Regarding the effect of nitrogen forms, the results obtained in Table (2), showed
that, yield components were higher by the application of the ammonium sulfate (N1)
and ammonium nitrate (N2) forms than applying urea form (N3). Additionally, nitrogen
61
as (N1) resulted in higher yield than (N2). Furthermore, the high levels of potassium
and sulfur fertilization significantly increased palm yield in both seasons (Table 2).
Fruit physical characters
The data of both seasons presented in Table (2) indicated that fruit weight was higher
by applying nitrogen in the ammonium sulphate form (N1) than urea. However, fruit
weight was significantly higher in the first season only by applying nitrogen as
ammonium nitrate (N2) than ammonium sulphate (N1) and urea (N3). Furthermore,
higher fruit length was recorded in both seasons by the application of nitrogen as the
sulphate form (N1) than N2 and N3. Fruit width was higher in the second season only
by applying nitrogen as sulphate (N1) and nitrate (N2) than as urea (N3). However, no
significant influence on fruit shape was obtained between the different nitrogen forms.
Application of ammonium sulphate (N1) significantly increased fruit color compared to
urea (N3) in both seasons. In the meanwhile, no significant difference in fruit color
was obtained between N1 and N2. In addition, the data of both seasons presented in
Table (2) showed that K2 and S2 fertilization rate significantly increased fruit weight,
length, width and color, while, fruit shape was not affected.
Fruit chemical characters
The data presented in Table (3) indicated that fruit acidity decreased by the
application of nitrogen as sulfur (N1) and ammonium (N2) forms compared to urea
(N3) in the first season. A significant increase in fruit TSS content was obtained by
applying ammonium sulphate compared to ammonium nitrate (in the second season)
and urea (in both seasons). Furthermore, data of the second season showed that fruit
non- reducing sugars content was higher by N1 and N2 than N3 application. Also, N1
and N2 increased fruit reducing and total sugars content comparing with N3 in both
seasons. The application of ammonium sulphate (N1) lowered fruit tannins compared
to urea application (N3) in both seasons. However, the lowest value of tannins content
was obtained in the second season by applying ammonium nitrate (N2). Anthocyanin
content was higher by applying N1 and N2 than N3 in both seasons. Applying
ammonium sulphate resulted in higher fruit dry matter content as compared with
ammonium nitrate and urea.
62
In addition, the data presented in Table (3) showed a significant increase in fruit
acidity by K2 rate, whereas, acidity was not influenced by S2 level. Moreover, fruit
TSS, anthocyanin, dry matter, reducing and total sugars content increased significantly
by K2 and S2 application rate. Fruit non reducing sugars content increased by K2
fertilization, whereas, it was not affected by S2 application rate. An obvious decrease
in fruit tannins was observed by potassium fertilization in both seasons, while sulfur
application lowered fruit tannins content in the first season only.
Leaf mineral content
Regarding the influence of nitrogen forms on leaf mineral content, data presented in
Tables (4&5) showed that leaf nitrogen increased by the application of N1 and N2
compared to N3. Ammonium sulphate (N1) resulted in higher leaf P, K, Ca, Fe and Zn
content than urea (N3). However, ammonium nitrate (N2) gave the highest leaf Mg
and Mn content in comparison with N1 and N3. Sodium concentration in the leaves
was not affected by any N form.
Furthermore, the data presented in Tables (4&5) showed that leaf N, P, K, Fe and
Mn content increased significantly by K2 and S2 fertilization. However, Ca and Mg
content decreased by K2 application, while Ca was not affected by applying S2
fertilizer. In the meantime, leaf Na and Cu content decreased by S2 application and
was not affected by K2 fertilizer.
Fruit mineral and nitrate content
The obtained results (Tables 4&5) showed that nitrogen added as ammonium
sulphate (N1) increased fruit N, Fe and Zn content in comparison with ammonium
nitrate (N2) and urea (N3). Fruit P and Ca content was higher by applying N2 than N3
in the second season. In addition, fruit potassium content increased significantly by the
application of N1 and N2 as compared to N3. On the other hand, fruit Mg, Mn, Cd and
NO3 content was lower by the N1 and N3 forms than N2. Fruit Pb content decreased
by the application of the sulphate form (N1) in comparison with N2 and N3. However,
nitrogen fertilizer form had no influence on fruit Na and Cu content.
With regard to potassium and sulfur fertilization , data presented in Tables (4&5)
indicated that fruit N, K, Fe, Mn, Cd, Pb and NO3 content increased while, Ca and Mg
content decreased by the application of K2 fertilizer. On the other hand, fruit P, Na, Zn
63
and Cu content was not affected by K2 fertilization. In addition, the application of
elemental sulfur (S2) significantly increased fruit N, Fe, Zn, Mn, Cd and Pb while,
decreased Cu and NO3 contents. On the contrary, fruit P, K, Ca, Mg and Na content
was not influenced by S2 application.
Discussion
Similar increase in the yield of date palms grown in Egyptian environment by
fertilization is previously recorded (Hussein and Hussein, 1983; El-Hammady et al.;
1994; Kassem et al., 1997 and Marzouk and Kassem, 2011). Palms fertilization has
been found to increase shoot and leaf growth (Hussein and Hussein, 1983 and AlGhamidi et al, 1999), which might have direct influence in improving palm
productivity and fruit quality.
Applying nitrogen as a sulphate form indicated the highest yield, fruit weight and
fruit dry matter content. Guelser (2005) stated that ammonium sulphate (NH 4) SO4 as
N- form decreases soil pH, which might favor elements availability and uptake by
plants in slightly alkaline soils. Also, ammonium assimilation into plant metabolites
requires less energy than nitrate assimilation, as it does not need to be reduced. Plants
may save energy by taking up reduced nitrogen and energy saved may be used for
increase production of secondary metabolites (Elwan and Abd El-Hamed, 2011).
Additionally, the application of nitrogen as the sulphate and nitrate forms resulted in
higher fruit quality characteristics than application of urea. This may be due to urea is
not suitable for fertilization under the conditions of the present study. Nitrogen validity
and uptake efficiency depends on the applied form (Sady et al., 2008). Urea -based
fertilizers are susceptible to volatilization losses of nitrogen especially under the
conditions of warm climate, light texture soil with pH levels greater than 7 (Nielsen,
2006), which are similar to that of the Zaghloul palms of the present investigation.
Recently, the possible human health effect due to high nitrate content in vegetables
and fruits is concerned. About 5-10% of the ingested nitrate is converted into the more
toxic nitrite by salivary or gastrointestinal reduction (Boink and Speijers, 2001).
Fertilization with ammonium sulfate and urea has been showed to lower the nitrate
content in the edible part of plants compared to ammonium nitrate (Sady et al., 2001).
Similar findings are obtained in the present study as nitrate content in the fruits was
64
decreased by applying ammonium sulphate and urea compared to ammonium nitrate.
The nitrate accumulation in plants is very low when nitrogen is applied in the (NH 4)2
SO4 form because it is slowly nitrified (Goh and Vityakan, 1986).
Regarding to the effectiveness of sulfur in increasing the yield and fruit quality, it is
well reported that plants assimilate inorganic sulphate into cysteine, which is converted
into methionine (Nicoforova et al, 2003). This amino acid and others (tryptophan or
phenylalanine) are precursors for glucosinolate production, a group of health-promoting
compounds (Schonhof et al., 2007). N and S interact to exert a strong effect on various
growth parameters such as biomass and yield (Salvagiotti and Miralles, 2008). S is an
essential constituent of enzymes involved in N metabolism (Campbell, 1999; Swamy et
al., 2005) and its availability could lead to an increase in N assimilation.
Also, fruit Cd and Pb content increased when N was applied in the nitrate form, as
well as it increased by K and S fertilization. The influences of different nitrogen
sources on Cd accumulation in plants are reported (Basta et al, 1998; Mair et al, 2002).
Also, many studies confirmed the positive effect of K when applied in the sulphate
form on Cd uptake by plants (Zhu et al, 2002 and Zhao et al, 2003). Additionally, the
sulfur anion weather applied as solo application or accompanied with K was also
found to increase Cd accumulation in plants (Bingham et al., 1986, Grant et al, 1999).
However, McLaughlin et al. (1998) showed no significant increasing effect of sulfur
on Cd uptake and suggested that sulfur would not have the same effect as the chloride
on Cd uptake. The strong relationship between soil salinity (Chloride concentration)
and Cd accumulation is reported (Norvell et al., 2000). Thus, studying the effect of K
and S fertilization and soil conditions on plant Cd and Pb accumulation is very
important to minimize their concentration in agricultural produce.
Conclusion
Hence, to produce higher yield and overall product quality of Zaghloul dates, in
particular lower nitrate content, the application of nitrogen fertilizer as ammonium
sulphate is advisable. Special considerations must be taken when fertilizing date palms
with K2 and S2 as, their applied rate, form and soil salinity might affect the uptake of
heavy metals, thus producing dates with quality and no human health risks.
65
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70
Table (1): Analysis of the experimental orchard soil and irrigation water.
Properties
Orchard Soil
EC
dS/m
pH
HCO-3
meq/L
++
Ca
meq/L
++
Mg
meq/L
+
K
meq/L
+
Na
meq/L
Fe
ppm
Mn
ppm
Zn
ppm
Cu
ppm
Cl 2
meq/L
Organic matter (%)
CaCO3
(%)
0-50 cm
50-100 cm
3.3
8.2
10
21.3
8.6
2.4
12.6
20.9
32.8
11.6
14.8
21
1.3
32.1
4.1
7.9
12
24.7
10.5
1.3
18.2
16.3
15.5
6.7
11.8
17.3
0.4
35.4
Irrigation water
3.8
7.2
10.8
14
7.6
1.1
17.5
0.75
0.21
0.62
0.6
17.6
-
Table (2): Main nitrogen forms, potassium and sulfur effect on yield and fruit
physical characters of Zaghloul date in 2008/2009 and 2009/2010 seasons.
Treat
ment
s
Yield
kg/palm
a
kg/bunch
a
N1
N2
N3
K1
K2
S1
S2
218
200 b
180 c
188 b
211 a
192 b
207 a
22
20 b
18 c
18 b
22 a
17 b
23 a
N1
N2
N3
K1
K2
S1
S2
191 a
179 a
166 b
160 b
197 a
170 b
187 a
19 a
18 ab
17 b
15 b
21 a
16 b
20 a
Fruit
Weight
(g)
Fruit
Length
(cm)
2008/ 2009
25.6 b
6.6 a
a
26.7
6.1 b
24.4 c
5.6 c
24.5 b
5.6 b
a
26.7
6.6 a
25.2 b
5.9 b
26.0 a
6.4 a
2009/ 2010
28.8 a
6.9 a
b
26.1
6.6 b
25.3b
5.7 c
23.3 b
5.8 b
30.2 a
7.0 a
b
26.5
6.0 b
27.0 a
6.8 a
Fruit
width
(cm)
Fruit
shape
Red
color
2.8
2.6
2.4
2.2 b
3.0 a
2.4 b
2.8 a
2.4
2.4
2.3
2.5 a
2.2 b
2.5
2.3
4.8 a
4.5 ab
4.3b
4.2 b
4.9 a
4.5b
4.6 a
3.0 a
2.9 a
2.5b
2.5 b
3.1 a
2.6 b
3.0 a
2.3
2.3
2.3
2.3
2.3
2.2
2.4
4.9 a
4.7 ab
4.2b
4.4b
4.8 a
4.3b
4.9 a
Values with different letters show significant difference as determined by L.S.D 0.05.
71
Table (3): Main nitrogen forms, potassium and sulfur effect on fruit chemical
characters of Zaghloul date in 2008/2009 and 2009/2010 seasons.
Treatments
N1
N2
N3
K1
K2
S1
S2
N1
N2
N3
K1
K2
S1
S2
Acidity
(%)
TSS
(%)
Sugars
(%)
Reduc-ing
Nonreducing
Anthocyanin
(mg/
100 g)
Tannins
(%)
Total
Dry
matter
(%)
2008/2009
0.33
27.5
5.5
27.2
32.7
0.48 b
22.3 a
32.8 a
a
ab
a
a
ab
a
0.35
26.4
5.6
26.5
32.1
0.50
21.8
29.7 b
0.41 b
25.3 b
5.0
24.4 b
29.4 b
0.60 a
17.5 b
27.8 c
0.32 b
25.8 b
5.0 b
25.1 b
30.1 b
0.68 a
19.1 b
29.2 b
0.40 a
27.0 a
5.7 a
27.0 a
32.7 a
0.37 b
22.0 a
31.0 a
b
b
b
a
b
0.35
25.5
5.0
25.5
30.5
0.59
19.1
27.8 b
0.37
27.3 a
5.7
26.5 a
32.2 a
0.47 b
22.0 a
32.4 a
2009/2010
a
a
a
a
0.36
28.2
5.9
26.4
32.3
0.29 b
22.9 a
35.6 a
b
a
a
a
c
b
0.33
27.2
5.8
25.6
31.4
0.24
20.4
32.6 b
0.34
25.9 c
4.0 b
22.5 b
26.5 b
0.46 a
16.5 c
31.7 b
0.32 b
24.9 b
4.6 b
22.6 b
27.2 b
0.43 a
18.3 b
31.7 b
a
a
a
a
a
b
a
0.37
29.3
5.9
27.1
33.0
0.23
21.6
34.9 a
0.34 a
26.5 b
4.6 a
24.1 b
28.7 b
0.31 a
18.3 b
31.3 b
0.35 a
27.7 a
5.9 a
25.6 a
31.6 a
0.35 a
21.6 a
35.5 a
Values with different letters show significant difference as determined by L.S.D 0.05.
a
a
a
a
Table (4): Main nitrogen forms, potassium and sulfur effect on leaf and fruit
macronutrients content (%) of Zaghloul date in 2008/2009 and 2009/2010 seasons.
N
forms
N
P
leaf
Fruit
leaf
K
fruit
leaf
Ca
Fruit
Mg
Na
leaf
Fruit
Leaf
Fruit
leaf
Fruit
2008/2009
a
a
a
N1
2.45
N2
2.38a
1.38b
0.27b
b
b
b
N3
K1
K2
2.24
2.28
b
2.43
a
b
1.64
1.25
1.33
b
1.52
a
b
S1
2.23
S2
2.48a
1.58a
a
a
1.27
0.36
0.29
0.25
b
0.30
0.28
a
b
0.21
1.96
a
0.98a
1.48a
0.76
0.40b
0.31b
0.38
0.18
0.17
1.82ab
0.94a
1.32b
0.82
0.57a
0.45a
0.33
0.21
b
b
b
b
b
0.38
0.25
0.38
0.19
0.35
0.24
a
0.24a
0.19
0.18
0.20
1.70
1.70
b
1.95
a
b
0.17
1.73
0.33a
0.21
1.92a
a
a
a
0.81
0.81
b
1.01
a
1.28
1.42
1.30
a
b
0.78
0.87
0.70
a
b
0.42
0.56
0.37
a
b
b
0.35
0.43
0.32
a
b
0.33
a
0.84
1.38
0.87
0.40
0.42
0.98
1.34
0.70
0.53a
0.42a
0.31b
0.19a
0.90a
1.55a
0.83ab
0.48b
0.40b
0.38a
0.22a
2009/2010
N1
2.56
N2
2.58a
1.24b
0.31b
0.20ab
1.74b
0.93a
1.53a
0.93a
0.62a
0.55a
0.32a
0.19a
N3
2.42b
1.28b
0.26c
0.18b
1.62b
0.73b
1.33b
0.79b
0.42b
0.38b
0.34a
0.26a
K1
2.40b
1.21b
0.29b
0.20a
1.62b
0.68b
1.70a
0.94a
0.58a
0.54a
0.39a
0.25
K2
2.64a
1.48a
0.34a
0.21a
1.94a
1.00a
1.24b
0.76b
0.43b
0.35b
0.30a
0.20
b
b
b
b
a
a
b
a
a
0.25a
0.22b
0.20a
S1
2.40
S2
2.64a
1.51
1.22
1.47a
0.37
0.26
0.37a
0.24
1.98
0.19
1.69
0.22
1.87a
0.68
1.03a
1.55
1.39a
0.89a
0.42
0.81a
0.59a
0.33
0.56a
0.47
Values with different letters show significant difference as determined by L.S.D 0.05.
72
Table (5): Main nitrogen forms, potassium and sulfur effect on leaf and fruit
micronutrients, fruit heavy metals and nitrate contents (ppm) of Zaghloul
date in 2008/2009 and 2009/2010 seasons.
N
Fe
forms leaf
Zn
Mn
Cu
Cd
Fruit leaf fruit leaf Fruit leaf Fruit Fruit
Pb
NO3
Fruit
Fruit
2008/2009
a
49
40
42
28b
29b
18a
0.012c
0.98b
57b
59b
51c
44b
36c
32b
30b
52a
44b
39a
31b
30b
37a
23a
17a
0.021a
0.016b
1.21a
1.14a
76a
58 b
101b
128a
53b
66a
36b
50a
32
36
42b
50a
27b
38a
26a
38a
17a
22a
0.012b
0.021a
1.02b
1.20a
52b
76a
105b
124a
52b
67a
40b
46a
22b
46a
33b
59a
27b
38a
35a
29b
22a
17b
0.010b
0.023a
1.04b
1.18a
68 a
60b
a
a
44
a
32
a
50b
32b
39a
23a
0.018b
1.02b
61b
N1
124
N2
N3
113ab
106b
K1
K2
S1
S2
68
a
a
a
b
2009/2010
N1
133
74
N2
128a
62b
43a
26b
66a
41a
32b
19a
0.028a
1.32a
81a
N3
111b
58b
30b
27b
53b
28b
34b
23a
0.019b
1.21a
60b
K1
K2
116b
132a
56b
73a
34b
44a
25
32
51b
62a
29b
38a
32
38
20a
23a
0.011b
0.032a
1.06b
1.31a
60b
75a
S1
S2
115b
123a
53b
76a
32b
46a
23b
34a
50b
63a
23b
44a
39a
31b
26a
17b
0.017b
0.026a
0.99b
1.38a
78a
67b
Values with different letters show significant difference as determined by L.S.D 0.05.
73
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74
OP 06
Effect of Gibberellic acid on fruit quality of Samani and
Zaghloul date palm cultivars.
Ghazawy, H.S1 ; Bakr, E.I2,S.EL-Kosary2 and A.EL-Bana1
1- Central Laboratory for Date Palm Researches and Development.
2- Pomology Dept., Faculty of Agriculture, Cairo University
Abstract
The present investigation aims to study the effect of Gibberellic acid (GA3) application
on fruit quality of Samani and Zaghloul cultivars in Egypt. Fruit quality estimated as
value of fruit set percentages, fruit physical characteristics (fruit weight and volume) and
fruit chemical characteristics (total soluble solid, total soluble sugars, reducing sugars,
non-reducing sugar and fruit colouration). A high fruit quality was recorded by the
application of Gibberellic acid (GA3) at 50 ppm concentration. Concerning the interaction
between GA3 concentration and time of spraying, the application of GA3 at 50 ppm after
75 days from blooming time gave the highest fruit quality.
Key words: Date palm - Gibberellic acid - (Phoenix dactylife- Fruit quality –
Samani - Zaghloul
Introduction
Date palm (Phoenix dactylifera. L.) has a great economical importance and
agricultural uses throughout human’s history. Also, it is one of the oldest cultivated
fruit trees in the world. Date palm is a very important crop in the Middle East, since it
can grow well in both semi-dry desert areas and the newly cultivated land. In Egypt,
date palm trees distribution covers a large area extends from Aswan to north Delta.
Egypt is one of the most productive countries of dates in the world, the number of
fruitful female palms in Egypt is about 12 million bearing female palms produce
1,300000 tons of dates (FAO DATA, 2005).
The commercial season of fruits have important economic factor and several trials
have been done to improve yield and fruit quality of date palm. El- Nabawy et al.,
(1977) observed that Gibberellic acid treatment of Samani dates results in greater fruit
size. Gibberellic acid application produced long bunch stalk and spiraling in Deglet
75
Noor, but not in other cultivars fruits became elongated and cylindrical. However, at
high concentration of Gibberellic acid, fruits of Deglet Noor shriveled. Fruits did not
develop normal Khalal color and ripened late (Asif et al, 1985). In case of dry dates of
Sakkoti, Gibberellic acid increased fresh weight of fruits and bunch but at the expense
of composition and quality (Nixon, 1959).
The present investigation was designed to study the effect of Gibberellic acid (GA 3)
application on fruit quality of Samani and Zaghloul cultivars in Egypt.
Materials and Methods
The present study was carried out during two successive seasons, at the
Experimental Research Station, Faculty of Agriculture, Cairo University, Giza
Governorate. Samani and Zaghloul date palm cultivars were used in this investigation.
Female palms of both cultivars (15 years old) were pruned at 8:1 leaf /bunch ratio (ElShazly, 1999). The palms were received normal agricultural practices. Samani and
Zaghloul palm cultivars were pollinated by the same source of pollen grains after
fourth days of spath cracking in both seasons.
Nine palm trees of each of Samani and Zaghloul cultivars were selected similar in
age, growth and pruning, received the similar orchard management and pollinated by
the same source of pollen grains. Twelve bunches were left on each palm tree and
labeled as groups, each group contains three bunches (replicate). Then each bunch
group was treated with one concentration of GA3 (0, 50, 100 and 150 ppm). GA3
applied after 75, 95 and 115 days from blooming time. All bunches were covered
before and after treatment to avoid the mixing between treatments. Samples of 30 date
fruits from each replicate were randomly picked from each bunch in intervals times
until harvesting time for the determination of physical and chemical properties.
1-Fruit set:
The average fruit set was calculated using the following equation:
Fruit set % =
Total number of setting fruits per bunch
× 100
Total scares number per bunch
2- Physical and chemical characteristics:
2-1- Fruit physical characteristics:
2-1-1- Fruit weights: Fruit weight was obtained in grams as average of 25 samples of
each treatment.
76
2-1-2- Fruit volume: Average fruit volume was determined by immersion samples
(five fruits) in a known quantity of water in a graduated jar. By replacement, the
difference between the new reading of water on the jar and the initial reading indicates
the volume of the fruits. Then the average fruit size was calculated in cm3.
2-2- Fruit chemical characteristics:
2-2-1- Total soluble solids (T S S): The (TSS) content was determined in the fruit
juice using Zice refractometer as described in (A.O.A.C.1985).
2-2-2-Fruit content of sugars: Total soluble sugars were determined according to
Smith et al. (1956) in the methanolic extract using the phenol sulphuric acid method.
Reducing soluble sugars was determined in the ethanolic extract according to Nelson
and Somogyi (1944) as described in A.O.A.C. (1985).
2-2-3-Fruit content of pigments: Carotene (in Samani cultivar fruit) and anthocyanin
(in Zaghloul cultivar fruit) content were determined according to Wettstein, (1957).
Statistical analysis: The obtained data were subjected to analysis of variance. The
mean values were compared using LSD method at 5% level. The data were tabulated
and statistically factorial analyzed according to the randomized complete block design
method (Snedecor and Cochran, 1980). Also, the percentages were transformed to the
arcsine to find the bionomial percentages according to steel and Torrie (1980).
Results and discussions
The effect of Gibberellic acid (GA3) application at 0, 50, 100, 150 ppm after 75,
95,115 days from blooming time on fruit quality during two successive seasons
summarized as follows:
A-Physical characteristics:
1- Fruit set percentage:
Cultivars Samani and Zaghloul fruit abscission was significantly increased
gradually with the increasing of GA3 concentration (50,100,150 ppm) during two
seasons (table1). In the view of interaction between the GA 3 concentration and
spraying time the concentration 50 ppm applied after 75 days from blooming time give
the most acceptable reduction in fruit abscission in Samani (15.05 & 14.81 in first and
second seasons respectively) and Zaghloul (12.91 & 12.73 in first and second seasons
77
respectively). This result is combatable with Aziz et al (1985), with Hussein et al
(1986) and Ghaffor and Rahman(1986).
2-Fruit weight:
Samani and Zaghloul cultivars recorded the highest fruit weight at 50 ppm of GA 3
followed by 100 ppm and 150 ppm in both seasons (table2). Concerning the
interaction between GA3 concentration and time of spraying, The application of GA3
at 50 ppm after 75 days from blooming time gave the heist fruit weight in Samani
(36.8 & 43g in first and second seasons respectively) and Zaghloul (37.7 & 40g in first
and second seasons respectively) than 95 and 115 days.
The above-mentioned results are agree with Hussein et al. (1976 a) on Barhi
cultivar, El-Kasassas (1983) on Zaghloul cultivar and Azia et al. (1985) on Sewy
cultivar.
3-Fruit volume:
The highest fruit volume was recorded at 50 ppm of GA3 in both cultivars Samani
and Zaghloul of both seasons than 100 and 150 ppm (Table 3).
Regarding the
interaction between GA3 concentration and time of spraying, 50 ppm of GA3 applied
after 75 days from the blooming time gave the highest fruit volume of Samani cultivar
(38.08 &42.99 in first and second seasons respectively) and in Zaghloul cultivar
(36.62 & 40.06 in first and second seasons respectively) than other times.
The results are accordance with the findings of El-Nabway et al.(1981),
Shehata(1986),Husein et al.(1993), Mostafa and Sief (1993), El-Hodiri et al.(1994),
Ben-Abdalla and Lepoive (2001) and Bassel and El-Deeb (2002).
B-Chemical constituents:
1-Total Soluble Solids (TSS)
The fruit contents of TSS recorded the highest percentage with 50 ppm of GA3
followed by 100,150 ppm for both cultivars in both seasons (table4).
Regarding the interaction between the time of application and GA 3 concentration,
the fruit contents of TSS was the highest percentage when applied after 75 days after
blooming time in Samani cultivar (29.9 &28.93 in first and second seasons
respectively) and in Zaghloul cultivar (37.35& 39.27 in first and second seasons
78
respectively) than 95,115 days from blooming time. These results were in line with the
Kamal (1995).
2- Fruit content of sugars:
Total soluble sugars, reducing sugars and non-reducing sugars were the highest
value with 50 ppm of GA3 followed by 100 and 150 ppm for both cultivars in both
seasons (table 5, 6 &7). As general means, the spraying time has no significant effect
on the total soluble sugar, reducing sugar and non-reducing sugar contents for both
cultivars in both seasons.
Concerning the interaction between GA3 concentration and spraying time, 50 ppm
of GA3 gave the highest total soluble sugars when applied after 75 days from blooming
time in Samani cultivar (30.3 & 30.33 in first and second seasons respectively) and in
Zaghloul cultivar (30.53 & 31.1 in first and second seasons respectively) (Table 5).
The same trend was recorded for non- reducing sugars content in Samani cultivar (25.1
& 25.55 in first and second seasons respectively, Table 6) and in Zaghloul cultivar
(25.96 & 25.98 in first and second seasons respectively, Table 6) and for reducing
sugars content in Samani cultivar (5.67 & 4.22 in first and second seasons
respectively, Table 7) and in Zaghloul cultivar (5.23 & 4.87 in first and second seasons
respectively, table 7). The results are coincided with the findings of El-Kassas(1983).
3- Fruit content of pigments:
The highest percentage of pigments of cultivars Samani (Carotene) and Zaghloul
(anthocyanin) was recorded at 50 ppm of GA3 in both seasons.As general means, the spraying
time has no significant effect on the pigment percentages for both cultivars in both seasons.
Conclusion
Concerning the interaction of GA3 concentrations and spraying time, 50 ppm of
GA3 was recorded the highest pigment content when sprayed after 75 days from
blooming time in Samani (1.83 & 1.84 ) and in Zaghloul (0.31 & 0.3) in first and
second seasons in both cultivars, respectively (table 8).Generally fruit quality
estimated as value of fruit weight and volume, total soluble solid, total soluble sugars,
reducing sugars, non-reducing sugar and fruit colouration was affected by
GA3(50ppm) applied after 75 days from blooming time.
79
REFERENCES
[1] Asif,M.I.:D.S.Al-Tahir and Y.M.Makki(1985).Effect of some growth chemicals on
fruit morphological characteristics of Gur and Khalas dates.In proceedings of the
first symposium on the date palm in Saudi Arabia. Al-Hassa,Saudi Arabia;King
Faisal Univ.(1985)270-275(En,ar,9 ref.).
[2] Association of Official Agricultural Chemists (1985): Official Methods of Analysis.
Published by A.O.A.C Benjamin Franklin Station, Washington, D. C., U. S. A.
[3] Aziz, A. B. A.; S. S. Maximos; I. A. Desouky and N.R.E. Samra (1985): Effect of
GA3 and hand pollination on the yield and quality of Sewi dates .In proceedings of
the first symposium of the date palm in Saudi Arabia.
[4] Bassal, M.A. and M.D. El-Deeb. 2002.
Effect of thinning and some growth
regulators on yield and fruit quality of Zaghloul date palm. Zagazig J. Agric. Res.
29 (6): 1815-1837.
[5] Ben-Abdalla, A. and P. Lepoivre (2001): Production of date palm (phoenix
dactylifera L.) fruit and diploid embryos following gibberellic acid treatment of
unpollinated female inflorescence. Cahiers Agric., 9 (6), 467- 473. Hort. Abst.
Vol.71 No. 11 (9412).
[6] Bulletin of Agricultural Economic, Central Management of Agricultural Economic,
Ministry of Agricultural, Egypt, 2005, ( In Arabic ).
[7] El-Kassas, Sh.E. 1983. Manual bunch and chemical thinning of “Zaghloul” dates.
Assiut J. Agric. Sci. 14 (2): 221-233.
[8] El-Nabawy,S.M.:ElHammady,A.M,;Marei.N.S.andBondok.A.Z.(1977):Effect
of
some growth regulators on growth and development of Samani date fruits.
Research Bulletin. Faculty of Agriculture, Ain shams University (1977), No. 729,
23 pp. (En, ar, 32 ref.) Cairo, Egypt. Agriculture. Ain Shames University
No.729.23pp.
[9] EL-Nabawy, S. M.; N. S. A. M. El-Hamady Marei and A. Z. Bondok (1981): effect
of some growth regulators on growth and development of “Samany” date fruits.
Research Bulletin, faculty of Agriculture, Ain shams University (1977), No. 729,
23 pp. (En, ar, 32 ref.) Cairo, Egypt. Agriculture. Ain Shames University
No.729.23pp.
80
[10] EL-Hodairi, M. H., A. A. El-Barkouli and O. Bawa (1994): The effects of some
growth regulators on fruit set of date palm Phoenix dactylifera L. trees. Acta
Hort. No. 321, 334- 342 Hort. Abst. Vol. 64 NO. 12 (9913).
[11] El-Shazly, S.M. 1999. Effect of fruit thinning on yield and fruit quality of
“Nabtet Ali” Saudi date palm. The International Conference on date palm Nov.
9-11, Assiut Univ., Egypt, pp.17-33.
[12] Gaffor, A. and Rahman, S. F. (1986): Effect of different concentration of
Naphthalene acetic acid on fruit drop, yield and quality of date palm Phoenix
dactylifera L.) cultivar DHAKKI .Abs-tract of the second symposium on date
palm, March 3-6, 1986, King Faisal Univ. Saudi Arabia.
[13] Hussein, F.; Moustafa, S. and El-Samaraea, F. (1976) Size, quality and ripening
of “Barhi” dates as affected by fruit thinning. Zagazig J. Agri. Res. 3 (2): 125142. Egypt.
[14] Hussein, M. A., H. M. Mahmoud and K. A. A. Amin (1986): Effect of GA3, fruit
thinning and some pollen application on fruit quality of Zaghloul dates. Abstract
of the second symposium date palm, March 3- 6, 1986 King Faisal Univ. Saudi
Arabia. Assiut J. of Agric. Sci. 23 (2): 335-347. Egypt
[15] Hussein, M. A., M. M. Hammdy and k. I. Amen (1993): Changes in the physical
and chemical characteristics of Zaghloul dates during development and maturity
as affected by GA3 and CCC under Assuit Governorate conditions. The Third
Symp. In Date Palm, Saudi Arabia: 369.
[16] Kamal. H. M. (1995): Effect of some growth regulators on the physical and chemical
properties of date fruits. Bull. Fac. Agric., Univ. Cairo, 46 (1995): 215-228.
[17] Moustafa, A. A.and S. A. Seif (1993): Effect of Ethrel and GA3 treatments on
yield and fruit quality of Sewi date palms, grown in El-Fayoum Governorate.
The Third Sym. on Date Palm in Saudi Arabia.
[18] Nelson N., Somogy, I., (1994): Colourimetric method for determination of
Reducing sugars related substances. J. Bio. Chem. 153. 375- 379. (Manual of
analysis of fruit and vegetable products 1978: 9-17.
[19] Nixon, R. W. (1959): Effect of gibberllin on of date palm. Date grows Inst. 98: 7- 9.
81
[20] Shehata E. E, (1986): Manual bunch and chemical thinning of Zaghloul dates the
second Symposium on Date Palm. Pp: 179-186.
[21] Smith,F.,Gilles,m.a.,Hamilton,J.K.and Gedes,P.A.(1956).Colorimetric methods
for determination of sugar and related substances,Anal.Chem.,23:350.
[22] Snedecor, G.W. and Cochran, W.G. (1980) Statistical methods, Oxford and
J.B.H pup co. Publishing 6th edition. Press Ames Iowa. U.S.A, PP.593
[23] Steel, R.G.D., And Torrie, J. H. (1980): Reproduced from principles and
procedures of statistics. Printed with the permission of c. 1. Bliss, pp. 448-449.
[24] Wettstein, D. (1957): Chlorophyll lethal faktoren under submikroskopoch for
mvechsel der plastide Explt. Gell. Res. 12: 427-433.
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ء٥٠ آ+' م٧٥ A' GA3 رشO< F DON ا8ر آC + / T وا+ وا
. F ا8 آ9 ا ن9
82
OP 07
Effects of water stress and arbuscular mycorrhizal fungi on
reactive oxygen metabolism and antioxidant production by date
palm seedlings (Phoenix dactylifera L.)
Kasrati A.1, Oussouf F.M.1, and Qaddoury A.1
1- Lab. Plant Biotechnology and Agro physiology of Symbiosis, FST-gueliz
Marrakech, Morocco
qadahmed@gmail.com
Abstract
Water deficit is known to induce formation of reactive oxygen species (ROS) in
higher plants. To maintain growth and productivity plants have to prevent
accumulation of these harmful species as rapidly as possible. The effect of water stress
on biomass production, reactive oxygen metabolism and osmoregulation substances
was investigated in mycorrhizal date palm (Phoenix dactylifera L.) seedlings grown
under well-watered (75% of field capacity) or water-stressed (25% of field capacity)
conditions. After 30 days of water treatments, plant height, root length and shoot dry
weight and root dry weight were recorded. Biochemical changes including superoxide
dismutase (SOD), catalase (CAT), guaiacol peroxidase (G-POD) and ascorbate
peroxidase (APX) activities were determined. ROS production (malondialdehyde and
hydrogen peroxide) as well as osmoregulation matters (proline, soluble protein and
soluble sugar) accumulation were analysed. Result showed that growth parameters
(plant height and root length) and biomass production (shoot and root dry weights)
were higher in mycorrhizal (AM) than in non-AM seedlings regardless of water
treatments. The highest biomass production and plant growth was measured in date
palm seedlings colonized by Glomus intraradices as well as under well watered (WW)
and water stressed (WS) conditions. Moreover this association showed the highest
mycorrhizal dependency value. There was less malondialdehyde (MDA) and high
soluble sugar and high proline concentrations in leaves of AM seedlings under WW
and WS treatments, while AMF inoculation did not affect hydrogen peroxide (H2O2)
concentration. Whether WS or not, AM symbiosis notably increased the guaiacol
peroxidase (G-POD) and ascorbate peroxidase (APX) activities of leaves. AM
83
infection also markedly increased the SOD activity and decreased the catalase activity
of WS leaves. Our results suggest that the increased activity of antioxidant enzymes
and decreased concentration of ROS compounds found in AM plants may serve to
protect the date palm seedlings against oxidative damage, enhancing drought
tolerance. The order of the ability of drought tolerance is GI > GM > Non-AM by
using index of drought tolerance (IRS) and oxidative damage (estimated as the ratio of
malondyaldehide to proteins) in leaves of date palm seedlings.
Key words: Arbuscular mycorrhizal fungi (AMF); date palm; drought; antioxidant
metabolism; oxidative damage.
Introduction
In many arid and semi-arid areas, the lack of adequate soil moisture leading to water
stress is the major limiting factor in crop production because it affects almost all plant
functions [22]. It well established that Arbuscular mycorrhizal fungi (AMF) can form
symbiotic association with the vast majority of land plants including those of the arid
areas [12]. Once established, AMF symbiosis improved plant growth, particularly
through enhanced water and mineral nutrient uptake thereby enhancing host plant
protection against the detrimental effects of drought [12, 29, 30]. Mycorrhizal plants
have higher water and nutrients uptake due to hyphal extraction [14] and higher root
hydraulic conductivity [7] than non-mycorrhizal plants. Several studies describing the
effect of AMF on growth and nutrition of date palm seedlings were reported [3, 20, 23,
25]. In earlier studies we described the positive effect of AM fungi on growth and
development of pot-grown date palm seedlings under greenhouse conditions. We have
shown that AM fungi allow for greater uptake of nutrients and play an important role in
water relations thereby stimulating date palm growth under water deficiency [5, 8, 9, 10,
11, 16]. The objective of the present study is to evaluate the effect of AMF on biomass
production and antioxidant metabolism in date palm seedlings under water deficiency.
Materials and methods
Pre-germinated Seeds of date palm, collected from the date palm grove of
Marrakech, were transferred in pots containing 1 kg of sterilized soil (collected from
date palm grove) without or with inoculum of arbuscular mycorrhizal fungi (AMF)
and grown under greenhouse conditions. Two Glomus species - Glomus intraradices
84
(GI) and Glomus mosseae (GM) supplied by the Estación Experimental del Zaidín
(Granada, Spain) were assessed. AM fungal inoculum consisted of 10g of a mixture of
rhizospheric soil from trap cultures containing spores, hyphae and mycorrhizal root
fragments of the corresponding AMF. The same amount of autoclaved mixture of
inoculum was added to non-inoculated plants. Water stress treatments consisted of two
watering regimes: 75% of field capacity (75% CF) and 25% of field capacity (25%
CF). Water status of the pots was daily examined and the amount of water loosed was
refilled into each pot. The experiment was arranged in a completely randomized block
design. Each treatment was replicated twenty times. Thirty days after water stress
application, plants were harvested and roots were washed free from soil under a stream
of cold tap water. Root colonization was evaluated according to Trouvelot et al. (1986)
[32]. Plant height, root length, shoot dry weight and root dry weight were recorded.
Biochemical changes including superoxide dismutase (SOD), catalase (CAT), guaiacol
peroxidase (G-POD) and ascorbate peroxidase (APX) activities and Osmoregulation
matters including proline, soluble protein and soluble sugar were determined
according to balsam et al., (2009) [8, 9]. Leaf antioxidant enzyme activities SOD [17],
CAT [1], APX [6] and GPOX [21] were determined. Malondialdehyde was measured
by the thiobarbituric acid method as described by Heat and Packer (1981) [19] and
H2O2 by using titanium method according to Patterson and al. (1984) [26]. Shoot dry
weight (SDW) was recorded by drying in oven at 70ºC to constant weight. All data
were analyzed statistically by an analysis of variance using ANOVA modules of the
Statistica software program (Statsoft, 1995). Mean comparisons were conducted using
Newman-Keuls test at P < 0.05.
Results
Root colonization by Glomus mosseae and G. intraradices varied from 54% to 61%
(Table 1). Water stress markedly decreased root colonization, well watered (WW)
seedlings showed 30% higher root colonization than water stressed (WS) seedlings.
Water stress significantly decreased biomass production (shoot dry weight, root dry
weight) and plant growth (shoots height and root length). AM inoculation notably
increased shoot dry weight, root dry weight, shoot height and root length in both WW
seedlings and WS seedlings (Table 1). The highest biomass production and plant
85
growth was measured in date palm seedlings colonized by Glomus intraradices as well
as under well watered and water stressed conditions. Moreover, this association
showed the highest mycorrhizal dependency value.
Water stress increased the MDA concentration of leaves (Table 2). Compared with
that of non-AM seedlings, the MDA concentration of AM seedlings decreased by 16%
and 28% under WW and WS respectively. There was high soluble sugar and high proline
concentrations in leaves of AM seedlings under WW and WS treatments, while AMF
inoculation did not affect hydrogen peroxide (H2O2) concentration (Table 2). Whether
WS or not, AM symbiosis notably increased the guaiacol peroxidase (G-POD) and
ascorbate peroxidase (APX) activities of leaves (Table 3). AM infection also markedly
increased the SOD activity and decreased the catalase activity of WS leaves (Table 3).
Our results suggest that the increased activity of antioxidant enzymes and decreased
concentration of ROS compounds found in AM plants may serve to protect the date palm
against oxidative damage, enhancing drought tolerance. According to the index of
drought tolerance (evaluated as the ratio of biomass production in WS seedlings to
biomass production in WW seedlings) and the oxidative damage (estimated as the ratio of
malondyaldehide to proteins) AM seedlings showed higher ability of drought tolerance.
Discussion
AM symbiosis increased growth and biomass production of date palm seedlings in
both the presence and absence of water stress, confirming earlier findings [5, 8, 9, 10,
11, 16]. The positive effect is likely attributed to the improvement of phosphorus
nutrition [31], the enhancement of water uptake by hyphae [15] and the increase of
root length density [13].
The enhanced sugar content in AM roots under well-watered conditions may be due
to the sink effect of the mycorrhizal fungus demanding sugars from shoot tissues.
Schellembaum et al. (1998) suggested that the AM fungus can be a strong competitor
for root-allocated carbon under conditions limiting photosynthesis. These authors
proposed that the lower hexose accumulation in leaves of mycorrhizal plants in
drought could be due to a lower availability of photosynthates for storage in these
tissues. The lower accumulation of compatible solutes may indicate that the plants
more successfully avoided drought stress [7]. In fact, proline, the other osmoregulator
86
measured in this study, also accumulated less in plants grown under drought condition
than optimal condition plants.
Water deficit is known to induce formation of reactive oxygen species (ROS) in
higher plants. To maintain growth and productivity plants have to prevent accumulation
of these harmful species as rapidly as possible. Under amply watered conditions, ROS
production and removal are strictly controlled [4]. When higher plants are subjected to
water stress, the equilibrium between production and scavenging of ROS is broken,
resulting in oxidative damage. The oxidation of membrane lipids is a reliable indication
of uncontrolled free-radical production and hence of oxidative stress [24]. In date palm
seedlings, Water stress increased the MDA and H2O2 contents of leaves (Table 2). In
the presence of AMF, the MDA concentration decreased by 16% and 28% under WW
and WS respectively, while AMF inoculation did not affect hydrogen peroxide H2O2
concentration. Accordingly, the oxidative damage in date palm seedlings inoculated
with GI and subjected to drought was 69 % and 44 % lower than in non inoculated
seedlings under WS and WW respectively.
In higher plants enzymatic defense is an important component of the protective systems
that minimize the deleterious effect of water stress. SOD catalyses the dismutation of O2
to H2O2, CAT dismutates H2O2 to oxygen and water, and APX reduces H2O2 to water
[18]. In date palm, compared to non-AM seedlings, AM inoculation notably increased the
G-POD, APX and SOD activities in of WS seedlings. Our results suggest that the
increased activity of antioxidant enzymes and decreased concentration of ROS compounds
found in AM plants may serve to protect the date palm against oxidative damage,
enhancing drought tolerance. The increases in the antioxidant enzymatic activities were
associated with plants resistance to water stress [2, 27]. Using the index of drought
tolerance and the oxidative damage in leaves, the order of the ability of drought tolerance
of date palm seedlings based on their AMF statut is GI > GM > Non-AM.
Acknowledgements
This study was supported in part by AECI: PCI MAROCO-ESPAGNOLE
N°A/5367/06.
87
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mycorhization
VA
d’un
système
radiculaire.
Recherche
de
méthodes d’estimation ayant une signification fonctionnelle. In : Gianinazzi
S, editor. Les mycorhizes : physiologie et génétique, 1er séminaire européen sur
les mycorhizes, 217-221.
90
Table 1: Mycorrhizal colonization, Plant height, Root length, shoot dry weight
and root dry weight of non-mycorrhizal (Non-AM) or mycorrhizal date palm
seedlings grown under well watered (75% field capacity) or water stress (25%
field capacity) conditions. GM = Glomus mosseae, GI = G. intraradices.
Water
statute
75% FC
25% FC
AMF
statute
Shoot
Root
dry
colonisa- weight
tion (%)
(g)
Root
dry
weight
(g)
Plant Root
Mycorhizal
height length
dependency
(cm)
(cm)
Non-AM
0
3,16c
1,76d
22,5c
27,9d
GM
54,3b
4,90b
2,68bc
33,8b
51,6b
34,97
GI
61,43a
6,41a
3,38a
44,7a
60,5a
49,74
Non-AM
0
1,43e
0,81e
13,3d
18,9d
GM
35,5d
2,83cd
2,35c
23,4c
42,5c
56,77
GI
42,6c
3,21c
2,97b
26,6c
46,9c
63,73
Values within each column followed by the same letter are not significantly different (p ≤ 0.05).
Table 2: Malonyldialdehyde (MDA), hydrogen peroxide (H2O2), soluble sugar and
proline contents and Oxidative damage in leaves of non-mycorrhizal or mycorrhizal
date palm seedlings grown under well watered (75% field capacity) or water stress
(25% field capacity) conditions. GM = Glomus mosseae, GI = G. intraradices.
Water
statute
75% FC
25% FC
Soluble
sugar
Prolin
(nmol.g-1
DM)
Oxidative
damage
(nmol MDA
mg-1 proteins)
54.6c
4105.2c
12.42b
25.2b
68.4b
7307.3b
8.57c
38.61d
24.2b
78.7a
9193.2a
6.88c
Non-AM
65.51a
28.4a
47.5d
3570.2d
20.47a
GM
44.37c
27.9a
52.9c
3986c
10.53b
GI
42.10c
27.3a
68.5b
4070.4c
6.35c
AMF
statute
MDA
H2O2
(nmol.g-1
DM)
(μmol.g-1
DM)
Non-AM
53.23b
24.1b
GM
41.42cd
GI
(mg.g-1
DM)
Values within each column followed by the same letter are not significantly different (p ≤ 0.05).
91
Table 3: Catalase, Superoxide dismutase (SOD), ascorbate peroxidase (APX) and
guaiacol peroxidase (G-POD) activities in leaves of non-mycorrhizal or mycorrhizal
date palm seedlings grown under well watered (75% field capacity) or water stress
(25% field capacity) conditions. GM = Glomus mosseae, GI = G. intraradices.
Water
statut
75% FC
25% FC
AMF
statut
SOD
(USOD g-1
DM min-1)
G-POD
(μmol.g-1
DM min-1)
Catalase
(nmol g-1
DM min-1)
APX
(μmol g-1
DM min-1)
Non-AM
652,3a
3,07c
116,7cd
2,11d
GM
458,9c
3,71bc
124,1cd
2,99d
GI
447,7c
4,20b
136,8c
2,89d
Non-AM
596,1b
4,44b
225,5a
4,68c
GM
693,5a
4,90ab
157,5b
6,59b
GI
667,4a
5,87a
104,41d
7,05a
Values within each column followed by the same letter are not significantly different (p ≤ 0.05).
92
* "< 2ت ا 'را ا7ر ا? و ا* 5Gد ا */( 6
إ!*1ج آ*ت اJوآ %ا? Iو ا آ*ت وا* !Gت ا *.دة
Nآ %ة M1Dت ! ا 1
ا أ ب ،أ ف اد ،آ م ر ،أ وري
١
ا ا وqadahmed@gmail.com :
ا
' ا &وف أن !ض ا " ! ت إ وف ا إ د
ا & ء 2/ ،دي إ ز /دة إ ج آ ت ا,وآ* ' ا ")(
Reactive oxygen species
) .(ROSو @Aأ& 9ا ?ن > أن ا 78ا = ر ;وف ا :د 9/إ ! 6 78ا & 5أو
ا & .(ROS) '/C! > 5و &KLار IJا "& و ا HFظ > دة و آ& ا &EFل
! OFوف ا :د !& ا " ! ت > Nو !اآLP ' (ROS) Mل !") ( إ ج ا &اد
ا &= دة Qآ*@ة و ا /دة ) IJط ا &/,ت ا &"; Hأو ا * Cل ).(ROS
M&XهVا ا @ UFرا 78! Kات ا :د ا & / > ITا "& )إ ج ا >Cا ، / Fل
و وزن ا )Lت ،ا زن ا ف >Vر و ا * ق( ،و و [ ا &آ ت وا &/:ت ا &= دة
Qآ*@ة ا * أآ* @ د(SOD) ! /واCK,ر \ اوآ* @(APX) /وا اC/ل
اوآ* @ (GPO) /و ا L! Cز )(CATو ا & ن داى أ @@ /
ا & ا &> /_H FNت ا Vر /ا ) /
)L5 @"(MDAت ^
)(AM-plantو ا &رو OF! I&F O IJ
وف @ NKة ' ٪75 ،ا * ا ، (WW) >NFأو ! OFوف a5ا & ' ٪25 ، 6
ا * ا f _! ' / ١٢٠ @ .(WS) >NFا :د ا & ، ITأ ت ا " eTأن /ا "&
)ل و وزن ا )Lت( وإ ج ا >Cا ) / Fا زن ا ف >Vر و ا * ق( آ Oأ> "@
ا )Lت ا &> _H FNا & Cر/ي ) & (AM-plantه @" g > Iا )Lت ا &>( FN
[h (Non-Amا "; ' وف ا *. INو! *! Mأ> إ ج > >Cا / Fو أ> &
"@ L5ت ا "^ ا &>FN
_Hا & Cر //ا ) ي آ>س أارادi */
)(GI
.intraradices GlomusوLوة > ذ \ أ ا J Cا & Cر/ي ' 5ات ^
ا & و ا (GI) _Hأ> * > ا & Cر . mycorrhizal dependency//آ& أ ت
ا " eTأن ه" ك ا^ Hض F IJى ا & ن داى أ @ ،(MDA) @ /و ار! Hع F IJى ا *C
93
و ا و ' IJأوراق ا )Lت ا &> ' ? IJ ،FNأن Fى وآ* @ ا @رو ' )M (H2O2
" 78/ا > .a NوKاء ! OFوف WWأو ،WSأ ا J Cا & Cر/ي ار! H
> ) IJ Fط أ &/ت ا / hآل وآ* @از )(GPOواCK,ر ت وآ* @از )(APXو
وا,آ* @ د(SOD) ! /وا^ ) IJ n Hط أ M/ا L! Cز )! (CAT) .ز /دة ) ط
ا &/:ت ا &= دة Qآ*@ة ،وا^ Hض *ى ) IJ (ROSأوراق ا )Lت ا &> /_H FNت
ا & Cر //ا ) /إ دور ه 6Vا /_Hت 5 / &? IJات ا "^ ' ا > oا 8آ*@ي .هVا
و' LPل ا@^Kام ا & 52ا "* &F Iا Hف ) (IRSو در ا > oا 8آ*@ي q !! 'C&/
@Aرة ا J Cا & Cر/ي ' ^ ا & و ا /_Hت ا & Cر //ا ) @A q !! 'C&/ ،/رة
هVا ا &F! > J Cا Hف آ .GI > GM > Non-AM I
94
OP 08
Relationship between yield and fruit quality and leaf and fruit mineral
content of Zaghloul dates in the Egyptian calcareous soil as a result of
flower boron spray and soil application with some micronutrients,
A.M. Attalla*, A.M. El-Kobbia* and S.M. El-Nawam**
*Pomology Dept., Fac. Of Agric. (El-Shatby), Alex. Univ.,Alexandria, Egypt
Fax: +2035922780, Email : profabozeidattalla@hotmail.com
**Agric. Research Center, Ministry of Agric., Giza, Egypt
Abstract
The present study was carried out on 11- years- old Zaghloul date palms grown in
calcareous soil located at El-Nahda region, Alexandria, Egypt to study the relationship
between yield & fruit quality and leaf & fruit mineral content as a result of flower
boron spray and soil application with some chelated micronutrients (Fe, Mn and Zn).
The present results showed that there were a significant positive relationship between
pinnae nitrogen, phosphorus potassium, iron, manganese and boron and fruit set were
significantly positive. There were significant positive correlation between fruits
weight/spathe and pinnae iron, manganese, zinc and boron. However, no significant
correlations were found between pinnae mineral content and fruit weight. The
relationships between pinnae nitrogen, phosphorus, iron, zinc and boron and fruit
sugar content were significantly positive, while the relationships were not significant
between pinnae mineral content and fruit tannins, except pinnae phosphorus content
was significantly negative. There were significantly positive correlations between
nitrogen, iron manganese and zinc in both pinnae and fruits.
Keywords: Date palms – Boron spray – Micronutrients – Pinnae mineral content.
Introduction
Date palms is one of the oldest cultivated fruit trees in the world .Egypt is
considered to be the leading of the Arab countries producing dates ( FAO, 2005 ). The
number of fruitful female was 11.88 million planted on about 85187 ha. and total
production reached 1.33 million metric tons of fresh, semi-dry and dry date cultivars(
Statistics of the Egyptian Ministry of Agriculture, 2006)
95
Date palms are
well adapted to the difficult conditions and high yields
accompanied with irrigation, good water availability and agricultural practices. The
plantations are spread all over the country, but the main producing area located at Nile
Valley, Nile Delta, New Valley and new reclaimed lands.
Dates are considered the main product of date palms for its highly nutritional
values).Dates ( at Tamr stage ) contains 70.6% carbohydrates, 1.9% protein,2.5%
Fat,13.8% water, 1.9% ash (mineral salts) and 9.3% Fibers (Popenoe,1974). Mineral
elements are necessary for life as they act as catalysts or structural components of
large molecules which have specific functions in the biological system (Mertz, 1981).
Egypt is in need to increase dates production to meet the demands of rapidly
expanding population and export. In the same time, there are insufficient factories
capacity to process and cure big date crop. Thus, date palm group has been set up to
devise a plan to develop the date palm sector and produce dry dates in order to extend
the period of fruit exposure in the markets to meet the consumer demands.
In date palm orchards, the main problems include soil fertility decline, increasing
land and water degradation, sanding up, diseases and pest infestation. The
experimental soil is high in CaCO3 content (40- 50%), where the pH is high (8.3) , the
micronutrients (Fe, Mn, Zn and Cu) are deficit, the ammonium fertilizers are subject to
losses and phosphate fertilizers are low efficiency . Also, the plants suffer from, the
basic reaction and the precipitation of micro nutrients and phosphate plus the losses of
ammonium fertilizers.
The present investigation aimed to study the relationship between yield & fruit
quality and leaf & fruit mineral content as a result of flower boron spray and soil
application with some chelated micronutrients ( Fe, Mn and Zn) for Zaghloul date
palm cultivar grown in calcareous soil at El-Nahda region , Alexandria, Egypt.
Materials and Methods
The present investigation was carried out during three successive seasons ( 1999, 2000
& 2001) on Zaghloul palms to study the relationship between yield & fruit quality and
leaf & fruit mineral content as a result of flower boron spray and soil application with
some chelated micronutrients ( Fe, Mn and Zn). Eleven-years-old of Zahloul palms were
selected for this study , planted at 10 x 10 meters apart and grown in Rasheed private
96
orchard located at El-Nhada region, Alexandria, Egypt. Fifty- two palms as uniform as
possible were randomly selected for this experiment, including 13 treatments with 4
replicates (one palm for each replicate). All selected palms were grown in calcareous soil
(40-50% CaCo3 and pH=8.32 ) and usually received the same cultural practices in this
orchard. The selected palms were subjected to the following treatments :
1- o.2 % flower boron spray.
2- o.2 % flower boron spray +200 g Fe / palm.
3- o.2 % flower boron spray + 200 g Fe +100 g Mn/ palm.
4- o.2 % flower boron spray+ 200 g Fe +100 g Zn/ palm.
5- o.2 % flower boron spray+ 200 g Fe +100 g Mn/ +100 g Zn/ palm
6- o.2 % flower boron spray+100 g Mn +100 g Zn/ palm
7- o.4 % flower boron spray.
8- o.4 % flower boron spray+200 g Fe / palm
9- o.4 % flower boron spray+ 200 g Fe +100 g Mn/ palm
10- o.4% flower boron spray+ 200 g Fe +100 g Zn/ palm.
11- o.4 % flower boron spray+ 200 g Fe +100 g Mn/ +100 g Zn/ palm
12- o.2 % flower boron spray+ 200 g Fe +100 g Mn/ +100 g Zn/ palm
13- Control (untreated palms).
Fe, Mn and Zn treatments were applied in the following Forms: PRO.SOL 20%IRON
CAC, SIDA Mn- CHELATE 12% SP and SIDA Zn-CHELATE 12% SP, respectively.
Three spathes on each palm were sprayed once with borax (11.3% soluble boron)
during 2-3 days after spathe cracking (before pollination) during April in the three
experimental seasons. The quantity of chelated macronutrients were added in three
halls around each palm trunk, 40-50 cm depth from soil surface in 2 equal doses in the
first week of April and June. The experimental palms were immediately irrigated after
treatments. The same pollen source was used to pollinate the experimental palm trees
during the three seasons of study. During the last week of June in the three
experimental seasons, fruit set percentage was determined by the following formula:
The total number of fruits/ spike
________________________________________ x 100
The total number of flower positions in the same spike
97
At the end of Khalal stage (harvest time), the average fruits weight/ spathe (as index
to yield/palm) was estimated using the average number of mature fruit weight. At
harvest time, a sample of thirty fruits was taken at random from each replicate for
determination of physical and chemical characteristics of fruits. The fruits of each
sample were washed with distilled water and the flesh was cut into small pieces by
clean knife, mixed well and then oven- dried at 70ºC until a constant weight. Chemical
properties of dry fruit samples including total sugars, soluble tannins and mineral
content ( nitrogen, phosphorus, potassium, iron, manganese, zinc and boron ) were
determined. Total sugars of each fruit sample were extracted from 0.5g ground dried
material by distilled water, then determined by phenol sulfuric method according to
Malik and Singh (1980). Soluble tannins were determined in each sample by Swain
and Hilis (1959)method. Total sugars and soluble tannins were expressed as
percentage (on dry weight basis). In order to determine pinnae mineral content, twenty
pinnae per each replicate were collected in November, from the middle pinnae of the
full developed leaves around the axis above fruiting zone as recommended by
Embleton and Cook (1947). Pinnae samples were washed several times with tap water,
distilled water and dried at 70 ºC to a constant weight to determine N, P, K, Fe, Mn,
Zn and for fruit and pinnae samples, the dried materials were ground with stainless
steel rotary knife mill, and 0.3g of each ground sample was digested with sulfuric acid
and hydrogen peroxide according to Evenhuis and Deward (1980).
Suitable aliquots were then taken for nitrogen phosphorus, potassium, iron,
manganese and zinc. Nitrogen and phosphorus were determined colorimetrically
according to Evenhuis (1976) and Murphy and Riley (1962), respectively. Potassium
was determined by flame photometer, while iron, manganese and zinc were
determined by Perkin Elmer Atomic Absorption Spectrophotometer 305B. Boron in
each dried ground sample was determined colorimetrically according to Hatcher and
Wilcox (1950). The concentrations of nitrogen, phosphorus and potassium were
expressed as percentage, while those of iron, manganese, zinc and boron were
expressed as parts per million (ppm).
The experimental treatments were arranged in Completely Randomized Design and
the data were statistically analyzed. Combines analysis for the three seasons data was
98
carried out according to Gomez and Gomez (1984). Correlation coefficient within the
elements in the pinnae as well as between the elements of pinnae on one side and fruit
set, fruits weight/spathe, quality and mineral content of fruits on the other side were
calculated for the three seasons average according to Snedecor and Cochran(1980).
Results and Discussion
The data illustrated in Fig(1) indicated that the correlation between pinnae mineral
content ( nitrogen, phosphorus, potassium, iron, manganese and boron) and fruit set ( r
= 0.823,
r=0.705, r=
242, r= 0.578, r= 0567 and r= 0.634, respectively) were
significantly positive. Also, there were a significant positive correlation were found
between fruits weight/spathe and pinnae iron (r = 0.769) manganese(r=0.785 )
zinc(r=0.729) and boron (r= 0.706). The present correlations indicated the important
role of micronutrient applications on increasing fruit set and yield. The present
correlations were in harmony with those reported by Wojcik et al (2000) who found
that boron application increased fruit set of apple trees, while Ubavic et al (1983)
concluded that soil application with some Fe-chelate preparations showed significantly
increased pigments content( chlorophyll a and b and carotenoids) and found high
correlation between chlorophyll (a and b) content and yield in apple trees. Also, Devi
et al. ( 1997) found that micronutrient applications significantly increased crop yield of
orange trees as compared with the control treatment.
The correlation between pinnae nitrogen, iron and zinc contents and both total
sugars and mineral content of fruits were significantly positive. There were
significantly positive correlations between pinnae phosphorus & boron and fruit sugar.
Also, the correlation coefficient between pinnae phosphorus and fruit tannins was
significantly negative (r =0.582), but the correlation between pinnae and fruit mineral
content were significantly positive (r=0.642, r=0.948, r=0.685 & r=0.881) for nitrogen
,iron, manganese and zinc, respectively. No significant correlations were found
between pinnae mineral content and fruit weight (Table 1 & Fig 2). The data of
correlation presented in Table 1 and Fig 1 showed that pinnae nitrogen, phosphorus,
iron, zinc and boron content increased yield and fruit sugar, and phosphorus decreased
fruit tannins. These results indicated the importance role of these elements to increase
yield and enhance fruit ripening under such condition.
99
The present correlation seemed to be in line with many investigators such as:
Sandhu et al. (1993), on pear trees, found that there were no significant differences in
fruit weight as a result of zinc sulfate application. Metha and Jindal (1986), on
Japanese plum, found that total sugars were highest in fruits from trees which were
sprayed with H3BO3 at 125 ppm. Also, Haggag et al. (1995) concluded that foliar
application of boric significantly increased the percentage of total sugars content in
mango fruits (cv. Hindi Be-Sinnara).
Concerning the correlation within pinnae mineral content, there were significant
positive correlation between nitrogen and potassium, iron, manganese and boron
(r=0.76, r=0.71, r=0.64 and r=0.59, respectively). Also, the correlations between both
manganese and zinc and iron were significantly positive (r = 0.79 and r= 0.80,
respectively). Also, there were significant positive correlations between both zinc &
boron and manganese (r= 0.78 and r= 0.69, respectively). The other correlations within
pinnae nutrient elements were not big enough to be significant (Table 2).
From the present results, it is can be concluded that suitable treatment to increase
yield & fruit quality of Zaghloul cv was: B spray at 0.4% plus (200gm Fe+100gm Mn
+ 100gm Zn) /palm /year.
100
References
[1] Agriculture statistics (2006). Ministry of Agriculture and Reclamation, Economic
Affairs Sector. Arab Republic of Egypt. October 2007, Vol.2: 326-327.
[2] Devi,D.D.; P.S. Srinivasan and K. Balakrishnan. 1997. Leaf nutrient composition
chlorosis and yield of Sathgudi orange as affected by micronutrient applications.
Department of pomology, Horticulture college and Research Institute, Coimbatore,
641003, India. On sex ratios and fruit quality of mango (Mangifera indica L.) cv.
Hindi Be-Sinnara. Annals of Agric. Sci., Cairo, 40 (2):753-758.
[3] Embleton, T.W. and J.A. Cook. 1947. The fertilizer value of date leaf and fruit
stalk prunings. Date Growers Inst.,24: 18-19 (C.F. Melouk et al.,1999).
[4] Evenhuis, B. 1976 Nitrogen determination. Dept. Agric. Res. Royal Tropical
Inst.,Amsterdam.
[5] Evenhuis, B. and P.W. Deward. 1980. Principles and practices in plant analysis.
FAO Soils Bull., 38(1): 152-163.
[6] F.A.O. (2005). Annual report (http://www. FAO. org/ag/ar).
[7] Gomez, K.A and A.A. Gomez (1984). Statistical procedures for Agriculture
Research 2nd Ed., Wiely, New York.
[8] Haggag, L.F.; M.A. Maksoud and F.M.Z. El- Barkouky. 1995. Effect of Boron
sprays on sex ratios and fruit quality of mango (Mangifera indica L.) cv. Hindi Be
–Sinnara. Annals of Agric. Sci., Cairo, 40(2): 753-758.
[9] Hatcher, J.T. and I.V. Wilcox. 1950. Colorimetric determination of boron using
carmine. Analyt. Chem., 22:567-569.
[10] Malik, C.P. and M.B. Singh. 1980. Plant enzymology and histoenzymology. And
histoenzymology. A. Text Manual, Kalyani publishers, New Delhi.
[11] Mertz, W. 1981. The essential trace elements. Science, 312, 1332.
[12] Metha, K. and K.K. Jindal. 1986. Effect of some nutrient sprays on fruit maturity
and quality of Japanese plum (Prunus salicina Lindl ) cv. Santa Rosa.
Proceedings of the National Symposium on Temperate Fruits, 15-18 March,
1984, Himachal Pradesh Agricultural University, Solan India, 1986, 203-207.
[13] Murphy, J. and J.P. Riley. 1962. A modified single solution method for the
determination of phosphorus in natural water. Anal. Chem. Acta, 27:31-36.
101
[14] Popenone, W. 1974. Manual of tropical and subtropical fruits. Hafiner press. A
Division of Macmillan Publishing Co., Inc. New York Collier Macmillan
Publishers, London.
[15] Sandhu, A.S., K. Singh and R. Singh. 1993. Effect of foliar and soil application
of zinc on yield and fruit quality of Patharnakh pear. Punjab Horticultural
Journal, 33 (1-4): 48-51.
[16] Senedecor, G.W. and W.G. Cochran. 1980. Statistical Methods. 7th ed., Iowa
State Univ. Press, Ames.
[17] Swain, T. and W.E. Hills (1959). The phenolic constituents of Prunus domestica
L. 1. The quantitative analysis of phenolic constituents. J. Sci. Food Agric.,
10:63-68.
[18] Ubavic, M., R. Kastori, N. Petrovic, A. Verb, B. Nikolic and N. Babic. 1983.
Effeciency of some Fe-chelate preparations for eliminating Fe-chlorosis apples.
Arhiv-za-poljoprivredni Nauke, 44 (154): 135-143.
[19] Wojcik, P.P., G. Cieslinski, J.V. Possingham and G.H. Neilsen. 2000. Effect of
boron fertilization on yield and fruit quality of "Elstar" and "Sampion" apple
culivars. Acta Horticulturae, No. 512: 189-197.
102
Table (1). Correlation Coefficient (r) between pinnae mineral content and
both soluble tannins and mineral content of Zaghloul cv. fruits
Pinnae mineral content
Soluble
tannins
Mineral
Content
N
P
K
Fe
Mn
Zn
B
-0.213
-0.582*
-0.419
-0.301
-0.073
-0.252
-0.201
0.642*
0.490
0.208
0.948*
0.685*
0.881*
0.540
* Significant at 0.05 level of probability
Table (2). Correlation coefficient (r) Within pinnae nutrient elements of Zaghloul cv.
Nutrient
N
P
K
Fe
Mn
Zn
elements
N
P
0.38
K
0.76*
0.30
Fe
0.71*
0.25
0.35
Mn
0.64*
0.23
0.29
0.79*
Zn
0.44
0.20
0.14
0.80*
0.78*
B
0.59*
0.47
0.37
0.45
0.69*
* Significant at 0.05 level of probability
103
0.31
B
0.9
0.8
0.7
0.6
Fruit
0.5
parameters 0.4
0.3
0.2
0.1
0
N
P
K
Fe
Mn
Zn
B
Pinnae mineral content
Fruit set %
Fruits weight / spath
Fig (1): Correlation coefficient (r) between pinnae mineral
content and both fruit set % & fruit weight (kg) / spathe.
1
0.9
0.8
0.7
Fruit
parameters
0.6
Fresh weight
0.5
T otal sugars
0.4
Mineral content
0.3
0.2
0.1
0
N
P
K
Fe
Mn
Zn
B
Pinnae mineral content
Fig (2): Correlation coefficient (r) between pinnae mineral
content and fruit parameters
104
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106
OP 09
Physical characteristics of fruit of different date palm cultivars
during various maturity stages
Imran Ullah
Agricultural Research Institute, Dera Ismail Khan.KPK – Pakistan
imranullah1981@yahoo.co.uk
Abstract
The present study was conducted at Agricultural Research Institute, Dera Ismail
Khan using six date palm varieties i.e. Azadi, Dhakki, Gulistan, Khudravi, Shakri and
Zahdi to see the physical characteristics of fruit at four maturity stages i.e. Kimri stage,
Khalal stage, Rutab and Tamr stage. The objective of the study was to record and
document the physical behavior of fruit of six important locally well-known date palm
cultivars. The results for all the physical parameters were found significant at four
individual maturity stages. At Kimri stage the maximum fruit length (5.022 cm) was
found in cultivar Dhakki, while fruit diameter (3.16 cm), fruit weight (26.449 gms),
pulp weight (25.1 gms) was found maximum at Rutab stage. The seed weight (1.53
gms) was found maximum at Tamr stage in cultivar Shakri. Similarly, the fruit of
cultivar Dhakki took longest time of 24.72 week while the fruit of cultivar Shakri took
minimum time of 21.82 weeks to reach a final maturity stage. The yield of 96 kg palm1
was recorded in cultivar Dhakki. Therefore, Dhakki cultivar is recommended for
general plantation in Dera Ismail Khan.
Key words: Date Palm, cultivar, physical characters, maturity stages, yield
Introduction
Date palms (Phoenix dactylifera L.) provide a good source of rapid energy due to its
high carbohydrate content i.e. 70-80 % [1]. In addition to carbohydrates, dates contain
minerals, protein, fats and vitamin etc. Pakistan is the fourth largest dates producing
country in the world. Date is the third major fruit of our country and is commercially
grown in Multan, Muzafargarh, Bahawalpur, Dera Ghazi Khan, Khairpur, Sakhar,
Khairpur, Makaran and Dera Ismail Khan. In Pakistan, the area and production of
dates was 90700 ha and 566500 tons respectively, while in Dera Ismail Khan dates
were grown on 622 ha with production of 11000 tons [2]. Date palm takes about 200
107
days from pollination to full maturation (Tamr stage). During its development and
ripening, the fruit passes through four distinct maturity stages; each one is
distinguished by one or more particular characteristics physically. These four stages
are named by their Arabic denomination; Kimri stage, Khalal stage, Rutab stage and
Tamr stage. Hababauk is the term used for the female flower and the period just after
pollination when the young fruit is still creamy white before gradually turning green at
the Kimri stage. The white creamy stage (Hababauk) remains for four weeks after
which the fruit turns into green that is the start of Kimri stage, At the Kimri stage,
there is rapid increase in size and weight of fruit. At this stage the fruit is botanically
mature. Kimri stage last for more than fourteen weeks by turning of fruit from green to
yellow or red according to the variety and that is the initiation of Khalal stage. At this
stage the fruit weight gain is slow but sucrose content is increased, moisture content
goes down, tannins precipitate and lose their astringent [3]. In some varieties this latter
process completes rapidly which make the fruit palatable at the Khalal stage. When
(normally) the tip of fruit turns brown this indicates the end of Khalal stage and
initiation of the Rutab stage. The Rutab stage is characterized by decrease in weight
due to moisture loss, a partial inversion of sucrose into invert sugar and a browning of
skin and softening of the tissues. The moisture content goes down to 35% and the
dates at this stage are sold as fresh fruit. Only when the dates are left on the palm will
turn into complete brown color with moisture ranging from 18.8 to 27.8% and total
sugar ranging from 73.8 to 84.2 % represent the Tamr stage [4]. Different researchers
have characterized dates on the bases of fruit length, weight, diameter etc at full
maturation stage, but no one has referred to the characterization of date palm fruit at
the individual maturity stage. Depending on the variety and growth conditions, the
physical characteristics of fruit of Date palm vary on different maturity stages. The
present investigations were undertaken to study the physical characteristics of the fruit
at different maturity stages of date palm cultivars grown at Dera Ismail Khan.
Materials and Methods
The investigations were carried out Agricultural Research Institute, Dera Ismail
Khan, Khyber Pakhtunkhawa, Pakistan. The cultivars included in this study were
Azadi, Dhakki, Gulistan, Khudrawi, Shakri and Zahdi grown under similar
108
environmental condition and were of the mostly same age (about 20 years). The
experiment was laid out in completely randomized design with a total of thirty-six
plants (two plants per treatment/variety) replicated three times. All the plants were
applied with recommended doses of fertilizers and were irrigated respectively. Four
different growth stages were identified and physical parameters of fruit were studied
during all four stages i.e. Kimri, Khalal, Rutab and Tamr. Data were recorded on;
1. Time taken in weeks to complete individual maturity stages.
2. Fruit length, fruit diameter (cm).
3. Fruit weight, pulp weight (gm).
4. Fruit pulp percentage.
5. Seed weight (gm).
6. Seed length (cm).
7. Yield per palm (Kg).
Data was analyzed statistically using analysis of variance techniques [5]. Least
significance difference (LSD) test was used to see the difference of treatment means.
Results and Discussions
1.Time taken by fruits of different cultivars at individual maturity stages
Time taken by each development stage was calculated in weeks, which is presented
in Table-1. Difference among the cultivars and maturity stages were found statistically
significant. Among the cultivars, the total time to reach at the final maturity stage was
found maximum in Dhakki (24.72 weeks), which differed significantly from all other
cultivars. The minimum total time to reach a final maturity stage was recorded in
cultivar Shakri (21.82 weeks). The cumulative effect of varieties and maturity stages
was also significant. The maximum number of weeks (16.40 weeks) was recorded for
Gulistan to complete its Kimri stage and minimum weeks (1.42 weeks) were taken by
Shakri at Tamr stage. Similarly, time taken by cultivar at each individual maturity
stage was also found significant. All the cultivars took maximum period to complete
Kimri stage, ranging from 11.86 weeks for cv. Shakri (Minimum period) to 16.40
weeks (maximum) for cv. Gulistan. The cultivar Khudrawi took statistically minimum
time of 14 weeks to complete the Kimri stage, however cultivar Zahdi, Gulistan,
Dhakki and Azadi took shortest period of 3-4 weeks to get through Khalal stage.
109
Shakri took statistically longest period of 6.11 weeks followed by Khudrawi taking
5.19 weeks to complete Khalal stage. Data showed that the period between Khalal and
Rutab stage was statistically maximum i.e. 3.05 weeks for cv. Azadi, while minimum
2.24 weeks for cv. Zahdi. Cultivar Azadi took maximum time of 2.10 weeks to
complete Tamr stage, while a period of 1.42 weeks was recorded cv. Shakri. The
results showed that fruit of all cultivars took maximum time at Kimri stage, which
were supported by authors [6] who explained that at Kimri stage the fruit is quite hard,
the color is apple green and it is not suitable for eating. This stage start from a small
green berry to full size green date. It is the longest stage of growth and development of
dates and last for a total of 9-14 weeks, depending on varieties.
2.Fruit length (cm) of Different date cultivars at individual maturity stages
Data regarding fruit length of date palm cultivars at individual maturity stages is
shown in Table-2, which showed that the fruit length of date palm was significantly
affected by varieties and maturity stages. Among the cultivars, fruit length of all four
maturity stages was significantly maximum (4.91 cm) in cultivar Dhakki, which
differed significantly from all other cultivars. The minimum fruit length (3.44 cm) was
found in cultivar Zahdi. The cumulative effect of varieties and developing stages on
fruit length was statistically significant. The maximum fruit length (5.09 cm) was
recorded in cv. Dhakki at Kimri stage, while the minimum fruit length (3.42 cm) was
recorded in cv. Zahdi at same stage. The data for fruit length of six date palm cultivars
was statistically significant at all maturity stages. It has been observed that the fruit of
all cultivars completed its redevelopment in term of fruit length at Kimri stage. No
further development was recorded during the rest of all stages. Perusal of the data
indicated that cv. Dhakki had significantly maximum fruit length of 5.09 cm at Kimri
stage followed by Gulistan (4.70 cm), Azadi (4.08 cm) and Khudrawi 3.87 cm at the
same stage. The minimum fruit length (3.45 cm and 3.42 cm) was found in Shakri and
Zahdi, respectively. All the cultivars showed similar trend at Khalal, Rutab and Tamr
stages. Dhakki remained at top while Zahdi at the bottom. Little decrease was noted in
fruit length at almost all three stages, which was probably due loss of moisture and
shrinkage. These results are generally in accordance with author [7] who explained that
fruit length of date fruit increase from kimri to Khalal stage than it decreased at Tamr
110
stage. Similarly [8] reported that if the female palm of cv. Assel are well fertilized and
well pollinated with superior males, the fruit could reach to a length of 5 cm.
3.Fruit diameter (cm) of six date cultivars at individual maturity stages
Data regarding fruit diameter of six date palm cultivars at individual maturity stages
is depicted in Table-3, which showed that the fruit diameter was significantly affected
by varieties and maturity stages. The cumulative effect of varieties and developing
stages on fruit diameter was significant. The maximum fruit diameter of (3.16 cm) was
recorded in cv. Dhakki at Rutab stage, while the minimum fruit diameter (2.07) was
recorded in cv. Shakri at Kimri stage. The data for fruit diameter of six date palm
cultivars was observed significant at all maturity stages. It was observed that the fruit
of all cultivars reached to its development in term of fruit diameter at Rutab stage. No
further development was recorded in the last maturity stage. Perusal of the data
indicated that cv. Dhakki had significantly maximum fruit diameter of 3.16 cm at
Rutab stage followed by Azadi (2.71 cm) and Khudrawi (2.44 cm). Cultivar Zahdi,
Gulistan and Shakri showed statistically same fruit diameter 2.44 cm, 2.34 cm, 2.32
cm respectively at Rutab stage. The minimum fruit diameter was recorded at Kimri
stage, where in the Dhakki cultivar had the maximum fruit diameter of 3.065 cm while
Shakri had minimum fruit diameter of 2.07 cm at the same stage. All the cultivars
showed similar trend at Khalal and Tamr stage during which cultivar Dhakki remained
at top while Shakri at bottom. Similarly, among the cultivars the fruit diameter of all
four maturity stages was significantly maximum (3.072 cm) in cultivar Dhakki, which
differed significantly from all other cultivars. The minimum fruit diameter (2.25 cm)
was found in cultivar Shakri. Little increase in fruit diameter was observed at almost
all three stages, which was probably due to the fact that fruit complete its full growth
before maximum inversion at the Rutab stage. These results are in accordance with [9]
where authors determined the Physico-chemical characteristics of fruits of four
Arabian date palm cultivars and stated that the date palm fruit attained its minimum
diameter at Kimri stage, which increased during the rest of stages. Similarly according
to [10] the diameter of five major date palm cultivars namely Khudrawi, Barni, Sullaj,
Ruzeiz and Sifri, ranged from 2.75-4.26 cm. However, it has been reported by author
111
[11] that the pollination at different times significantly affected fruit length and
diameter of cultivar Dhakki.
4.Fruit weight (gm) of six date cultivars at individual maturity stages
Results of table-4 pertaining to fruit weight of six date palm cultivars showed
significant variation in varieties as well as different maturity stages. Among different
cultivars the mean fruit weight (21.46 gms) was found maximum in Dhakki, which
differed significantly from all other cultivars. The minimum fruit weight (10.06 gm)
was found in cultivar Shakri. The cumulative effect of varieties and developing stages
on fruit weight was found significant. The maximum fruit weight of (26.45 gm) was
recorded in cv. Dhakki at Rutab stage, while the minimum fruit weight (8.35 gm) was
recorded in cv. Gulistan at Tamr stage. The results for fruit weight of six date palm
cultivars were found significant at all maturity stages. It was observed that the fruit of
all cultivars completed its development in term of fruit weight at Rutab stage. No
further development was recorded during the rest of all stage. Perusal of the data
indicated that cv. Dhakki had significantly maximum fruit weight of 26.45 gm at
Rutab stage followed by Gulistan (15.16 gm), Azadi (12.57 gm) and Zahdi (12.02 gm).
The cultivar Khudrawi and Shakri showed statistically same fruit weight of 9.53 gm
and 9.41 gm respectively, at Rutab stage. The minimum fruit weight was recorded at
Tamr stage, wherein the Dhakki cultivar had significantly maximum fruit weight of
12.70 gm while Gulistan had minimum fruit weight of 8.35 gm. All the cultivars
showed similar trend at Kimri and Khalal stage. Abrupt decrease in fruit weight was
observed at Tamr stages, which was probably due to the fact that fruit lost its
maximum moisture content at this stage. These results were supported by [7] who
obtained higher fruit weight of date palm in Kimri to Khalal stage than it decreased at
Tamr stage. [9] Also obtained similar findings in five date cultivars grown in UAE.
Similarly according to author [10] the fruit weight of five major date palm cultivars
namely Khudrawi, Barni, Sullaj, Ruzeiz and Sifri, ranged from 7.83-16.96 gm.
5.Seed weight (g) of six date cultivars at individual maturity stages
Data depicted in Table-5 showed that the fruit seed weight of date palm was
significantly affected by varieties as well as different maturity stages. Among the
cultivars, significantly maximum fruit seed weight of all four maturity stages (1.41
112
gm) in cultivar Zahdi, which differed significantly from all other cultivars. The
minimum fruit pit weight (1.14 gm) was found in cultivar Khudrawi. The cumulative
effect of varieties and developing stages on fruit weight was statistically significant.
The maximum fruit seed weight (1.5 gm) was recorded in cv. Shakri at Rutab stage,
while the minimum fruit seed weight (0.98 gm) was also recorded in cv. Shakri at
Kimri stage. The data for fruit seed weight of six date palm cultivars was statistically
significant at all maturity stages. It has been observed that the fruit of all cultivars
completed its development in term of fruit seed weight at Rutab stage. No further
development was recorded during the rest of all stage. Perusal of the data indicated
that cv. Shakri had significantly maximum fruit seed weight of 1.53 gm at Rutab stage
followed by Gulistan (1.51 gm), Zahdi (1.49 gm) and Dhakki (1.193 gm) at the same
stage. The minimum fruit seed weight at the Rutab stage was recorded in cv. Khudrawi
1.14 gm. The minimum fruit seed weight was recorded at Kimri stage, wherein the
Gulistan cultivar had the maximum fruit seed weight of 1.44 gm while Shakri had
minimum fruit seed weight of 0.91 gm. A little increase was noted in fruit seed weight
at almost all three maturity stages, which was probably due to the fact that fruit
completed its development in term of seed weight at the last maturity stage. These
results are best supported with the findings of [12] where the fruit seed weight of 15
date palm cultivars ranged from 0.68-1.43 gm. The date palm fruit complete its seed
development at Rutab stage [9]. Similarly author [10] calculated the fruit seed weight
of five major date palm cultivars namely Khudrawi, Barni, Sullaj, Ruzeiz and Sifri,
ranging from 0. 71-1.39 g.
6.Fruit pulp weight (gm) of six date cultivars at individual maturity stages
Table-6 results on fruit pulp weight of six date palm cultivars revealed significant
variation in varieties as well as different maturity stages. Among different cultivars the
mean fruit pulp weight (19.97 gms) was found maximum in Dhakki, which differed
significantly from all other cultivars. The minimum fruit pulp weight (8.78 gm) was
found in cultivar Shakri. The interactive effect of varieties and developing stages on
fruit pulp weight was found significant. The maximum fruit pulp weight of (25.09 gm)
was recorded in cv. Dhakki at Rutab stage, while the minimum fruit pulp weight (7.17
gm) was recorded in cv. Gulistan at Tamr stage. The results for fruit pulp weight of six
113
date palm cultivars were found significant at all maturity stages. It was observed that
the fruit of all cultivars completed its development in term of fruit pulp weight at
Rutab stage. No further development was recorded during the rest of all stage. Perusal
of the data indicated that cv. Dhakki had significantly maximum fruit pulp weight of
25.10 gm at Rutab stage followed by Gulistan (12.35 gm), Azadi (11.14 gm) and
Zahdi (10.14 gm). The cultivar Khudrawi and Shakri showed statistically same fruit
pulp weight of 8.33 gm and 8.16 gm respectively, at Rutab stage. The minimum fruit
pulp weight was recorded at Tamr stage, wherein the Dhakki cultivar had significantly
maximum fruit pulp weight of 11.28 gm while Gulistan had minimum fruit pulp
weight of 7.17 gm. All the cultivars showed similar trend at Kimri and Khalal stage.
Abrupt decrease in fruit pulp weight was observed at Tamr stages, which was probably
due to the fact that fruit lost its maximum moisture content at this stage. These results
were supported by author [7] who obtained higher fruit pulp weight of date palm at
Rutab stage than it decreased at Tamr stage. [9] Also observed similar growth pattern
of date palm fruit in term fruit pulp weight in five date cultivars grown in UAE. The
fruit pulp weight of fifty date palm cultivars ranged from 13.87-17.30 g [13].
7.Yield per palm (kg)
The personal of Table-7 revealed the yield per palm at full maturity stage, which
was affected significantly due to varieties. Among the cultivars, significantly the
maximum yield was recorded in cv. Dhakki (96 kg) followed by Gulistan, Khudrawi,
Azadi and Shakri which gave 88, 63, 60 and 45 kgs yield per palm respectively. The
minimum yield per palm (32 kg) was recorded in cv. Zahdi. These results were
supported with the finding of author [14] who obtained 112.4 kg yield per palm in cv.
Dhakki. A heavy bearing tree can give a yield of 119.96 kg [15].
Conclusion
The physical measurements of date fruits of six cultivars grown in Dera Ismail
Khan showed that fruits differ in length, weight, diameter, seed weight and yield. All
the date fruits completed their physical maturity at Rutab stage. However, all the
fruits took maximum time to complete their Kimri stage. The cultivar Shakri was
found early maturing variety, while Dhakki was late but superior in many important
physical characters.
114
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palm (Phoenix dactylifera L.) on the quantitative characters and ripening of the
fruit of Asell variety. M. Phil. Thesis, Shah Abdul Latif Univ. Khairpur.
[9] Al-Hooti, S., J. S. Sidhu and H. Qabazard (1997). Physicochemical characteristics
of five date fruit cultivars grown in the United Arab Emirates. Plant Food for
Human Nut. 50 (2): 101-113.
[10] Khatchadourian, H. A., W. N. Sawaya, J. K. Khalil and A. S. Mashadi (1983).
Processing of five major varieties of Saudi Arabian Dates into Date Butter and
date in syrup. Date palm. 2 (1): 103-19.
[11] Iqbal, M., A. Ghafoor and S. Rehman (2004). Effect of Pollination times on fruit
characteristics and yield of date palm cv. Dhakki. Int. J. Agric. Biol. 6 (1).
115
[12] Ahmed., S. and W. A. Farooqi (1972). Studies on some distinguishing characters
of Iraqi Dates at Jhang. Pb. Frt. J. Date palm. 33 (4): 136-147.
[13] Muhammad, S. H., R. Shabana and E. A. Mawlod (1983). Evaluation and
identification of Iraqi dates cultivars. Date palm J. 2 (1): 27-55.
[14] Khan, H. (1988). Effect of different manures on the yield of Dhakki Dates. Pb.
Frt. J. Date palm. 38 (5): 251-259.
[15] Ahmed, S and H. M. Sheikh (1972). Effect of fruit thining on the fruit yield,
maturity and quality of date fruit. Pb. Frt. J. date palm. 33 (4): 242-252.
116
Table 1.
Time taken by fruits of different date palm cultivars at individual maturity stages
Maturity Stages
Varieties
Kimri
Khalal
Rutab
Tamr
Azadi
14.43 b
4.00 c
3.05 a
2.10 a
Dhakki
16.10 a
3.91 c
2.71 b
2.00 a
Gulistan
16.40 a
3.90 c
2.48 d
1.95 ab
Khudrawi
14.00 b
5.19 b
2.43 c
1.76 ab
Shakri
11.86 c
6.11 a
2.43 c
1.42 c
Zahdi
16.00 a
3.52 d
2.24 cd
1.71 ab
LSD0.05
5.769
2.317
1.680
2.169
Table 2.
Fruit length (cm) of different date palm cultivars at individual maturity stages
Maturity Stages
Varieties
Kimri
Khalal
Rutab
Tamr
Azadi
4.08 c
3.96 b
4.02 b
4.04 c
Dhakki
5.09 a
4.76 a
4.85 a
4.94 a
Gulistan
4.70 b
4.52 a
4.53 a
4.51 b
Khudrawi
3.87 d
3.82 b
3.84 bc
3.82 c
Shakri
3.45 e
4.04 b
4.14 b
4.12 c
Zahdi
3.42 e
3.46 c
3.47 c
3.43 d
LSD0.05
0.181
0.315
0.377
0.330
117
Table 3.
Fruit diameter (cm) of different date palm cultivars at individual maturity stages
Maturity Stages
Varieties
Kimri
Khalal
Rutab
Tamr
Azadi
2.27 c
2.67 b
2.71 b
2.71 b
Dhakki
3.07 a
2.98 a
3.16 a
3.05 a
Gulistan
2.33 c
2.30 c
2.34 c
2.33 c
Khudrawi
2.28 c
2.36 c
2.44 bc
2.45 c
Shakri
2.07 d
2.27 c
2.32 c
2.34 c
Zahdi
2.48 b
2.36 c
2.44 c
2.45 c
LSD0.05
0.128
0.190
0.287
0.222
Table 4.
Fruit weight (g) of different date palm cultivars at individual maturity stages
Maturity Stages
Varieties
Kimri
Khalal
Rutab
Tamr
Azadi
13.39 b
12.01 c
12.57 c
8.94 bc
Dhakki
23.36 a
23.31 a
26.45 a
12.70 a
Gulistan
12.48 b
14.53 b
15.16 b
8.35 c
Khudrawi
12.03 bc
14.78 b
9.53 d
9.50 b
Shakri
9.20 d
12.07 c
9.41 d
9.58 b
Zahdi
10.61 cd
11.34 c
12.02 c
12.11 a
LSD0.05
1.507
1.776
0.831
0.780
118
Table 5. Seed weight (gm) of different date palm cultivars at individual maturity stages
Maturity Stages
Varieties
Kimri
Khalal
Rutab
Tamr
Azadi
1.11 b
1.05 d
1.32 b
1.17 b
Dhakki
1.35 a
1.23 c
1.19 bc
1.14 b
Gulistan
1.44 a
1.47 a
1.51 a
1.02 c
Khudrawi
1.07 b
1.20 c
1.14 c
1.13 b
Shakri
0.91 c
1.42 ab
1.53 a
1.53 a
Zahdi
1.43 a
1.29 bc
1.49 a
1.44 a
LSD0.05
0.140
0.140
0.152
0.099
Table 6. Fruit pulp weight (gm) of different date palm cultivars at individual maturity stages
Maturity Stages
Varieties
Kimri
Khalal
Rutab
Tamr
Azadi
11.65 b
10.76 c
11.14 c
7.80 c
Dhakki
21.58 a
21.93 a
25.10 c
11.28 a
Gulistan
11.49 b
12.92 b
12.35 b
7.17 b
Khudrawi
10.84 b
12.84 b
8.33 c
8.32 c
Shakri
8.24 c
10.57 c
8.16 c
8.16 c
Zahdi
9.05 c
9.88 c
10.14 c
10.20 c
LSD0.05
1.602
1.859
1.619
4.555
Table 7.
Varieties
Yield
Azadi
60.80 c
Dhakki
96.00 a
Gulistan
88.00 b
Khudrawi
63.67 c
Shakri
45.00 d
Zahdi
32.00 e
LSD0.05
6.798
Yield per palm (kg)
119
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120
PP 01
Effect of amount of pollen on anatomy and quality of Zagloul date
palm fruit (Phoenix dactylifera, L.)
Alaa El-Din Kh. Omar1, 2
1
Department of Plant Production, College of Food and Agric. Sciences
King Saud Univ., P.O. Box 2460, Riyadh 11451, Saudi Arabia
2
Hort., Dept., Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh 33516,
Egypt
E-mail address: aomer@ksu.edu.sa
Abstract
This investigation was carried out during two successive seasons (2009/2010) in a
private orchard-Egypt, with the aim to investigate the influence amount of pollen (AP)
on fruit quality and anatomy of “Zaghloul” fruit. One male date palm (Meghal) and
four date palm healthy were selected, 12 bunches for each female were leaved (3
bunches for each treatment). Four treatments i.e. Control (AP0-without pollination),
AP1 (4 strands / bunch), AP2 (8 strands/bunch) and AP3 (16 strands/bunch).
8
strands/bunch (AP2) gave the highest bunch weight, fruit weight, fruit length, TSS and
lowest fruit tannin content and total acidity as compared the other treatments. Also in
fruit anatomy, AP2 recorded the lowest layers in epiderm cells, stone cells, outer
mesocarp cells and tannin cells ( 14.67 μ, 63.33 μ, 1041.67 μ and 533.33 μ ,
respectively ), additional highest layer in inter mesocarp (4500.67 μ). On the other
hand, Control fruits had the highest layers in epiderm, hypoderm and tannin and
recorded 20.00 μ, 48.22 μ and 1072.22 μ, respectively. Applications (AP1 and AP2)
recorded the highest layers outer mesocarp cells and stone cells and recorded 1700.00
μ and 90.00 μ, respectively. Through this investigation, Results showed that amount of
pollen treatments can be play a role in fruit quality of “Zaghloul” date palm fruits
through some changes of layers cells which correlated with fruit anatomy.
Key words: Fruit quality, fruit anatomy, Date palm, Amount Pollen.
Introduction
Dates are an extremely important subsistence crop in most of the desert regions. For
millions of people, dates are an important nutritional element contributing to food
121
security. “Zaghloul” date palm cultivars is one of the most important soft dates in
Egypt . According to the statistics book published by the Ministry of Agriculture
(2010) there are 11.829.410 female date palms in Egypt and total production of date
fruits amounts to 1.300.000 tons/year (FAO, 2010). Since date palm is a diocious
plant, pollen has to be transported from the male to the female tree. Fruit set is a wellknown problem; much attention was given to studying different aspects of pollination
which is the first crucial stage of fruit development. Several investigators have
evaluated some date palm varieties Godara et al(1994), Nail(1994), El-Gamdi (1996),
Metwaly (1999), Hussein et al.,(2001), Soliman(2002), Rizk et al(2003) and ElAgamy et al(2003).
Date palm growers believe that different cultivars require
different amount of pollen in order to get satisfactory fruit set.
Dowsen, 1982
concluded that a lower fruit set was achieved when a lower pollen deposit was
observed in the inflorescence area.
Studies on the anatomy of the date fruit
development were focused by different workers on a certain development stage (Long
1943; Reuveni 1967 ; Reuveni 1970 ; Osman et al 1974; Al-Jarrah and Al-Ani 1981)
or a certain part or tissue of the fruit (Schroeder and Nixon 1958; Hass and Bliss 1935;
Turrell et al 1940 and Coggins et al 1967)
In a transversal section at the equatorial zone of a development fruit , three main
regions are found :exocarp, mesocarp and endocarp. The exocarp includes one layer of
epidermal cells beneath which two to three layers of hypodermal cells containing tannin
are found. Next to it is a layer of three to four parenchymatic cells ( Gefen 1966 ).The
endocarp consists of one layer of small cells which can be distinguished in fertilized
fruit only at the first stages of fruit development. At a later stage these cells die
together with few layers of the inner mesocarp and after being stretched form the
papery envelope of the mature seed.(Long 1943) who studied mainly the pericarp
development, divided it into six (or seven when including the period of softening
which follows maturation ).
Reuveni 1967 studding the pericarp and seed development in fertilized and
unfertilized fruit (Zaghloul cv.), divided it into six stages, but the length of each was
different to some extent from that of (Long 1943). As there might be differences
between cultivars growing conditions. The majority of researchers in this field were
122
interested in the physical characteristics of the fruits in one or two seasons, information
about date fruit as affected by pollination amount on fruit anatomy is scarce.
Therefore, the objective of this investigation is studying amount of pollen effects
on fruit quality and
anatomical characteristics for date palm fruits (Phoenix
dactylifera L.) , Zaghloul cv. during 2009 and 2010 seasons.
Materials and Methods
2.1. Plant Material: This investigation was carried out during two successive seasons
(2009/2010), in a private orchard, Egypt. One date palm male (Meghal) and five date
palm healthy, 17 years old Zaghloul female date palms were selected, 12 bunches for
each female were leaved (3 bunches for each amount pollen level/palm) and the
pollination was done after 2 or 4 days from spathes opening. Four amounts of pollen
(AP) at i.e. Control (AP0 - without pollination), 4 strands/ bunch (AP1), 8 strands/
bunch (AP2) and 16 strands/ bunch AP3) for Zaghloul bunch were evaluated as to their
effect on anatomy fruits that will be mentioned thereafter . The palms were grown in
loamy soil and planted 8 meters apart and receiving the regular horticultural practices
recommended by the Ministry of Agriculture.
2.2. Average bunch weight: was estimated in kg.
2.3. Fruit physical characters: Samples of 60 fruits per each treatment, 20 fruits were
taken randomly from each bunch (replicate) to determine fruit weight, flesh weight
and fruit dimensions (length and diameter "cm”).
2.4. Fruit chemical characters: Fruit acidity, total soluble solids and tannins content:
were determined according to (A.O.A.C.1995)
2.5. Fruit anatomy: For the histological studies, samples were picked at fruit at the
first week of October during both seasons. The individual sample contains three
replicates, six fruits/replicate in each treatment. The samples were kept in vials. The
ordinary paraffin method was followed for preparing the samples using the formalin
acetic alcohol fluid i.e. FAA (70%) as a killing and fixing agent, the normal butyl
alcohol for dehydration and paraffin wax (56E - 58EC m.p.) for infiltration and
embedding Johansen (1940). Serial longitudinal and cross sections of 10 micron were
carried out by rotary microtome. Staining of sections were stained using safranine and
light green technique according to the schedule of Sharman and mounted in Canada
123
Balsam. The obtained slides were microscopically examined and the following
measurements were detected. Epidermis cells dimension (μ), Hypodermal cells
dimensions (μ), Stone Cells dimension (μ) , Outer mesocarp cells dimension (μ) ,
Tannin Cells dimension (μ) and Inner mesocarp cells dimension (μ).
2.6. Experimental design and statistical analysis: The experiment was set up in a
randomized complete block design and each treatment was represented by five
replicates, Data were subjected to Duncan’s Multiple Range Test (Snedecr and
Cochran, 1980).
Results
3.1.-Effect of amount of pollen on some physical characters of Zaghloul fruit:Data concerning the quality of the fruits in both seasons are presented in Tables 1
and 2 indicated that amount of pollen had effect on Zaghloul date palm fruit as
following:3.1.1. Bunch weight (kg)
Data in table (1) showed that, in both seasons the highest bunch weight recorded by
treatment 3 (8 strands/bunch). The bunch weight of those treatments ranged from
16.52 to 23.67 kg in the first season and from 17.73 to 24.87 kg in the second season.
Differences in this respect among those treatments were highly significant. On the
other hand, the lowest values were recorded by the control and recorded 16.52 and
17.73 kg in both seasons, respectively.
3.1.2. Fruit weight (gm)
The obtained data cleared that the tested treatments significantly affected fruit
weight in the both seasons .The uppermost value was 40.99 gm for the treatment
AP2(8 strand /bunch) and the lowest value was 26.32gm for the control , Table(1).
3.1.3.Flesh weight (gm)
The data in table(1) reveal an obvious promoting effect of amount of pollen on flesh
weight in both seasons. Thus, the highest values were recorded by AP2 (8
strands/bunch)in the first season and recorded 36.78 gm. On the other hand, the lowest
value recorded by the control in both seasons.
3.1.4. Fruit length (cm)
124
Table (1) showed that amount of pollen had a slightly effect on fruit length. The
uppermost value was 8.63 cm for AP3 (16 strands/bunch) in the second season, and
the lowermost value was 5.80 cm for AP0 (control) in the first season
3.1.5. Fruit diameter (cm)
Data
in table(1) reveal that amount of pollen at the rate of 4, 8 and 16
strands/bunch increased the fruit diameter in comparison with control in both seasons
but the differences were insignificant among treatments.
3.2.-Effect of amounts of pollen on some chemical characters of Zaghloul fruit:3.2.1. Total acidity (%):
The obtained data in table(2) clarify that treatments significantly affect total acidity
in Zaghloul dates in both seasons. The uppermost value was 0.071% in the first season
by control treatment (without pollination). On the other hand, 8 strands/bunch
treatment recorded the lowest value 0.011 in first season by 8 strand/bunch (AP2).
3.2.2. TSS(%)
Table (2) showed that in both seasons were graduations in TSS values as the rate of
amount of pollen increased. In the first season, the lowest TSS value (24.57 %) was recorded
by the control, while the highest value (36.85%) was recorded by the (8 strands/bunch), the
differences were highly significantly in both seasons. The same trend could be detected in the
second season (8 strands/bunch), control recorded the lowest fruit in both seasons.
3.2.3. Tannin (%)
Tannin content of the considered date fruit reveal highly significant response to all
tested treatments in both seasons. However, slight and gradual reductions in fruit
tannins content were observed as the rate of amount of pollen were increased. Control
treatment recorded the highest tannin content in both seasons, table (2).
3.3. Effect of amount of pollen on fruit anatomy of Zaghloul fruit
Data concerning the histological (anatomy) of the fruits in the two seasons are
presented in Figures 1,2 and 3,indicated that amount of pollen has effective on
Zaghloul date palm fruit anatomy as following:3.3.1. Epidermis cells dimension (μ)
Fig (1and3) showed that, applications of amount of pollen(AP 1, AP2and AP3) were
decrease in the Epidermal cells (μ ) as compared with control (AP 0) and recorded
125
(20.00 μ ) . On the other hand, AP2 (8 strand/bunch) recorded the lowest value in
epidermis cells and recorded (14.67 μ ).
3.3.2. Hypodermal cells dimensions (μ)
The micrographs in Fig ( 1 and 3) showed that different applications of pollination
significantly decreased the cells of Hypodermal of Zaghloul fruit. AP 1 recorded the
lowest value (38.33μ). There is no-significant differences between treatments AP 2
and (AP0 ) but there is a significant deference among of other treatments.
3.3.3. Stone Cells dimension (μ)
In cross sections of all pollination treatments were highly significant effect on
Stone Cells dimension of Zaghloul dates and noticed that AP 3-(16 strand/bunch)
recorded the highest value (90.00μ) as compared with other treatments specially
control (66.10μ), AP2 recorded the lowest value(63.33 μ ).
3.3.4. Outer mesocarp cells dimension (μ)
As shown from micrographs in Fig ( 2and3) pollination treatments highly
significant effects in outer mesocarp cells dimension,
AP1 recorded the highest
value(1700.00 μ ) as compared with other treatments recorded 138.92, 1041.67 and
1658.67 μ for AP0,A P2 and AP3, respectively.
3.3.5. Tannin Cells dimension (μ)
The micrographs in Figs( 2and3) showed that pollination treatments had a highly
significant effect on tannin cells dimension of Zaghloul fruit . AP 0 treatment recorded
the highest value (1072.22 μ ) and followed (1041.67 μ ) by treatment
AP1(4
strand/bunch). On the other hand treatment AP2 recorded the lowest value (533.33μ )
and followed treatment AP3 and recorded 816.67 μ.
3.3.6. Inner mesocarp cells dimension (μ)
In cross sections of all applications of amount of pollen were highly significant
effects on inner mesocarp cells dimension of Zaghloul fruit , and noticed that AP 2
recorded the highest value (4500.67μ) as compared with other treatments ,followed
(3481.97, 2700.00 and 2366.67μ ) by treatments AP0, AP1 and AP3, respectively.
Discussions
Concerning
the results, noticed that many factors can be make a changes on
anatomy fruits and it is correlated with fruit quality (physical and chemicals properties)
126
such as temperatures(Summerville ,1944 ,Fahn et al. 1961, Fortescue and Turner 2005),
thinning of the fruit clusters or bunches(Nixon and Crawford 1937,1942), high and
standard crop load levels(S a l v a d o r et al 2006). Also, amount of pollen can be play
the same role in effect on fruit quality through increase the bunch weight, fruit length
and fruit weight, improve TSS, reduce tannin and total acidity content specially with
treatment 3 (8 strands/bunch) as compared with other treatments (4 ,16 strands/bunch)
and control in must fruit characters in both seasons, all this changes correlated with
fruit anatomy. Where the same treatment AP2 recorded the lowest layers in Epiderm
cells, Stone cells, Outer mesocarp
cells and Tannin cells and 14.67 μ, 63.33 μ,
1041.67 μ and 533.33 μ , respectively, additional highest layer in Inner mesocarp
(4500.67 μ). On the other hand, Control fruits had the highest layers in Epiderm,
Hypoderm and Tannin and recorded 20.00 μ, 48.22 μ and 1072.22 μ, respectively.
Omar (2004), who reported that 6 strands/bunch improve of fruit quality comparing
the 3,9 strands/bunch in Hayany fruits. These changes in fruit quality through change in
some
promoters , enzymes or processes (El-Mardi et al 2007 ), who found that low
pollen concentration can be produce the same effect on the pectin content as high pollen
concentration does. Thus indicating no effect of the amount of pollen on the activation
of pectinase enzymes and providing a clue for economizing on pollen application on
Farad date palm fruits. Both pollen and developing seeds contain plant hormones, and
they may serve as sources of some of these hormones Talon et al. 1990; GarciaMartinez et al. 1991; Ben-Cheikh et al. 1997; Ozga et al. 2002). However, movement
of hormones from pollen and seeds directly into fruit progenitor tissues has not to our
knowledge, been demonstrated. Anatomy Zaghloul date palm anatomy showed that
increase or decrease of some cells in some layers such as increase of stone and tannin
cells in control and lower amount of pollen
as compared with other pollination
treatments and it is reflect on fruit quality , these results agree with Shabana et
al(2001), John Burt (2007) who reported that pollination of date palm cultivars gave
the highest fruit set percentage and quality , also Howpage et al (2001) found that the
lack of
amount of pollen in kiwifruit reduce of fruit quality under Australian
conditions, Yong Seub Shin et al(2007) investigate the effect of amount of pollen on
development and sugar content of melon fruits.
127
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pollination period for
Khalas date palm cultivar. Ministry of
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[39] Talon, M., Zacarias, L. and Primo-Millo, E. (1990) .Hormonal changes
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mandarins differing in their
parthenocarpic ability. Physiol. Plant 79: 400–406.
[40] Turrell ,F.M. , W.B.,Sinclair and D.E.Bliss(1940).Structural and chemical factors
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[41] Yong Seub Shin, So Deuk Park and
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131
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132
PP 02
Evaluation of pollination ability and metazenic effects of seedlings
date palm males, on two female cultivars Mishrig Wad Laggai
and Mishrig Wad Khatiab under Khartoum state condition
Dawoud H.D., and Fatima A.El-Rauof
dawoudhussien@hotmial.com
fatimaraouf.ahmed0@gamail.com
Abstract
One of the main objectives of this study was to select highly potent male palm to
raise standard of male varieties in the Sudan. This evaluation experiment involved ٧٠
male palms located in 21 private orchards in 5 districts at Khartoum state. The male
evaluation experiment took 3 successive seasons 1997/98, 98/99, and 99/2000.Then
another experiment was conducted to study the metazenic effect of Seven selected
males (from the above ٧٠ males) on Mishrig Wad Laggai and Mishrig Wad Khatiab
date palm cultivars (MWL, MWK) for three successive seasons 2000/2001, 2001/2002
and 2002/2003.
Strong metazenic effects of those pollens of Seven selected males were found on
fruit characteristics, fruits maturity period and yield per kilogram per tree of MWL and
MWK cultivars, and recorded, As Khartoum Male, A , KM,B, KM,C up to KM,G in
Shambat Horticultural Research section files as Males for pollination of Mishrig Wad
Laggai and Mishrig Wad Khatiab
According to highly significant differences in all studied parameters. We can
recommend Male C and Male F as high compatible males for pollination MWL and
MWK date palm cultivars under Khartoum state
Introduction
Date palm (Phoenix dactylifera L.) is one of the most important fruit crop grown in
the Sudan, which needs a lot of research efforts to increase its yield. Therefore, it is
essential to study the factors influencing productivity and improving their fruit quality.
One of the most important factors is pollination of female palms by the compatible
male at a suitable time with the best pollen type. At Khartoum area the date palm
133
industry has not been able to attain any commercial status simply because its ripening
time during July and August synchronizes with Autumn rain which cause rot and
fermentation of the 'Rutab' fruits on the palm tree due to high humidity and high
temperature during that period.
Many varieties have been tested but no variety can escape in the rainy season ,
except Medina which ripen at mid May, However, Nixon et al. (1978) was able to
produce mature fruits 20 days earlier by metaxenic effects of pollination. They also
found that the Fard pollen induced early maturity while that of Mosque delayed fruit
ripening. Most of date palm males available for pollinating different female cultivars
are mainly originated from seed propagation. Therefore, different males vary in their
ability to pollinate and in their metaxenic effect on certain variety (El_ Sabrout, 1979;
Hussein et al. 1979; El – Gheyaty, 1982; Higazy et al., 1982; Nasser et al., 1986; El
Amer et al., 1993; and El – Salhy et al., 1997). This variability is due to the degree of
compatibility among certain males and females (Higazy et al., 1982). Evaluation of
male palm trees includes, generally, morphological aspects of reproductive organs,
such as average number of spathes/each male, weight of whole spathe, number of
strands/spathe, number of flowers per each strand and average weight of pollen
grains/spathe in relation to percentage fruit set.
In the Sudan little or no consideration has been given to the metaxenic effect under
dry and hot environmental conditions. In addition, date palm grower's use any pollen
grains are readily available at the time of pollination. As a result, yield, fruit quality and
maturity period of the fruits of the same variety, differ greatly from one year to another.
For these reasons, the growers are now beginning to realize the value of selection of
males. The objectives of the present study are to evaluate male seedlings morphological
characters, pollen quality and quantity, and to study the metaxenic effects on fruits and
yield of female date palm cultivars Mishrig Wad Laggai and Mishrig Wad Khatiab, and
finally to identify superior males to improve yield and quality.
Materials and Methods
Two experiments were carried out during 1997 through2003
Experiment 1: Phenotypic variation between seedlings male - palm used in
pollination at Khartoum.
134
Experiment 2:The metazenic effect of seven seedling date males (selected from the
above study) on two female cultivars (Mishrig Wad Laggai (MWL) and Mishrig Wad
Khatiab (MWK).
An experiment was established to test the phenotypic variation between seedling
male - palm used in pollination.
The evaluation covered seventy males used in pollination a (MWL and MWK)
located in 21 date palm orchards. The evaluation survey started in 1997/98 to select
highly potent male palms.
The seedling males were classified into seven groups similar in their morphological
characteristics, then randomly selected one from each group for vigor and for being
disease free. It also necessary to select males having off- shoots to allow vegetative
propagation of promising males. Males without off- shoots were not evaluated.
Selected palms were of more or less the same age and vigor i.e. the survey continued
for one year followed by selection, classification and evaluation for two years
.According to the method adopted by Nixon (1950)
Morphological character of seedling males:
Leaf characteristics
The number of leaves in each seedling male was counted, a one year old leaf was
removed from each male and its length, and number of pinnae was counted Lengths
and width of pinnae and spines were measured.
Spathe characteristics:
At blooming time the male palms, were visited every morning, five mature spathes
from each male, were collected and morphologically studied for spathe characteristics.
Flowering time and duration period were recorded and the pollen grain characters,
even the viability percent of pollen grains (Fresh and stored for 1 year), according to
Shajeem et.al, (1986). These parameters were recorded as an average of three years
1997/98, 98/99, and 99/2000. To eliminate the effect of alternate bearing phenomenon
A second experiment was established to test the metazenic effect of seven seedling
date males on two female palms of the cultivars Mishrig Wad Laggai and Mishrig
Wad Khatiab.
135
This experiment was carried out in Hag Basheir private orchard during the years
(٢٠٠٢/٢٠٠٣و
٢٠٠١/٢٠٠٢ و٢٠٠٠/٢٠٠١)The female date palm cultivars namely:
Mishrig Wad Laggai and Mishrig Wad Khatiab were pollinated with pollen grains
from seven male – palm trees marked as Male A, Male B, Male C, Male D, Male E,
Male F and Male G.
The seedlings male – palm lines under study have been recorded as individual's
pollinizers for date palm trees, and have been identified as Khartoum Male, A , KM,B,
KM,C up to KM,G in Shambat Horticultural Research section files as Males for
pollination of Mishrig Wad Laggai and Mishrig Wad Khatiab .
Forty two trees from each variety of MWK and MWL were selected for this
purpose, uniform in age and height. All the trees were planted in a randomized
complete block design, with three replications and two trees per plot. Prior to
pollination, unopened spathes in all trees were covered with cloth bags 60 x 40 cm.
The bags were removed during the time of pollination and covered again soon after
pollination for four weeks to prevent cross pollination.
Each spathe was labeled just on emergence and its date was recorded. The date of
opening of each spathe and its pollination was also noted, the method and technique of
the pollination was in principle similar to that of Nixon (19 53).
At blooming, the female trees were visited every morning and spathes, were
pollinated from specific pollen using a brush. A great care was carried exercised
against mixing of different pollens with each other or contamination by any means.
The pollinated spathes were covered immediately after pollination with (40 x 60
cm) paper bags for four weeks. Number of set fruits and total number of flowers in 20
randomly selected strands were recorded to calculate fruit set percent in pollinated
bunches as follows:Fruit-set percentage
=
No of Set fruits
Total number of flowers
When fruits reach khalal stage also at Rutab stage fifty fruits (full yellow colored),
were taken to determine physical characters of the fruits, average (fruit weight, flesh
weight, fruit dimensions, seed weight and total soluble solids (TSS) at the base, middle
and terminal parts of each fruit by hand refract meter.
136
Moisture contents
Five grams of the chopped pulp taken from 50 fruits(khalal and Rutab stages) in a
china dishes was placed for draying in vacuum oven at 70 C for 24 hours after drying
the samples were weighed and percentage water contents was worked out Yield was
calculated as combined yield of 2 seasons as kg /tree Data were analyzed using new
Duncan's multiple range tests at 0.5 (Snedecor and Cochran 1980)
Results and Discussion
The seventy studied males, phenotypically (According to the Leaves, Spathes,
and Pollen grains characters) divided to seven groups using Nixon 1956)
methodology for date palm classification one male from each group was selected to
study the phenotypic variability as Number, length and width of the leaves, pinnae,
spines and spine area per leaf, the data portrayed in Table (1) which showed clear
evidence of a significant differences between the phenotypic characters of the seven
males used in pollination.
Pinnae and spines characteristics
The leaves number of the 7males ranged between (85 and 189). Significantly higher
number of the leaves and pinnae were produced by Males A, B, C and F. While the
lower number producers No. of leaves and pinnae were produced by Male G,
The number of spines varied significantly and with males A and B gave
significantly higher number of spines compared with the other males.
All these phenotypic variations were in line with Nixon (1956) El-Sabrout (1979)
Nasser et al. (1986) and El – Amer (1993).
Spathe characteristics
The number of spathe was significantly variable amongst the males (Table 2)
Spathe characters such as length, width and weight with male A, B, C, and F were
significantly higher than the other males (Table 2) the heaviest sheath weight was
produced from Males F, A, and B which were significantly higher than the other
males. The internal parts of the spathes (length of strands, number of strands and
number of flowers per strand) were significantly higher on males A, B and F (Table 2)
137
Flowering time of the 7 males varied from late December to mid February and
the flowering duration ranged from 21 (males A, and E) to 50 days (male C) as
shown in Table (3).
There were variations in pollen grain size and weight. The largest size of pollen
grain were found on male A and G while the smallest sizes ones were produced by
males C and F as shown in Table (3). Generally Date palm scientists prefer the most
lightly and smallest size pollen grains to be carried by winds or by insects, Nixon et al.
(1978) These results were in line with Higazy et al. (1982) who stated that no two
seedling palms are alike each other. Along with this statement, Nixon et al. (1978)
reported that fruit tree raised from seeds is greatly heterozygous.
Pollen viability
Male C and Male F had the highest pollen viability compared with other Males in Table
(3).The viability of the pollen grains decreased after storage for one year, Fruit setting
The combined data of fruit setting for 3 years illustrated significant differences
among the males used for pollinating Mishrig Wad Laggai and Mishrig Wad Khatiab
(Table 3). The highest percentages of fruit setting were produced when Males C and F
were used as source of pollen grains for pollinating Mishrig Wad Khatiab and Mishrig
Wad Laggai. While the lowest percentages of fruit setting were produced when Males
D, and E were used as source of pollen grains.
The highest percentage of fruit set reflected successful pollination and fertilization
or high compatibility among the certain males and MWK and MWL cultivars, these
results are in agreement with those reported by Swingle (1928), Nixon and Carpenter
(1978), El- Sabrout (1979), El- Ghayaty et al (1982) and Higazy et al (1982b).
Fruit characters
Data in Tables (4, and 5) indicated significant differences in all studied parameters
for the two cultivars in the two stages of the fruits (Rutab and Khalal) for three years
(2000/2003) as affected by the 7 males. Male B, C and F produced significantly higher
fruit size, weight, volume, TSS, and moisture content in Khalal and Rutab stage for the
two cultivars for the three seasons.
Seed weight:
138
The data in Table (6) indicated a highly significant difference in the seed weight of
the fruits as affected metaxinically in the three seasons for the two cultivars at Khalal
and Rutab stages. The highest seed weight was produced when Males G and E were
used as pollinators, and the lowest seed weight was produced when Males C, F and B,
were used.
Maturity period of fruits (from pollination to harvest)
The maturity period was metaxinically affected by the males, significant differences
between the males were found on the two cultivars at Khalal and Rutab stages (Table
6).The shortest maturity period observed on Male C and F while the longest maturity
period obtained from Males D, E and G, for the two cultivars, as average of 3 years for
Khalal and Rutab Stage.
Yield per kg per tree
Data in Table (٧) indicates that significant difference between the males and their
metazenic effect on the yearly and combined yield of the two cultivars of date palm.
The highest yield was produced from male pollinator's F, C, and B.
The date palm cultivar Mishrig Wad Laggai produced slightly higher yield than
cultivar Mishrig Wad Khatiab. While the lowest average yield produced from, G
and D. All the results obtained in this study, are in agreement with findings of ElHammady et al. (1977), Khalifa et al (1979) Higazy et al. (1982a) and El – Amer
et al. (1983).
139
References
[1] El-Amer, M., M. Fayed, M Gehgah and E EI-Hammady (1993) pollinators on fruit
set and qualities of some date cultivars. Proceeding of the third symposium on date
palm (17-20 Jan )King Fisal Univ.Saudia Arabia, pp.250-260.
[2] EI-Ghayaty, S.H. (1982). Effects of different pollinators on fruit setting and some
fruit properties of "Siwi" and "Amhat" date varieties. Proceedings of the first
symposium on date palm (March 23-25) King Fisal Univ.Saudia Arabia, pp.72-82.
[3] EI- Hammady, .M,M A.S. Khalifa and A.M. (1977). The effect of date pollen on
some physical and chemical characters of Hayani Variety. Res.Bull. No.733 fac.
Agric Ain-Shams Univ., Cairo.
[4] EI-Sabrout, M. B. (1979). Some physiological studies on the effect of pollen type
on fruit-setting and fruit quality in some varieties M.Sc. Thesis Fac. Agric Alex
Univ Egypt.
[5] EI-salhy. A.M., A.Y. Abdalla, and R.A.A Mostafa (1997) Evaluation of some date
palm male seedlings in pollination of Zaghloul and Samany date palms under
Assiut conditions Assiut JOUR. agric. SCI. 28(2): 79-89.
[6] Higazy, M.K., S.H. EI- Ghayatya and, F.B. Al Makhton (1982a).Effects of pollen
type on fruit-setting yield and Some physical fruit properties of some date
varieties. Proceedings of the first symposium on the on date palm(March23-25) )
King Fisal Univ.Saudia Arabia, pp:74-93.
[7] Higazy, M.K. , S.H.El Ghayaty, and F.B. Al- Makhton (1982b) Effects of different
pollen types on fruit chemical properties of some date varieties. Proceedings of the
first symposium on the date palm (March 23-25) King Fisal Univ.Saudia Arabia,
pp: 94-101
[8] Hussein F, M.S Al-kahtani, and Y.A. Wally (1979) Date palm cultivation and
production in the Arabic and Islamic world. Ain Shama Univ .press, Egypt,
576pp.(In Arabic)
[9] Khalifa, A.S., Z.M. Hamdy, S. Azzouz, H.El-Masry, and M Yousef (1979).Effect
of source of pollen on the physical and chemical quality of,, Amhat.. Date variety.
Agric Res. Rev. 58 (3): 15-23.
140
[10] Nasser T.A., M.A. Shaheen and M. A Bacha (1986). Evaluation of date palm
males used in pollination in the central region, Saudi Arabia. Proceedings of the
second symposium on the date palm (3-6 March) ) King Fisal Univ.Saudia
Arabia, pp337-345.
[11] Nixon, R. W. (1950) Imported varieties of Dates in the United States. United
States Department of Agriculture Washington, D.C., July 1950
[12] Nixon, R. W. (1953) Metaxenia in dates . Proc.Amer.Soc.Hort.Sci.32:221-226
[13] Nixon, R. W. (1956) Effect of Metaxenia and fruit thinning on size and checking
of deglet noor
[14] Nixon, R. W.
And J. B. Carpenter (1978).Date growing in the United
States.Agric.Info.Bull.207, Washington (DC)
[15] Shajeem, M. A., T. A., Naser, and M. A Bacha (1986). Date palm pollen viability
in relation to storage conditions. Proceedings of the second symposium on date
palm (March 3 – 6) King Fisal Univ. Saudi Arabia. Pp 331 – 336.
[16] Swingle, W.F.(1928) Metazenia in the date palm. Jour. Hered 19:257-268.
[17] Snedecor, G. W. and W. G. Cochran (1980). ″Statistical Methods″ Oxford and
J.B.H. Publishing Com.6th Edition (1980).
141
Table 1. Phenotypic character of seven seedling date males used in pollination of
MWL and MWK palm tree.
%
Spine
area
per leaf
Length
Male
Number
Leaves
Width (cm
(cm)
Pinnae Spines
Leaves
Pinnae Spines
Pinnae
Spine
A
175a
193a
28a
429a
64a
12a
5.0a
0.2c
11
B
181a
200a
24a
432a
61a
17a
4.8a
0.2c
12
C
189a
210a
22b
436a
65a
13a
4.8a
0.2c
10
D
165b
180b
14c
382b
48b
9b
3.4b
0.8a
18
E
161b
182b
18b
334b
46b
7b
3.4b
0.8a
18
F
183a
204a
12c
434a
66a
15a
4.8a
0.2c
8
G
91c
122c
16b
280c
36c
8b
2.8c
0.6b
16
* Means followed by the same letters are not significantly different at p≤:0.05 according to
Duncan's Multiple Range test.
Table 2.Variation between spathe characters of seven seedling date males used in
Pollination of MWL and MWK palm tree
Male
Spathe
Sheath
weight
(g)
Length of
strand
(cm)
No.
Length
(cm)
Width
(cm)
Wight
(g)
A
26a
120b
40b
920b
38a
B
23a
130b
42b
950b
C
28a
160a
46a
D
20b
90c
E
19b
F
G
No. of
Strands/
spathe
Flowers/
strand
36a
400a
91a
35a
37a
390a
48a
3150a
25b
38a
415a
95a
40b
430c
27b
23b
300b
74b
85c
20c
400c
20c
26b
330b
74b
27a
153a
48a
3090a
40a
38a
410a
92a
12c
87c
12d
350c
18c
18c
200c
60c
Means followed by the same letters are not significantly different at p≤:0.05 according to
Duncan's Multiple Range test.
142
Table 3.Time, duration of flowering, Pollen grain characters of different Date
males used in pollination and their effect on Fruit set of MWL and MWK cultivars
Males Flowering duration
time
of
flowering
94/9596/97
(days)
***
***
Pollen grain characters
♣
Size
(micron)
Wt. Weight
of
of
(P. g) pollen
grain
1. cc
per
spathe
(g)
Fruit set
(%)
Pollen
viability %
**
1997/ 981999/2000
♣
Fresh Stored
for
one
year
K
L
A
28 Dec3Jan.
21.3
545c
0.63c
60b
80.0b
60.0b
71.7b
67 b
B
16 -19
February
34.3
563c
0.65c
61b
82.0b
61.0b
81a
76.3a
C
13-15January
50.3
469d
0.58d
83.1a
99.0a
70.1a
81.3a 76.7a
D
25 -27
Dec.
34.3
660b
0.73b
34c
73.0c
52.0c
56.3c
E
29 -30
Dec.
21.3
720a
0.82a
25.3d
74c
54.0c
71.7b 66.7b
F
15 -16
February
48.3
470d
0.59d
82.2a
95.0a
71.0a
80.7a
76a
G
1-3
January
31.3
710a
0.81a
36.1c
74.0c
54.0c
45c
54.7c
55 c
*** Significant at 0.001 level.
*Means followed by the same letters are not significantly different at p≤:0.05 according to
Duncan's Multiple Range test.
** Stain with 0.7% tetrazolium sah (2:3:5 triphcnyl tetrazolium chlorides).
♣ Combined data 0f three years 1998/ 99-1999/2000)
143
Table 4. Effect of different pollen grains on fruit characters of MWK and MWL,
date palm cultivars (Khalal stage) ♣
Fruit
Length (cm)
Ma
le
Diameter
(cm)
Weight (g)
Volume (cc)
Moisture (%)
TSS (%)
K
L
K
L
K
L
K
L
K
L
K
L
A
3.6b
4.53b
2.51b
2.71b
11.1b
12.07b
11.0b
12.0b
44.0b
45.1b
32.14
b
35.1b
B
3.90a
4.81a
2.82a
2.93a
12.1a
12.99a
12.17a
13.07a
53.0a
53.1a
37.21a
40.2a
C
3.90a
4.82a
2.81a
2.92n
12.1a
12.98a
12.15a
13.01a
52.0a
53.2a
38.31a
41.3a
D
3.21c
4.24c
2.31c
2.45c
10.1c
11.02
10.44c
11.05c
33.0c
37.1c
20.4c
32c
E
3.62b
4.53b
2.52b
2.63b
11.1b
11.0b
11.12b
12.02b
45.0c
46.2b
32.4b
35.5b
F
3.91a
4.81a
2.82a
2.89a
12.1a
13.0a
12.14a
11.01a
53.0
53.2a
38.17a
41.3a
G
3.23c
4.24c
2.31c
2.43c
10.2c
11.2c
10.40c
11.06c
38.0c
38.1c
29.51c
32.6c
• Means followed by the same letters are not significantly different
• at p≤:0.05 according to Duncan's Multiple Range test.
♣ Combined data 0f three years 1997/98-1998/99
Table 5.Effect of different pollen grains on fruit characters of MWK and MWL date
palm cultivars (Rutab stage) ♣
Fruit
Ma
le
Length
(cm)
Diameter
(cm)
Weight (g)
Volume
(cc)
Moisture (%)
TSS (%)
K
L
K
L
K
L
K
L
K
L
K
L
A
3.30
b
4.13
b
2.28
b
2.29
b
8.09
b
8.15
b
0.2b
8.24
b
11.2
b
11.4b
63.0b
75.9b
B
3.54
a
4.41
a
2.42
a
240
a
9.05
a
9.26
a
9.16
a
9.23
a
12.3
a
12.5a
74.29a
84.3a
C
3.60
a
4.40
a
2.46
a
2.48
a
9.07
a
9.24
a
9.17
a
9.24
a
13.4
a
13.5a
75.3a
85.2a
D
2.20
c
3.84
c
2.10
c
2.14
c
2.01
c
7.03
c
7.5c
7.60
c
9.17
c
9.20c
59.2c
70.2c
E
3.30
b
4.12
b
2.22
b
2.26
b
8.05
b
8.19
b
8.2b
8.30
b
11.2
1b
11.25b
64.2b
76.3b
F
3.55
a
4.41
a
2.41
a
2.47
a
9.13
a
9.32
a
9.2a
9.31
a
12.0
a
12.14a
77.3a
85.3a
G
2.31
c
3.83
c
2.01
c
2.10
c
7.16
c
7.21
c
7.4c
7.46
c
9.23
c
9.23c
57.0c
71.3c
• Means followed by the same letters are not significantly different at
p≤:0.05 according to Duncan's Multiple Range test.
♣ Combined data 0f three years 2001/2002-2002/2003
144
Table 6. Effect of different pollen grains on maturity period and Seed
weight of MWK and MWL date palm cultivars
Maturity period
♣
Male
Seed weight
♣
2001/20022002/2003
Khalal
Rutab
K
L
K
L
K
L
A
162b
171b
0.90b
0.92b
0.90b
0.81b
B
161b
173b
0.71c
0.79c
0.71c
0.65c
C
150c
160c
0.72c
0.78c
0.72c
0.67c
D
173a
188.7a
0.90b
0.92b
0.90b
0.80b
E
172a
188.0a
1.0a
1.20a
1.0a
090a
F
152c
161c
0.70c
0.77c
0.70c
0.64c
G
174a
186a
1.0a
1.24a
1.0a
0.90c
• Means followed by the same letters are not significantly different at
p≤:0.05 according to Duncan's Multiple Range test.
♣ Combined data 0f three years 2001/2002-2002/2003
Table 7. Effect of different pollen grains on yield of MWK and MWL palm cultivars
Yield (kg/tree)
Combined yield
♣
2000/2001
2001/2002
2002/2003
K
L
K
L
K
L
K
L
A
285b
289b
282b
296b
292b
3033b
286.3b
301b
B
309ab
293ab
305ab
312ab
311ab
324ab
310.7ab
324.3ab
C
320a
337a
317a
332a
325a
343a
320.7a
328a
D
240c
252c
235c
249c
251c
360c
242c
253.7c
E
280b
305b
271c
300b
292b
315b
221b
306.7b
F
318a
330a
315a
326a
324a
340a
319a
332a
G
231c
257c
225c
251c
243c
262c
233c
256.7c
Male
•
Means followed by the same letters are not significantly different at
p≤:0.05 according to Duncan's Multiple Range test.
♣ Combined data 0f three years 2001/2002-2002/2003
145
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146
PP 03
Effect of some Artificial Ripening Treatments used as
Environmentally Safe on fruit quality and storage ability of
"Sewy" Dates.
El-Kafrawy, T.M. (1) and N. Abdel- Hamid (2)
(1) Central Laboratory For Date Palm Research and Development Agricultural
Research Center. Egypt.
(2) Faculty of Agric. Ain Shams Univ., Cairo, Egypt.
E-mail: tarek3lkafrawy@yahoo.com
Abstract
This study has been conducted to evaluate the effect of using different artificial
ripening treatments on "Sewy" dates fruits, and studying some physical and chemical
changes during different storage periods. Ripening treatments were applied by three
different methods: sun ripening, oven heat ripening and calcium carbide ripening
during 2004&2005 seasons. Results revealed that the three used ripening methods
improved obviously fruit quality, reduced the percentages of decayed fruits and weight
loss, during the different storage periods in all the studied treatments. Using the sun
drying method, obviously improved the studied physical qualifications especially flesh
weight %, seed weight % color and ripening stage. Also, it improved the chemical
properties like the total soluble solids, total tannins and the total sugars percentages.
Generally, we can advise that "Sewy" dates (semi dry cultivar) had high storage ability
lasted for one year with lesser percentages of decay and weight loss percent with good
quality and taste of the fruits. Treating the fruits by sun drying was the most promising
trial, as it was superior in improving fruit qualifications.
Introduction
Date palm “phoenix dactylifera” is one of the important fruits especially in the Arab
world. “Sewy” date is one of the semi dry dates and the most economic varieties in
Egypt. The traditional semi dry date fruits technologies often result in postharvest
losses because drying date fruits under a controlled conditions results in a
147
contamination with dirt, dust, attacks by insects rodents and prolonged drying time (1
– 2 week) Ali, 1999.
In addition, unsuitable climatic conditions for normal ripening (heat and relative
humidity ) , led us to find out a good methods for artificial ripening for this cultivar.
The need to artificial ripening is very important to improve fruit quality of “Sewy”
dates which harvested at maturity stage (El- Hammady et al, 2002),.They concluded
that ethrel or calcium carbide treatments hastened the ripening process of date fruits
and produced fruits with good quality of “Sewy” date fruits. Cold storage is essential
to minimize biochemical changes and enhancing quality, can extend the storage life of
date fruits (Bangamin 1985, Kamal 1995 and El-Zayat et al 2002).
The main objective of this work was study the effect of some safety artificial
ripening methods, i.e. (sun-dried, calcium carbide-ripened and oven-dried) on fruit
quality during cold storage at 5± 2 ºC and 85% relative humidity (R.H). on “Sewy”
dates (at kalal stage).
Materials and Methods
This study was conducted during two successive seasons of 2004 and 2005 on
“Sewy” dates (semi-dry cultivar) This work was to study the effect of some artificial
ripening agents, i.e. (sun-dried, calcium carbide-ripened and oven-dried ) on fruit
quality during cold storage at 5±2ºC and 85% relative humidity (R.H).
“Sewy” dates were harvested at maturity (Khalal stage) in the experimental
farm at Horticulture Research Institute, Agricultural Research Center, Giza
Governorate, Egypt.
148
Fruits were subjected to be ripened by one of the following treatments:
A group of six bunches were subjected to ripening by exposure to sun for nearly
three weeks, till the dates were semi dry, as the latest stage of the product.
A group of six bunches were put in the ripening box with calcium carbide at a rate
of 5 g per m3 of box space for 24 hr. then removed from the box to be held at room
temperature, and dried by heat oven at 50ºC till the dates were semi dry, as the
latest stage of the product.
A group of six bunches were ripened by exposure to heat oven at 50 ºC for 10 days
for 7 hr. ( nearly 72 hr.) and removed from the exposure to be held at room
temperature, till the dates were semi dry, as the latest stage of the product.
Post-harvest treatments:
Fruits packed in three boxes, representing three replicates, were used to record the
weight loss, before and during storage from each treatments.
Fruits packed in three boxes, representing three replicates, were used for the
determination of physical and chemical constituents, before and during storage from
each treatments.
Fruits packed in three boxes, representing three replicates, were used to record
decay Percentage, before and during storage from each treatments.
Fruits packed in three boxes, representing three replicates, were used during storage
from each treatment.
Cold storage experiment:
At each harvest date six cartons of fruits from the experimented treatments were
kept at 5±2ºC and 85% relative humidity (R.H).
Methods of analyses
Physical and chemical properties of the fruits were determined every (60) days
during cold storage, as follow:
Physical properties: for all treatments, 3 samples each of 50 fruits were taken from
each replicate for the determination of Decay Percentage: The discarded fruits
included all the injured or spoiled fruits resulting from fungus or bacteria, shriveling
and other various defects, were calculated and expressed as decay percentage.
Loss in fruit weight: percentage of loss in fruit weight was calculated as follows:
149
flesh weight %, seed weight %, and Fruit firmness were estimated in 60 dates by
(Lifra texture analyzer) instrument using a penetrating cylinder of 1 mm of diameter to
a constant distance of 3 mm inside the skin by a constant speed 2 mm parsec and the
peak of resistance was recorded per gram. Rind color was estimated by a Hunter
colorimeter type (Dp- 9000) and the data was expressed as colour concentration and
recorded as described by Mc Guire (1992).
• Chemical properties: titratable acidity %, was measured by titration against 0.1 N.
Sodium hydroxide using phenolphthalin as an indicator. A.O.A.C (1990). Tannins
content was determined according to the method of Yeshajahu and Clifton (1977)
(as gm gallotanninc acid / 100gm dry weight). Total soluble solids % (TSS) were
estimated by using the Abbe digital refractometer according to A.O.A.C (1990).
Reducing and total sugars: were colourimetrically determined as (g glucose / 100 g
dry weight ) However, Non-reducing sugars were calculated by subtract reducing
sugars from total sugars according to the method of Lane and Eynon described in
A.O.A.C (1985).
Statistical analysis: data obtained in the two studied seasons were subjected to the
analysis of variance according to Snedecor and Cochran (1980) and the least
significant ranges (L.S.R) were used to differentiate the obtained values.
Results and Discussions
1. Fruit decay%
Data tabulated in Table (1) show the effect of artificial ripening methods on fruit
decay % which increased with advanced in cold storage periods regardless of the used
treatments. No fruit decay % was observed till 90 and 120 days in first and second
seasons, respectively. However, oven-dried fruits exhibited the least values of fruit
decay during all cold storage periods followed by sun-dried fruits whereas calcium
carbide ripened-fruits exhibited the higher fruit decay %. Generally, it could be noticed
that the three methods of artificial ripening produced stored fruits for 360 days without
reaching 50 % decay. After 360 days of cold storage sun-dried fruits recorded 36.06 %
decay against 48.67 % for calcium carbide-fruits and 23.15 for oven-dried fruits (data
of first season). The reduction of fruit decay was observed with oven-dried fruits than
150
other treatments attributed the inhibition of enzymes activity and minimize fruit decay.
From the obtained data, it could be noticed that “Sewy” oven-dried fruits could be
stored for 270 days with less fruit decay than 10 % as compared with 180 days for sundried or calcium carbide ripened fruits. The same trend of results was also noticed in
the second season of the study.
The previous results are in partial agreement with those found by El-Hammady et al
(2002) on “Hayany” dates The same author (2003) reported that “Sewy” dates treated
with ethephon and calcium carbide of room ripening were effective in reducing decay
% fruits when compared with the control.
2. Weight loss%:
It is evident from data tabulated in Table (2) that different artificial ripening
methods greatly affected weight loss % as it generally increased with advanced cold
storage periods with all artificial ripening methods. After 360 days of cold storage
weight loss % reached to 5.82, 7.97 and 4.14 for sun-dried, calcium carbide-ripened
and oven-dried fruits, respectively (data of first season).However, oven-dried “Sewy”
dates exhibited the least values of weight loss followed by sun-dried fruits whereas
calcium carbide- ripened fruits recorded the highest values of weight loss with
significant differences between them. In some cases, differences between oven or sundried fruits in their weight loss were negligible. It is well known that in dried fruits
weight loss was mostly due to consumption of sugars and partially due to water loss
(Hulme, 1970). From the obtained data and fruit decay % previously discussed, it is
clear that oven-dried “Sewy” fruits were superior than sun-dried or calcium carbide
ripened fruits and stored well for 360 days without losses in fruit quality. The same
trend of results was also noticed in the second season of the study.
The previous results are in partial agreement with those found by El-Hadidy (2004)
who studied the effect of common ripening or of artificial ripening process on
handling and storability of “Sewy” semi dry dates. The change in weight loss % at 0 or
5ْ C ± 2 and 90 RH% for 5 months were determined. Generally, artificial ripening of
“Sewy” dates decreased weight loss. There was an evident increase in weight loss with
advanced cold storage periods.
151
3. Flesh weight%:
Data tabulated in Table (3) show the effect of artificial ripening methods on the
change of flesh %. It is clear that with advanced cold storage durations, a slight
decrease in flesh % was noticed with all treatments. However, high flesh % was
obtained with sun-dried fruits with significant differences with other two artificial
ripening methods. After 360 days of cold storage flesh % was decreased from 88.29 to
84.63, from 87.81 to 82.57 and from 86.72 to 81. 86 % for sun-dried, calcium carbideripened and oven- dried fruits, respectively (data of first season). The decrease in flesh
% with advanced in cold storage durations was attributed to both losses of sugars
during respiration pathway and water losses from fruit tissues. The same trend of
results was also found in the second season of study.
The previous results are in partial agreement with those found by Salah El Din
(1967) worked with “Sewy” dates from Giza and Siwa, and Amry dates from Sharkeya
and Kalyobeya were investigated during maturation and ripening. Any change in such
characteristics during and after either sun drying or dehydration, and throughout
prolonged storage at room temperature. A pronounced decrease was found in flesh
weight towards maturation and ripening and even after processing and during storage.
4. Seed weight %
Data illustrated in Table (4) show the effect of artificial ripening methods on seed
weight %. It is clear that with advanced cold storage durations a general increase in
seed weight % of “Sewy” dates was obtained. After 360 days of cold storage, the
highest seed weight % was obtained with oven-dried followed by calcium carbideripened fruits whereas sun-dried fruits recorded the least seed weight %. However, no
significant differences were obtained between calcium carbide-ripened fruits and
oven-dried fruits in their seed weight %.
The increase in seed weight % with advanced cold storage duration could be
attributed to the decrease in pulp % as a result of water loss and respiration process.
The same trend of results was also found in the second season of study.
The previous results are in partial disagree with those found by Salah El Din (1967)
who found that “Sewy” dates from Giza and Siwa, and Amry dates from Sharkeya and
Kalyobeya were investigated during maturation and ripening. No changes in such
152
characteristics during and after either sun-drying or dehydration, and throughout
prolonged storage at room temperature were detected. A pronounced decrease was
found in seed weight towards maturation and ripening and even after processing and
during storage.
5. Fruit rind colour attributes:
Data tabulated in Table (5) show the effect of artificial ripening methods on fruit
rind colour attributes (lightness, Hue- angle and chroma)
Regarding fruit lightness, it is clear that it decreased with advanced cold storage
periods although some increase was noticed in some cold storage durations. Sun-dried
fruit lightness was decreased from 28.85 to 24.31 after 360 days of cold storage,
calcium carbide-ripened fruits lightness decreased from 29.67 to 25.07 whereas ovendried lightness fruits decreased from 28.65 to 24.09 At the end of cold storage periods,
(first season) No significant differences were obtained between calcium carbideripened fruits and oven-dried fruits whereas, sun dried fruits exhibited the least values
of lightness. The same trend of results was also found in the second season of study.
Regarding Hue-angle data, it showed that an evident decrease in Hue-angle with
advanced cold storage with all treatments. The decrease in rind colour (Hue-angle)
was from 56.50 to 35.84, 67.66 to 39.97 and from 71.87 to 36.31 for sun-dried,
calcium carbide-ripened and oven-dried fruits, respectively. After 360 days of cold
storage, calcium carbide-ripened fruits exhibited the highest values of Hue-angle
compared with other treatments.
Data of rind colour chroma showed an evident decrease with advanced cold storage
durations with all treatments. The decrease in rind colour (Chroma) was from 8.89 to
2.59, 9.40 to 2.82 and from 5.58 to 1.18 for sun-dried, calcium carbide-ripened and
oven-dried fruits, respectively. The same trend of results was also found in the second
season of study.
The previous results are in partial agreement with those found by Nezam El-Din
(1996) on “Hayani”. Nezam El-Din, and Azza Kamal (1997) on “Amhat” dates. In
addition, Nezam El-Din (1998) on “Bent Aisha” dates and El-Hadidy (2004) reported
that the effect of artificial ripening process on handling and storability of “Sewy” semi
dry dates. The change in fruit colour during at 0 or 5ْ C ± 2 and 90 RH % for 5 months
153
was determined. Generally, it could be observed that artificially-ripened fruits had
good colour.
6. Fruit texture (g /cm2)
Data illustrated in Table (6) show the effect of artificial ripening methods on fruit
texture (g /cm2) at 3 mm It is clear that fruit texture of “Sewy” dates at 3 mm depth it
is evident that fruit texture was greatly decreased with advanced cold storage
durations. At first season (2004 season), sun dried fruit texture was decreased from
45.0 to 5.00 after 360 days in cold storage. However, calcium carbide-ripened fruit
texture was decreased from 52.33 to 10.67 against 53.36 to 7.33 in oven dried fruits.
Generally, it is evident that calcium carbide ripened fruits exhibited higher texture
values than oven or sun dried fruits during all cold storage duration. Significant
differences were clear between calcium carbide ripened fruits and oven or sun dried
fruits, however, significant differences were sometimes appeared between oven or sun
dried fruits. Losses of fruit texture with advanced cold storage durations were
attributed to the increase in enzymes activity including enzyme pectine substances
which turn insoluble pectine to soluble pectine leading to fruit softening. The same
trend of results was also obtained in the second season of study but values of fruit
texture generally were higher in the second season than in first one.
However, with advanced cold storage, significant differences between oven and
sun-dried fruits disapeared whereas calcium carbide dried fruits still recording the
higher significant values of fruit texture.
This finding confirmed the previous results obtained by El-Zayat et al (2002) when
testing some methods to preserve quality of semi dried dates after being dried
traditionally by exposure to sun. Fruit firmness tended to decrease gradually with time
advance in cold storage in both seasons beginning with (72.4 -51.4 gm) & from (36.1 27.1 gm) by end of storage at two seasons, “Sewy” dry dates could be kept for 3
months at 5ºC in good conditions. Moreover, El-Hammady et al (2003) and El-Hadidy
(2004) indicated that, the effect of common ripening or of artificial ripening process on
handing and storability of “Sewy” semi dry dates. The change in fruit texture during at
0 or 5ْ C ± 2 and 90 RH % for 5 months were determined. Generally, artificial ripening
of “Sewy” dates decreased artificial ripening produced fruits with high texture.
154
Chemical properties:
1 Total soluble solids %
Data tabulated in Table (7) show the change in T.S.S % as affected by artificial
ripening methods. At the beginning, the results showed many obvious effects of using
different artificial ripening methods either by using sun ripening or Calcium Carbide
application or by using the heat of the oven.
It showed that total soluble solids of the fruits during maturity and (khalal stage)
and when harvested, recorded the values of only (37.70 to 42.9%). After applying the
different treatments of artificial ripening, the results showed great variations in the
values of the total soluble solid contents as it reached the highest values when the
fruits were semi dry, as the latest stage of the product. We also found significant
variations between the three used methods of artificial ripening fruits treated by sun
ripening represented the highest contents of total soluble solids (69.25 %) of sun-dried
(66.70 %) while the least values (63.64 %) were represented when fruits were treated
with oven heat.
At the beginning of cold storage durations, the higher value of T.S.S (69.25 %) was
recorded by sun-dried fruits followed by calcium carbide ripened fruits (66.70 %) and
oven-dried fruits (63.64 %) with significant differences between them. However,
a slight increase in T.S.S % with advanced cold storage durations were noticed. At
the last time after 360 days T.S.S contents of sun-dried (69.88 %) was followed by
calcium carbide- ripened fruits (67.30 %) while the least values (64.68%) for ovendried fruits. This finding could be attributed to the reduction in dried fruit respiration
and reduction in sugar consumption. The same trend of results was nearly found in the
second season of study.
This finding confirm the previous results obtained by El-Zayat et al (2002) when
testing some methods to preserve quality of semi dried dates after being dried
traditionally by exposure to sun. TSS tended to increase with advance in storage tim.
This increase in cold store, TSS average value started at 76.7 % and reached 79 % by
end of stay “Sewy” dry dates could be kept for 3 months at 5ºC in good conditions.
Moreover, El-Hammady et al (2003) on “Sewy” semi dry dates.
155
2. Total acidity %
Data presented in Table (8) show the effect of artificial ripening methods on total
acidity % .A general increase in fruit acidity with advanced cold storage durations with
all treatments was obtained.
Fruit acidity of sun-dried fruits was increased from 0.130 % at the beginning of cold
storage to 0.43 % after 360 days of cold storage. However, acidity of calcium carbide
ripened fruits increased from 0.08% to 0.387% and oven- dried from 0.07% to
0.392%. In most cases, no significant differences were noticed between calcium
carbide-ripened fruits and oven- dried fruits in their acidity contents. The same trend
of results was also obtained in the second season.
The previous results are in partial agree with those found by El-Ghazali and Hussin
(1999) on “Sakkoti”, “Bartamuda” and “Gondalla” . In addition, El-Zayat et al (2002)
when testing some methods to preserve quality of semi dried dates after being dried
traditionally by exposure to sun, that acidity tended to increase with time in cold store,
and these respective values were (0.23 % and 0.30 %) “Sewy” dry dates could be kept
for 3 months at 5ºC in good conditions.
3. Total tannins (mg / 100g dry weight)
Data presented in Table (9) show the effect of artificial ripening methods on total
tannins content. At the beginning, the results showed many obvious effects of using
different artificial ripening methods either by using sun ripening or calcium carbide
application or by using the heat of the oven.
When comparing these results with the findings, it showed that total tannins of the
fruits during maturity (khalal stage) and when harvested recorded the values of only
1.86 to 2.19 (mg / 100g dry weight). In the second time, after applying the different
treatments of artificial ripening, the results showed great variations in the values of the
total tannins contents as it reached the lowest contents when the fruits were semi dry,
as the latest stage of the product.
It is clear that total tannins were greatly decreased with advanced cold storage
durations regardless of the artificial ripening methods. However, sun-dried fruits
exhibited the least values of total tannins than other treatments. In most cases, no
significant differences were noticed between calcium carbide-ripened fruits or oven156
dried fruits. The reduction of total tannins was from 0.362 to 0.034- mg / 100g dry
weight, from 0.394 to 0.067 mg / 100g dry weight and from 0.416 to 0.059 mg / 100g
dry weight for sun-dried, calcium carbide and oven dried fruits, respectively. The same
trend of results was also found in the second season of study.
This finding confirms, the previous results obtained by Elham, Z. Abd El-Motty
(1995) on “Hayany” and “Sewy” dates. In addition, El-Samahy et al (2006) for
controlling the loss during maturity stages of semi-dry date fruits and prolonging the
storage period at room temperature of these fruits, Egyptian (Amry and Sewy) date
fruits at Khalal stage were treated with steam for 15 min and potassium sorbate at
different concentration, then dried at 50º C for 48 h. Generally, during storage period
(6 months), there was no significant difference in tannins contents.
4. Reducing sugars% (g/100g dry weight)
Data tabulated in Table (10) show the change in reducing sugars % as affected by
artificial ripening methods of “ Sewy” dates stored at 5ºC ± 2 and 85% RH, during
2004 and 2005 seasons.
At the beginning, the results showed many obvious effects of using different
artificial ripening methods either by using sun ripening or calcium carbide application
or by using the heat of the oven.
When comparing these results it showed that reducing sugars % of the fruits during
maturity (khalal stage) and when harvested recorded the values of only 17.08 to 19.24
%. after applying the different treatments of artificial ripening, the results showed
great variations in the values of the reducing sugars % as it reached the highest
contents when the fruits were semi dry, as the latest stage of the product. We also
found significant variations between the three used methods of artificial ripening,
fruits treated by sun ripening represented the highest contents of reducing sugars %
(50.16 %) followed by those treated by calcium carbide (47.36%) while the least
values (44.42 %) were represented when fruits were treated with oven heat. The same
trend of results was nearly found in the second season of the study.
At the beginning of cold storage durations, the higher value of reducing sugars
percentage in the first season (50.16 %) was recorded by sun-dried fruits followed by
calcium carbide-ripened fruits (47.36%) and oven-dried fruits (44.42 %) with
157
significant differences between them. However, a slight increase in reducing sugars %
with advanced in cold storage durations was noticed. This finding could be attributed
to the reduction in dried fruit respiration and reduction in sugar consumption. The
same trend of results was nearly found in the second season of study.
These previous results are in accordance with those obtained by Elham, Z. Abd ElMotty (1995) on “Hayany” and “Sewy” dates, Moreover, El-Ghazali and Hussin
(1999) “Sakkoti”, “Bartamuda” and “Gondalla” are excellent dry date cultivars, grown
at Aswan Governorate. reducing sugars were determined in fresh date fruit (Tamar),
after sun-drying for one months and mechanical drying at 70ºC for 24 hrs. reducing
sugar was higher in “Sakkoti” than “Bartamuda” and “Gondaila”. After, sun-drying,
reducing sugar decreased.
5. Non- reducing sugars% ( g/100g dry weight).
Data tabulated in Table (10) show the changes in non-reducing sugars % as affected
by artificial ripening methods.
At the beginning, the results showed many obvious effects of using different
artificial ripening methods either by using sun ripening or calcium carbide application
or by using the heat of the oven.
When comparing these results with the findings show that non-reducing sugars % of
the fruits during maturity (khalal stage) and when harvested recorded the values of only
20.58 to 22.43 %. After applying the different treatments of artificial ripening, the results
showed great variations in the values of the Non- reducing sugars % contents as it reached
the slightly less contents when the fruits were semi dry, as the latest stage of the product.
At the beginning of cold storage durations, the higher value of non-reducing sugars
% in the first season (18.31%) was recorded by sun-dried fruits followed by ovendried fruits (18.10%) and calcium carbide ripened fruits (18.00%) with no significant
differences between them. However, a slight decrease in non-reducing sugars % with
advanced cold storage durations were noticed. This finding could be attributed to the
reduction in dried fruit respiration and reduction in sugar consumption. The same trend
of results was nearly found in the second season of the study.
These previous results are in accordance with those obtained by Elham, Z. Abd ElMotty (1995) who designed to improve the ripening methods of both “Hayany” and
158
“Sewy” dates, respectively. In addition, El- El-Hadidy (2004) found that The effect of
artificial ripening process on handing and storability of “Sewy” semi dry dates
decreased with the advance in non-reducing sugars at 0 or 5ْ C ± 2 and 90 RH % for 5
months were determined.
6. Total sugars% ( g/100g dry weight)
Data tabulated in Table (10) show the change in total sugars % as affected by
artificial ripening methods.
At the beginning, the results showed many obvious effects of using different
artificial ripening methods either by using sun ripening or Calcium Carbide application
or by using the heat of the oven.
When comparing these results with the findings show that total sugars % of the
fruits during maturity (khalal stage) and when harvested recorded the values of only
38.35 to 41.69 % g/100g fresh weight. After applying the different treatments of
artificial ripening, the results showed great variations in the values of the total sugars
% as it reached the highest contents when the fruits were semi dry, as the latest stage
of the product. We also found significant variations between the three used methods of
artificial ripening. Fruits treated by sun ripening represented the highest contents of
total sugars % (69.72 %) followed by those treated by calcium carbide (65.36%) while
the least values (62.52%) were represented when fruits were treated with oven heat
ones of the first season.
At the beginning of cold storage durations, the higher value of total sugars % in the
first season (69.72 %) was recorded by sun-dried fruits followed by calcium carbide
ripened fruits (65.36%) and oven-dried fruits (62.52%) with significant differences
between them. However, a slight increase in total sugars % with advanced cold storage
durations were noticed. This finding could be attributed to the reduction in dried fruit
respiration and reduction in sugar consumption. The same trend of results was nearly
found in the second season of the study.
These previous results are in accordance with those obtained by Salah El Din (1967)
on “Sewy” and “Amry” dates Moreover, El-Hammady et al (2003) on “Sewy” dates
and El-Hadidy (2004) when studied the effect of artificial ripening process on handling
and storability of “Sewy” semi dry dates.
159
References
[1] Ali, G.A. (1999) Future prospects of date palm by products and residues in Sudan. Tecx.
[2] Report, FAO. Proceeding of the Regional work shop Date palm postharvest processing
[3] Technology Tehar, Eran 11-14.
[4] A.O.A.C. (1985). Association of Official Agricultural Chemists, Official Methods of
Analysis, Benjamin Franklin Station Washington 14 th. Ed. PP 494-500 D. C,U.S.A.
[5] A.O.A.C. (1990) Association of Official Analytical Chemists. Official Methods of
Analysis. Benjamin Franklin Station, Washington, 4.D.C USA.
[6] Bengamin, N.D.; M.S.AL-Khalid; H.R.Shabana and A.S. Moroki,(1985) The
effect of cold storage conditions of six dates fruit cultivars at rutab stage. Date
palm Journal 4 (1): 1 – 18
[7] El-Hadidy, G.A.E.(2004) Safe environmental treatments for handling semi dry
date. M. Sc Thesis, Fac. Agric., Ain Shams University, Egypt.
[8] El-Hammady, A.M.; Youssef, M. and Abdallah, A.S. (2002) Yield and quality of
“Sewy” dates as effected by bunch thinning. Journal of Environmental Science
vol: 5 no 4 December, 2002 (52) Egypt.
[9] El-Hammady, A.M.; Montasser, A. S.; Abdel-Hamid, N.; Nageib, M. M. and
Elham Zinhoum (2002) Artificial ripening of “Hayany” date fruits. Journal of
Environmental Science vol: 5 no 3 December, 2002 (35) Egypt.
[10] El-Hammady, A.M. ; Montasser, A.S.;
Abdel-Hamid, N. ; Nageib M. and
Elham Zinhoum (2002) Ripening of “Seewi” date fruits as affected with postharvest application of ethephon or calcium carbide. Journal of Environmental
Science vol: 5, No. 3 December, 2002 (34) Egypt.
[11] El-Hammady, A.M.; Abdel-Hamid, N.; El-Orabi, S. and Gehan El-Hadidy (2003)
environmentally safe treatments for ripening and storage of “Sewi” semi dry
dates. Journal of Environmental Science vol: 7 no (1) December, 2003 (11) Egypt.
[12] Elham, Z. Abd El-Motty (1995) physiological studies on ripening and storage of
dates M.Sc. Thesis of Faculty of Agriculture Ain shams University, Egypt.
[13] El-Samahy, S. K.; Shatter, A. A.; Abd El-Had, E.A. and Youssef K.M. (2006)
Some Technological Treatments for Improving the Keeping Quality of Semi-Dry
160
Dates in Egypt. International Conference on Date Palm Production &Processing
Technology 9-11 May 2006 - Muscat, Oman. Book of Abstracts
[14] El-Zayat, H.H.; Alma, H. and Hassam, A.M. (2002) Testing some methods to
preserve quality of semi dried dates. Journal of Agriculture Research. Tanta
University, 28 (3/11).
[15] El-Ghazali, M. N. and Hussin, F. (1999) Effect of sun-drying and mechanical
drying on chemical composition and lipid, phospholipid fractions of Aswan dryDates Zagazig J. Agric. Res. Vol. 26 No. (5) 1303-1323. Egypt.
[16] Hulme, A.C. (1970) The Biochemistry of fruits and their products. Vol. 1, Chap.
16 pp: 475-514, Academic Press, London, and New York
[17] Kamal H.M. (1995) Effect of cold storage temperatures on storability and quality
of date palm fruits. Bull. Fac. Agric., Univ. Cairo, 46 (2): 265-276.
[18] Mc Guire, R.G. (1992) Reporting of objective color
measurements. Hurt
Science. Vol. 27 (12), Dec.
[19] Nezam El-Din, A. (1998) Effect of some technological treatments on ripening
and drying of soft date “Bent Aisha” variety. Egypt. J. Agric. Res., 76 (1), 1998.
[20] Nezam El-Din, A. (1996) Effects of Microwave Treatment on Khalal stage of
“Hayani” dates Egypt. Food Sci., 14, No. 1, pp. 61-69
[21] Nezam El-Din, A. and Azza, K. (1997) Chemical and technological study on
Egyptian Amhat date Effect of temperature on ripening of khalal dates. Egyptian
journal of Agricultural Research, 75(4): 1113-1122.
[22] Salah El-Din, I. K. (1967) Factors influencing the quality of dehydrated dates.
M.Sc. Thesis of Faculty of Agriculture Cairo University, Giza.
[23] Snedecor, G.W. and Cochran, W.G. (1980) Statistical methods, Oxford and
J.B.H pup co. Publishing 6th edition. Press Ames Iowa. U.S.A, PP.593
161
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162
PP 04
Effect of time and patterns of fruit thinning on yield and fruit
quality of Saidy date palm cultivar .
El-Salhy A.M.*, A.A. El-Bana**, H.A. Abdel-Galil* and E.F.S. Ahmed**
*Horticultural Dept., Fac. Agric., Assiut University, Assiut, Egypt.
**Central Lab. for Date Palm Res. & Develop Agric. Res. Centre, Giza, Egypt.
E-Mail: foudaemad@yahoo.com
Abstract
The present study was carried out during 2004, 2005 and 2006 growing seasons at
The Agricultural Research Station that located at El-Kharga Oasis, New Valley
Governorate the present experiment aimed to study the effect of different methods and
dates of fruit thinning on yield and fruit quality of saidy date palm cultivar.
The obtained results indicated that fruit thinning by either removing or cutting back
40% of strands after four weeks gave the least fruit set percentage. Also, fruit thinning
after eight weeks of pollination by any methods or rate decreased the fruit set
percentage compared to un thinned. The reduction in fruits weight/bunch was
increased as the thinning degree increased. However, thinning 20% of fruit after four
weeks from pollination by removing the entire strands, insignificantly decreased the
fruits weight/bunch as comparing to unthinning one (control). Increasing the thinning
intensity as cutting back or removing the spikelets of bunch was followed by
significant decrease in fruits weight/bunch. Cutting back at 20-40 % of the spikelet tips
of each bunch was more effective in fruits weight/bunch reduction than removing 2040% of the entire spikelet. The best results dealt with fruit physical properties were
obtained by thinning 40 % of fruit either cutting back or removing of strands after four
weeks of pollination. Such improvement of fruit physical properties due to increasing
the fruit weight and size that occurred as response to fruit thinning especially at early
stage of fruit growth and development. It could be stated that there is a positive
correlation between fruit weight and thinning time in the some cultivar. All fruit
thinning treatments significant increasing the total soluble solids percentage and total
reducing sugar percentages as compared to unthinning once. The highest values of
163
such traits were recorded when fruit were thinning at 40% either cutting back or
thinning out of strands after four weeks from pollination.
Introduction
Date palm has a great economical importance and agricultural uses throughout
humans history. In Egypt, distribution of date palms cover a large area that extends
from Aswan to the north Delta, in addition to the oasis of Kharga, Siwa, Bahrya,
Dakhla and Farafra (Abd El-Azim and Merie, 1961).
Among all the Egyptian Governorates, New Valley ranked the fourth position
after Sharkia, Behaira and Aswan according to the acreage and the number of
females, since they are 12736 feddans and 907694 palms, respectively. This area
produced 72418 ton of fruits. Saidy date palm is covered all the acreage of the total
cultivated area in such governorate where the present investigation was carried out.
It is the most important cultivar of semi-dry dates and is very demand in the local
and foreign markets.
Several efforts have been accomplished to improve date palm production through
facing production problems and improving agricultural practices, i.e. efficiency of
pollination process, horticultural practices and suitable fertilization program
Fruit thinning is one of the major practices that often helps in overcoming the
alternate bearing, enhances fruit quality of dates and reduces compactness of fruit
bunches, it also adequately increases the flowering of the following season (Hussein,
1970 and Nixon & Carpenter, 1978).
Materials and Methods
This study was conducted in date palm Research Farm in Agricultural Research
Station, at El-Kharga Oasis, New Valley Governorate, Egypt, during three successive
growing seasons 2004, 2005 and 2006, on 35 years old saidy date palm cultivar (as
semi dry date palm cv.)
Eight palms in the same age and uniform in growth and bearing of approximately
the same number of spathes were selected. Pollination was uniformed in respect of
source and method to avoid residues of metaxenia.
Nine thinning treatments are included in the present experiment arranged as follows:
Control (no thinning).
164
1- Removing 20% of the entire spikelet from bunch center after four weeks of pollination.
2- Removing 40% of the entire spikelet from bunch center after four weeks of pollination.
3- Cutting back 20% of spikelet tips of each bunch after four weeks of pollination.
4- Cutting back 40% of spikelet tips of each bunch after four weeks of pollination.
5- Removing 20% of the entire spikelet from bunch center after eight weeks of pollination.
6- Removing 40% of the entire spikelet from bunch center after eight weeks of pollination.
7- Cutting back 20% of spikelet tips of each bunch after eight weeks of pollination.
8- Cutting back 40% of spikelet tips of each bunch after eight weeks of pollination.
The previous fruit thinning treatments were applied on the same palm.
The
experiment was arranged in a complete randomized block design with eight
replications of one bunch each.
Measurements:
A – Yield:
1- Fruit set %
The number of fruit set was counted using 5 strands per spathe after ten weeks from
pollination. The percentage of fruit set was calculated according to the following
equations:
The percentage of fruit set was calculated using the following equation:
Fruit set % =
Number of setting fruits on the strand
x 100
Total number of flowers per the strand
2 - Bunch weight:
All bunches were harvested at tamar stage in the three experimental seasons, The
bunch weight (in kg) was determined then the average of yield /palm (Kg) for each
treatment. was recorded.
3- Fruit characteristics:
At harvest time, sample of 30 fruits were picked at random from each bunch for
determination of some physical and chemical fruit properties.
a- Physical fruit properties included:
- Fruit and seed weight (in g), then pulp percentage was calculated.
- Fruit length (L) and diameter (D) were measured by vernier caliper (in cm).
b- Chemical characteristics included:
165
- Percentage of total soluble solids by using a hand refractometer.
- Reducing, non-reducing and total sugars percentages were estimated according to
Land and Eynon official method outlined in the Official Methods of Analysis
(A.O.A.C., 1985).
Results and Discussion
1-yield index
Fruit set and Fruits weight/bunch are considered as index for date palm yield.
The effects of fruit thinning at different time and date on fruit set percentage of
saidy date palm cultivar during 2004, 2005 and 2006 seasons are given in Table (1). It
is worthy to mention that the results reacted almost similarly during the three studied
seasons. Nevertheless, it appears that there are differences between the three reasons.
Such finding might be attributed to the climatic and environmental conditions during
these seasons . These finding are in accordance with the results of Reuveni (1986) .
Date presented in previously table show clearly that fruit set percentage was
significantly affected by various fruit thinning treatments. Fruit thinning by either
removing 40% of entire strands (T2) or cutting back 40% of strand tips (T4) after four
weeks gave the least fruit set percentage during the three studied reasons.
Also, fruit thinning after eight weeks of pollination by any methods or rate had un
significant reduce on fruit set percentage when compared with unthinned ones
(control) during the three studied reasons.
That may be the outcome of some mechanical harms due to the removal or cutting
of some strands, and the effects of these harms on the physiological operations and
hormone stability, which leads to the decrease of the fruit set percentage.
The results indicated that fruit thinning at 40% after four weeks of pollination (T 2 &
T4) significantly decreased the fruit set percentage whereas fruit thinning either at 20%
after four weeks of pollination (T1 & T3) or 20-40% after eight weeks of pollination
(T5, T6, T7 and T8) had insignificant decrease compared to un thinned ones.
Fruits weight/bunch (Kg)
Fruits weight/bunch (Kg) is an indicator to the yield of palm trees because the
number of bunches on the tree was constant. Data regarding the effect of fruit thinning
at different time and rate on fruits weight/ bunch of Saidy date palm in 2004, 2005 and
166
2006 reasons are presented in Table (1) . The obtained data indicated that the different
fruit thinning treatments decreased fruits weight/bunch (Kg) than unthinning one
(control). The reduction in fruits weight/bunch was increased as the thinning degree
increased. However, thinning 20% of fruit after four weeks from pollination by
removing the entire strands (T1), insignificantly decreased the fruits weight/bunch as
comparing to un thinning one (control).
It could be concluded that this is a positive correlation between fruit set percentage
and fruits weight/ bunch.
So, the thinning of 40 % of fruit either by removing the entire strand after four
weeks of pollination (T2) , after eight week of pollination (T6) or by cutting back
strand tips after four weeks of pollination (T4) or after eight weeks of pollination (T8)
significantly reduced the fruits weight/ bunch composed with unthinning. Moreover,
all fruit thinning which done after eight weeks after pollination both 20 and 40% of
fruit , either removing or cutting back the spikelets of bunches had a significantly
reduction in fruits weight/bunch compared with unthinning one (control).
Generally, the reduction in the fruits weight/bunch due to thinning may be
attributed to the great reduction in number of fruits per strand, hence a total fruit per
bunch, In addition the insignificant reduce in the fruits weight/bunch due to the early
fruit thinning may be attributed to that the increase in fruits weight/bunch compensated
the reduction in number of fruits. This finding could be attributed to the increase in the
fruit retention number and increase the fruits weight/bunch as results of thinning which
reduce the competition between fruit as well as improve the supply of food material to
individual fruit and consequently improved fruits weight/bunch at harvest.
The positive effect of fruit thinning is improving the palm nutritional status gave
good food material, healthy and productivity palm, surely reflected on improving the
fruit retention percentage as well as increasing the fruit weight which consequently
improved the bunch and yield weight.
These findings are in agreement with those obtained by Hussein et al. (1992a&b),
Abdel-Hamid (2000) , Hammam et al , (2002, Bassal and El-Deeb (2002), Harhash
and Abdel-Nasser, 2007). and Marzouk et al. (2007)
167
2- Fruit quality
A- Physical fruit properties
Data present in tables (2) show the effect of fruit thinning methods and rates on
some physical fruit properties of saidy dates during 2004, 2005, 2006 and 2007
seasons It is clear from data that no major differences were detected between the
results that were obtained during three studied seasons It is clear from the previously
tables that all fruit physical characteristics in terms of fruit weight and fresh
percentage as well as fruit length were significantly increased than unthinning once
(control). Comparing the different time of thinning ,it was found that fruit thinning
early (after four weeks of pollination) was more effective compared to late fruit
thinning (after eight weeks of pollination) in increasing fruit weight as well as
improving other physical fruit traits.
It is clear from the prementioned table (2) that fruit weight was significantly increased
in response to any fruit thinning treatments compared with unthinning one ( control).
The heaviest fruit weight was significantly increased in response to fruit thinning by
cutting back 40% of strand tips after four weeks of pollination (T 4). Whereas the least
fruit weight was recorded on the unthinning bunches. These results were true during
the three studied seasons.
Such results could be due to the effect of thinning on reducing the number of fruits
per bunch without changing the number of leaves and consequently better supply of
food material (carbohydrates) that are manufactured in the leaves .
Moreover, the merits of the early fruit thinning in producing heaviest fruit weight
compared to the late one might be to the save of organic and mineral nutrients which
will exhaust by the thinned fruits.
Also, the date in Tables (2) show that the other physical fruit characteristics, i.e.
flesh percentage as well as fruit length ware reacted as fruit weight responded to fruit
thinning treatments during the three studied seasons .
The best results dealing with fruit physical properties were obtained by carrying out
fruit thinning by thinning 40 % of fruit either cutting back or removing of strands after
four weeks of pollination. Such improvement of fruit physical properties due to
increasing the fruit weight and size that occurred in response to fruit thinning
168
especially at early stage of fruit growth and development and development. It could be
stated that there was a positive correlation between fruit weight and thinning time.
These results are in line with those obtained by Hassablla et al (1983), Khalifa et al.
(1987), Hussein et al. (1992a&b), El-Kassas et al. (1995), Abdel-Hamed (2000),
Bassal and El-Deeb (2002), Hammam et al. (2002), El-Assar (2005) and harhash and
Abdel-Nasser (2007) .
The same finding was confirmed by Moustafa (1998b), Akl et al. (2004), Nirmaljit
et al. (2006), Tavakkoli et al. (2006) , Marzouk et al. (2007), and Behseresht et al.
(2007) who reported that thinned bunches at pollination or 20 days after pollination
with 20-40 % of total number of bunch strands by removing or reducing fruit set were
suitable for obtained heavy fruit with good physical quality in Sewy, Halawy, Haiany,
Khadrawi and Kabkab date palm cultivars
B - Fruit chemical constituents
The results obtained concerning the effect of time and methods of fruit thinning on
fruit chemical constituents of saidy date palm cultivar in 2004, 2005 and 2006 seasons
are listed in tables (3 & 4). It is noticed from these data that the chemical constituents
in dates pulp took similar trend in response to all tested fruit thinning treatments
throughout the successive seasons .It is evident from these data that the fruit thinning
treatments significantly improved the fruit chemical quality in terms of increasing the
total soluble solids and sugar contents compared to unthinning one.
Contrarily, data in table (3) reveal that the fruits produced from thinning bunches
had significantly lower moisture content than the unthinning ones In this respect
removing 40% of fruit by any methods gave the fruits which had the least moisture
percentage compared to removing 20% of fruits. So, removing of the entire strands
and cutting back strand tips had the same effect. These results were fruit during the
three studied seasons .
Such result is most important target since the reduce fruit moisture content in such
cultivar is very necessary due to improving the quality and resulted an increase in
pack able yield.
169
Such reduction might be attributed to adequate carbohydrates and other essentials
food fruits left to induce increase the fruit weight and size as well as hasten the
maturity which improved fruit quality and decreased the moisture content.
In addition, all fruit thinning treatments significant increasing the total soluble
solids percentage and total reducing sugar percentages as compared to un thinning
once. The highest values of such traits were recorded when fruit were thinning at 40%
either cutting back or thinning out of strands as well as at after four or eight weeks
from pollination compared with the other fruit thinning treatments.
As a general view from the previously data, it can noticed that the reducing and
total sugars took a similar tendency as response to fruit thinning treatments during the
three studied seasons. So, the fruit thinning treatments had no effect on the nonreducing sugar percentages in the fruits table (4).
So, it could be say that there is appositive relationship between fruit thinning time
and rates and total soluble solids and total sugar contents of dates pulp.
170
References
[1] A.O.A.C. Association of Official Agricultural Chemists. 1985. Official Methods of
Analysis A.O.A.C. Benjamin Franklin Station, Washington, DC, M.S.A., pp. 440-512.
[2] Abd El-Azim, A. and Merie, H. 1961. Date palm in Egypt. Ministry of
Agriculture, Egypt.pp 10-20.
[3] Abd El-Hamid, N. 2000. Effect of time, rate and patterns of thinning, leaf bunch
ratio and male type on “Zaghloul” date yield and quality. Arab, J. Agric. Sci. Ain
Shams Univ., Cairo, 8 (1): 305-317.
[4] Akl, A.M.; M.A. Ragab and A.Y. Mohamed. 2004. Yield and fruit quality of Sewy
date palms in response to some fruit thinning treatments. The Second Inter. Conf. on
Date Palm Faculty of Envir. Agric. Sci., El-Arish, Suez Canal Univ. Egypt. 6-8 Oct.
[5] Bassal, M.A. and M.D. El-Deeb. 2002. Effect of thinning and some growth
regulators on yield and fruit quality of Zaghloul date palm. Zagazig J. Agric. Res.
29 (6): 1815-1837.
1. Behseresht, R.R. Khademi and P. Bayat. 2007. The effects of bunch thinning
methods on quality and quantity of date palm cv .Kabakab the 4 th symosium on
Date Palm in Saudi Arabia. King Faisal Univ, Al-Hassa, 5-8 May p 117
[6] El-Assar, A.M. 2005. Response of "Zaghloul" date yield and fruit characteristics
to various organic and inorganic fertilization types as well as fruit thinning models
in a rich carbonate soil. J. Agric. Sci. Mansoura Univ., 30 (5): 2795-2814.
[7] El-Kassas, Sh.E.; T.K. El-Mahdy, A.A. El-Khawaga and Zynab Hamdy, 1995.
Response of Zaghloul date palms to certain treatments of pollination, flower
thinning and bagging .J. of Agric. Sci. 26 (4): 167-178.
[8] Hammam,M.S. Asma Sabour and sanaa Ebeed 2002 :Effect of some fruit thinning
treatments on yield and fruit quality of zaghloul date palm Arab Univ . J. Agric.
Sci. Ain Shams Univ., Cairo, 10 (1),261-271
[9] Harhash , M.M. and G . Abdel – Nasser (2007): Impacts of Potassium fertilization
and bunch thinning on zaghloul date palm The 4th symposium on date palm in
Saudi Arabia 5-8 May book of Abst. pp.70
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[10] Hassaballa, L.A., M.M. Ibrahim, M.M. Sharaf, A.Z. Abd El-Aziz and N.A, Hagagy.
1983 : Fruit physical and chemical characteristics of “Zaghloul” date cultivar in response
to some fruit thinning treatments. Annals. Agric. Sci. Moshtohor, Egypt, 20(3): 3-14.
[11] Hussein, F. 1970:
Effect of fruit thinning on size, quality and ripening of
"Sakkoti" dates grown at Aswan. Trop. Agric. 47 (2): 163-166.
[12] Hussein, M.A., S.Z. El-Agamy, K.A. Amen and S. Galal. 1992a. : Effect of
certain fertilization and thinning application on the yield and fruit quality of
Zaghloul date palm. Assiut J. Agric. Sci., 23 (2): 349-360.
[13] Hussein, M.A., S.Z. EL-Agamy, K.A. Amen and S. Galal. 1992b: Physiological
studies for prolonging harvest date of Samany date under Assiut Governorate
conditions. A: Effect of GA3 and fruit thinning. Assiut J. Agric. Sci., 23(2): 321-334
[14] Khalifa, A.S., A.I. El-Kady, K.M. Abdalla and A.M. El-Hamdy. 1987: Influence
of thinning patterns and leaf/bunch ratio on “Zaghloul” dates. Ann. Agric. Sci.,
Fac. Agric., Ain Shams Univ., Cairo, Egypt. 32(1): 637-647.
[15] Marzouk, H.M, A.M.Al- salhy, H.A.Abdel-Galil and A.E. Mahmoud. 2007 :yield and fruit
quality of some date palm cultivars in response to some flower thinning rates . the 4th
symosium on Date Palm in Saudi Arabia. King Faisal Univ, Al-Hassa, 5-8 May p 110
[16] Moustafa, A.A. 1998b. Studies on fruit thinning of date palm .The First International
Conference on date palm Al-Ain United Arab Emirates. March 8-10, pp.354-363.
[17] Nirmaljit, K., J.S. Josan and P.K. Monga. 2006 : Fruit thinning of dates in
relation to fruit size and quality. Abstract of the Third International Date Palm
Conf. Feb. 19th-21th, Abu Dhabi.
[18] Nixon, R.W. and J.B. Carpenter, 1978 : Growing dates in the United States. U.S.
Dep. Agric. Inform. Bull. No. 207, 56 p.
[19] Reuveni, O. 1986. Date. In: CRC Handbook of fruit set and development. Edited
by Shaulp. Monselise. Boca Raton, Fla. CRC Press, pp. 119-144.
[20] Tavakkoli, A., E. Tafazoli and M. Rahem. 2006 : Comparison of hand versus
chemical thinning on quality and quantity of fruits and alternate bearing of
"Shahani" date (Phoenix dactylifera L.). Abstract of the Third International Date
Palm Conf. Feb. 19th-21st, Abu Dhabi.
172
Table (1):Effect of thinning strands and time of thinning on fruit set %, and fruit
weight/ bunch (Kg),of Saidy date palm cultivar during 2004, 2005 and 2006 seasons.
Characteristics.
Treat.
Year
fruit set %
fruit weight/ bunch (Kg)
2004
2005
2006
Mean
2004
2005
2006
Mean
74.34
68.32
77.41
73.36
9.50
8.80
10.10
9.45
T1
71.51
65.24
75.40
70.72
8.75
8.60
9.00
8.78
T2
61.66
52.06
67.82
60.51
6.80
6.65
6.90
6.78
T3
68.47
61.00
72.54
67.34
8.16
7.90
8.16
8.07
T4
57.69
50.73
61.26
56.56
6.15
6.03
6.19
6.12
T5
70.62
63.35
74.50
69.49
8.56
8.08
8.50
8.38
T6
69.78
62.22
73.95
68.65
7.25
6.85
7.38
7.16
T7
70.38
63.42
73.95
69.25
8.60
8.15
8.63
8.46
T8
69.56
62.51
73.21
68.43
7.20
6.85
7.23
7.09
3.58
5.33
3.25
4.82
0.80
0.45
1.20
0.81
Control (unthinning)
L.S.D 0.05
T1- Remove 20%of strands from the center after four weeks; T2- Remove 40%of strands from the center after
four weeks; T3- Cut 20%of strand tips after four weeks; T4 - Cut 40 %of strand tips after four weeks; T5- Remove
20%of strands from the center after eight weeks; T6 - Remove20%of strand tips after eight weeks; T7- Cut
20%of strands from the center after eight weeks; T8 - Cut 20%of strand tips after eight weeks
Table (2 ): Effect of thinning strands and time of thinning on fruit weight (g), pulp
weight %,and fruit length (cm) of saidy date fruits during 2004, 2005 and 2006 seasons.
Characteristics.
fruit weight (g)
pulp weight %
Treat.
Year
2004
2005
2006
Mean
2004
control
(unthinning)
9.56
10.26
9.30
9.70
T1
11.08 12.01 10.59
T2
Mean 2004 2005 2006
Mean
86.76 86.71 86.41
86.63
3.58
3.68
3.59
3.62
11.23
87.61 87.56 87.24
87.47
3.86
3.93
3.89
3.89
11.88 12.58 11.62
12.03
87.62 87.28 87.61
87.53
3.92
3.99
3.96
3.96
T3
11.31 12.23 10.84
11.46
87.98 87.73 87.82
87.84
3.89
3.96
3.92
3.92
T4
12.17 12.77 12.00
12.31
87.92 87.55 87.92
87.81
3.98
4.05
4.01
4.01
T5
11.14 12.03 10.68
11.28
87.66 87.66 87.21
87.51
3.86
3.92
3.90
3.89
T6
11.33 12.06 11.04
11.48
87.72 87.34 87.72
87.59
3.91
4.01
3.91
3.94
T7
11.20 12.09 10.75
11.35
87.56 87.49 87.23
87.43
3.90
4.02
3.87
3.93
T8
11.31 12.06 11.00
11.46
87.79 87.77 87.39
87.65
3.89
4.01
3.88
3.93
0.58
0.41
0.69
0.58
0.12
0.13
0.16
0.17
L.S.D 0.05
0.60
0.45
173
2005
0.53
2006
fruit length (cm)
0.75
Table (3):Effect of thinning strands and time of thinning on total soluble solids percentage
and fruit moisture %, of Saidy date fruits during 2004,2005 and 2006 seasons.
T.S.S %
Characteristics.
fruit moisture %
2004
2005
2006
Mean
2004
2005
2006
Mean
Control
(unthinning)
77.75
78.00
76.58
77.44
13.98
13.50
14.98
14.15
T1
80.00
81.00
79.63
80.21
12.88
12.25
13.38
12.83
T2
82.19
83.50
81.50
82.40
10.73
10.38
11.50
10.87
T3
80.31
81.13
79.88
80.44
12.81
12.25
13.13
12.73
T4
82.50
83.50
82.00
82.67
10.53
10.25
11.00
10.59
T5
80.00
81.50
79.00
80.17
13.00
12.13
13.63
12.92
T6
82.31
83.38
81.75
82.48
11.33
10.50
11.25
11.03
T7
80.31
80.88
79.50
80.23
12.90
12.50
13.29
12.90
T8
82.19
83.38
82.00
82.52
11.28
10.25
11.42
10.98
L.S.D 0.05
0.68
0.73
0.75
0.81
0.46
0.52
0.70
0.61
Treat.
Year
Table (4):Effect of thinning strands and time of thinning on total sugars %, reducing sugars
%, and non-reducing sugars %, of saidy date fruits during 2004,2005 and 2006 seasons.
Chara
-cteristics.
Total sugars %
reducing sugars %
non reducing sugars %
2004
2005
2006
Mean
2004
2005
2006
Mean
2004
2005
2006
Mean
72.23
73.00
71.50
72.39
64.10
64.58
62.95
63.88
8.13
8.42
8.55
8.37
T1
74.75
75.35
74.70
74.95
66.60
66.80
66.20
66.53
8.16
8.55
8.19
8.30
T2
76.73
77.65
76.65
76.96
67.95
68.64
67.70
68.05
8.78
9.02
8.95
8.91
T3
74.83
75.38
75.10
75.07
66.95
67.25
67.15
67.06
7.88
8.13
8.03
8.01
T4
76.98
77.8
76.75
77.12
68.25
68.84
67.75
68.20
8.73
8.97
9.05
8.91
T5
74.78
75.78
74.3
74.98
66.88
67.60
66.10
66.95
7.90
8.18
8.03
8.03
T6
76.75
77.65
76.75
76.94
68.00
68.63
67.75
68.04
8.75
9.02
8.95
8.91
T7
74.95
75.15
74.50
74.91
66.97
67.25
66.43
66.96
7.98
7.90
7.96
7.95
T8
76.93
77.80
76.70
77.14
68.44
68.88
67.80
68.37
8.49
8.93
8.90
8.77
L.S.D 0.05
0.65
0.44
0.59
0.71
0.54
0.47
0.68
0.61
N.S
N.S
N.S
N.S
Treat.
Year
control
(unthinning)
174
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176
PP 05
Effect of some activators and wounding on rooting and
growth of Phoenix dactylifera L. cv. Haiany aerial offshoots
Lobna M. Abdel-Galeil; Tahani Y. Saber; M.A. Abd El-Baky and A.A. El-Bana
Central Lab. for Res. & Date Palm Develop., Hort. Res. Inst., ARC, Giza, Egypt.
Abstract
A trial was conducted in the open field at the nursery of Hort. Res. Inst., Giza,
Egypt during 2009 and 2010 seasons to detect the response of both wounded and
unwounded aerial offshoots of date palm (Phoenix dactylifera L.) cv. Haiany wrapped
in polyethylene bags filled with a mixture of sand+peatmoss+perlite (1:1:1, v/v/v) to
the following activators, which mixed with rooting medium four times with two
months interval: control (without treatment), nitrobien (a biofertilizer contains a
specific strain of Azotobacter chroococcum) at 30 g/offshoot, yeast (Saccharomyces
cerevisiae) at 500 ml/L, auxins mixture (I3AA+IBA+NAA) at 3000 ppm, humic acid
(HA) NPK (10:10:10) organic fertilizer at 30ml/L and sulphur (S, 98.5%) at
10g/offshoot, as well as the interactions between the previous activators and wounding
and unwounding treatments.
The obtained results showed that all activators, wounding treatment and their
interactions significantly increased rooting%, No. roots/offshoot, the longest root
length (cm), rooting efficiency index(REI%), the first new leaf length (cm), No. newformed leaves/offshoot, as well as fresh and dry weights (g) of the first new leaf and
roots. All activators markedly improved the leaf content of chlorophyll a, b and
carotenoids (mg/g F.W.), while wounding treatment significantly increased
chlorophyll a content only, but for chlorophyll b and carotenoids content, the
increasing was slight. Likewise, all activators greatly raised indoles content (ppm) in
the leaves, whereas wounding treatment does not effect on this parameter. Phenols
content was not affected by nitrobien and yeast treatments, but was slightly decreased
by HA and S treatments. Wounding treatment, however decreased it to the minimum
value. Data also revealed that N and K% reached the highest means by nitrobien at 30
g/offshoot treatment, which followed by HA at 30 ml/L. The opposite was the right
concerning P content, as it reached the maximum by HA treatment, that followed by
177
nitrobien one. Wounding treatment, on the other hand slightly declined N and K
content, but significantly reduced P content. In general, the dominance in all previous
measurements was for the interaction between HA at 30 ml/L and wounding treatment,
which gave the highest means in most cases.
So, to obtain the best rooting and growth from aerial offshoots of Phoenix
dactylifera L. cv. Haiany wrapped in polyethylene bags while still on the mother palm,
it is recommended to wound the bases of these offshoots plus drenching the rooting
medium with HA (10N:10P:10K) organic fertilizer at the rate of 30 ml/L, 4 times with
two months interval.
Introduction
Phoenix dactylifera L., Date Palm, is considered a common fruit grown in tropical
and subtropical areas. It is a dioecious feather palm belongs Fam. Palmae. Its delicious
fruits constitute the major component of the daily diet and play the main factor in
exportation in some countries. Date palms are intimately connected with the Egyptian
landscape, as the trees grow in everywhere (El-Hadidi and Boulos, 1979).
Propagation of date palm is commercially accomplished by offshoots, which are
produced in a limited number for a certain period in the life of a young palm. This low
number of offshoots usually subjects to failure if separated from mother palm due to
poor root formation (Al-Ghamdi, 1988). Decay of the detachment surface seems to be
the major reason for this failure (Corner, 1966). This truth was emphasized by Vij et
al. (1977) who stated that direct-planted offshoots in a nursery after dipping them in
IBA at 1000 or 3000 ppm for 1 minute failed to root. In addition, Al-Mana et al.
(1996) mentioned that the survival of small aerial offshoots, which require special
nursery conditions, is very low.
Recently, there has been a great demand for date palm offshoots already rooted well
to face the great widening in dates production. So, many efforts were done to detect
some effective methods for rooting these offshoots successfully. In this regard, Vij et
al. (1977) found that treating the bases of aerial offshoots of date palm cv. Majdool
that were still on their mother palm with IBA at 3000 ppm gave the longest, thickest
and heaviest roots. Similarly, where those results of Al-Mana et al. (1996) on cvs.
Shishi and Shahl, Singh et al. (2003) on cvs. Anbara, Barhi, Hilali, Khalas, Majdool,
178
Succari and Sultana and Al-Obeed (2005) who reported that date palm cvs. Segie and
Khalas produced well-developed root systems after 6 months from treating with
commercial NAA at 0.8%, while Succari and Seleg cultivars produced poorly
developed root systems. The cultivars varied in the number of small and large roots,
mean root length, weight of small and large roots, percentage of small and large roots
and total root weight. On date palm cv. Zaghloul, El-Sayed et al. (2010) confirmed
that the combined treatment between IBA and NAA at 1000 ppm for each gave the
highest survival and rooting percentages, root length, No. roots/offshoot, REI%, leaf
length, No. leaves/offshoot, fresh and dry weights of leaves and roots, as well as their
content of N, P and K%.
Humic acid (HA) is widely used to improve vegetative and root growth of most
crops. This was indicated by Abdel-Galeil et al. (2010) who postulated that HA at 10
ml/L greatly improved rooting%, root length, No. roots and rooting efficiency index
(REI%) of Phoenix dactylifera cv. Zaghloul offshoots. Leaf length, number of newformed leaves and their fresh and dry weights were also increased, as well as N, P and
K content in the leaves. On some ornamentals, Evans and Li (2003) revealed that HA
at 2500 and 5000 mg/L increased lateral root number, lateral root length and roots dry
weight of Catharanthus roseus, Pelargonium hortorum, Tagetes patula and Viola
tricolor. El-Sayed and El-Shal (2008) declared that HA at either 5 or 10 ml/L greatly
improved vegetative and root growth of Schefflera plants, as well as content of N, P,
K, Fe, Zn and Mn in the leaves. Similar observations were also gained by AbdelFattah et al. (2009) on Dracaena and Ruscus.
Since years ago, the use of biofertilizers was suggested to be a choice for restoring
the natural conditions of safe and clean environment. They are preparations containing
one or more of beneficial microorganisms that can release nutrients from rocks and
organic matter in the soil to become available for plants. Among of them, nitrobien
and yeast that were greatly used for improving growth and quality of various crops. In
this concern, El-Sayed et al. (2007) pointed out that nitrobien at 5 g/pot significantly
improved vegetative and root growth of Peperomia obtusifolia cv. Variegata, as well
as the content of chlorophylls a and b, carotenoids, total carbohydrates, N, P and K in
the leaves. Likewise, El-Sayed et al. (2009) mentioned that nitrobien at 5 g/pot gave
179
the highest number of roots, longest root and heaviest roots fresh and dry weights in
Nephrolepis exaltata plant. The leaf content of chlorophylls a and b, carotenoids, total
carbohydrates and N were also increased. On Euonymus japonicus, Abdel-Wahed et
al. (2006) claimed that yeast at 4g/L led to increasing of number of branches, stem
diameter, root number and length, fresh and dry weights of roots, carotenoids content
in the leaves and K% in roots. Similarly, El-Sayed et al. (2010) affirmed that yeast at 6
g/L as a foliar spray on Spathiphyllum plants caused a marked increment in root
length, number of roots and roots fresh and dry weights.
Several studies demonstrated that sulphur (S) is considered one of the most essential
nutrients required for growth and health of different plants. This was indicated by
Yadegari et al. (2008) who observed that treatment of Melissa officinalis plants with
400 kg S + Thiobacillus + organic matter resulted the heighest fresh and dry weight,
number of lateral stems and roots, oil content in dry matter and Cu, Zn, Fe and Mn
contents in the soil after harvesting. Likewise, Deshbhartar et al. (2010) found that
application of S at 40 kg/ha improved soil fertility and root growth of pigeonpea
(Cajanus cajan). Moreover, Ravi and Channal (2010) noticed that application of 30 kg
S/ha markedly increased vegetative and root growth of safflower plants, as well as
nutrient uptake of N, P, K, S, Zn and Fe.
As for wounding effect on rooting, Sing et al. (2009) found that wounding the stem
of litchi tree and treating them with IBA at 500 ppm helps in better rooting. Kasim et
al. (2009) reported that bitter almond hardwood cuttings treated with IBA at 8000 ppm
plus wounding resulted a high rooting % and a maximum number of roots/cutting. On
Leyland cypress, Collado et al. (2010) indicated that the highest values of rooting%,
number of roots and mean root length were obtained with wounding + 3000 ppm of
IBA combined treatment.
This work, however was done to explore the role of some activators and wounding
process, individually or in combinations, on rooting promotion and growth of aerial
offshoots of date palm cv. Haiany.
Materials and Methods
An investigation was consummated in the open field at the nursery of Hort. Res.
Inst., Giza, Egypt during the two consecutive seasons of 2009 and 2010 to promote
180
roots formation on the aerial offshoots of date palm (Phoenix dactylifera L.) cv.
Haiany using some activators, with or without wounding treatment.
Therefore, small size aerial offshoots, high and unrooted, aged about 3 years and
weighing about 8-10 kg, while still on the mother palm, were prepared and used in this
study. The outer and older leaves are carefully removed to leave 4 leaves only on each
offshoot. These 4 leaves were shortened to 75 cm long and tied together around the
apical meristem to protect it from damage. The bases of all offshoots on each palm
tree, that were in circle around the trunk, were well cleaned and sprayed with a 0.5%
solution of Topsin-M, 70% WP (Summitomo Chemical Co., Ltd. Osaka, Japan), and
half of them (3 shallow incisions) were wounded (3 wounds) by a sterilized stainlesssteel sharp cutter to get the following two treatments: (a) wounded, and (b) unwounded
offshoots. Afterwards, the wounded and unwounded offshoots on trunk of each palm
tree were wrapped on March, 15th for the two seasons using polyethylene bags filled
with a mixture of sand+peatmoss+perlite (1:1:1, by volume). Wrapping the bases of
the offshoots with polyethylene bags proved to be suitable for rooting of them since
the polyethylene bags maintained the moisture around the bases of offshoots. Some
physical and chemical properties of the used sand and peat moss in both seasons were
determined and illustrated in Tables (a) and (b), respectively.
Immediately after wrapping, the wounded and unwounded offshoots still on the
mother palm received the following activators:
1. No treatment, referred to as control.
2. A biofertilizer nitrobien, which contains a specific strain of Azotobacter
chroococcum bacteria (conc. 106cell/ml) at the rate of 30 g/offshoot.
3. An aqueous solution of the commercial yeast solution (Saccharomyces cerevisiae)
at the rate of 500 ml/L. Chemical composition of the commercial yeast solution is
shown in Table (c).
4. An aqueous solutions of 3 auxins, named indole-3-acetic acid (I 3AA), indole
butyric acid (IBA) and naphthalene acetic acid (NAA), products from Sigma
Chemical Co., USA, at the rate of 1000 ppm for each were mixed together to form
a mixed solution of 3000 ppm.
181
5. An aqueous solution of humic acid NPK (10:10:10) liquid organic fertilizer at the
rate of 30 ml/L. The different components of this liquid organic fertilizer are
averaged in Table (d).
6. Sulphur (a very fine powder, 98.5%) at the rate of 10g/offshoot.
All previous treatments were added to the rooting mixture 4 times with 2 months
interval, and each 500 ml of the various aqueous solutions mentioned above was set for
each 3 offshoots (i.e. 166.67 ml/offshoot). Moreover, the different activators treatments
were combined with each one of wounding to form 12 interaction treatments.
The experiments in both seasons were laid out in a complete randomized design
with factorial concept replicated thrice (Mead et al., 1993), as each palm tree carries 6
aerial offshoots was considered one replicate. The usual agricultural practices needed
for such plantation were followed, especially irrigation that was regularly done to keep
the rooting medium moist.
At the end of each season (on November, 15th), the following data were recorded:
rooting (%) from this equation: Rooting (%) = R/Tx100 (where R= number of the
rooted offshoots and T= total number of offshoots in the treatment), number of
roots/offshoot, the longest root length (cm) and the rooting efficiency index as a
percentage (REI%), which was calculated from the equation described by Ruter et al.,
(2004) as follows:
REI (%) = Mean root length of the treated offshoot/mean root length of control x 100.
Besides, the first new leaf length (cm), number of new formed leaves/offshoot, as
well as the first new leaf and roots fresh and dry weights (g) were also determined.
In fresh leaf samples, the photosynthetic pigments (chlorophyll a, b and carotenoids,
mg/g F.W.) according to the method of Saric et al. (1976), total indoles and total
phenols content (ppm) as indicated by A.O.A.C. (1990) were measured, while in dry
leaf samples, the percentages of N, P and K were assessed according to the methods
recommended by Jackson (1973).
The data were then tabulated and statistically analyzed according to SAS program
(1994) using Duncan's Multiple Range Test (Duncan, 1955) for elucidating the
significance between various treatments.
182
Results and Discussion
Effect of activators, wounding and their interactions on:
1- Rooting parameters:
It is clear from data averaged in Table (1) that both activators and wounding
treatments significantly increased rooting (%), No. roots/offshoot and the longest root
length (cm) comparing with control in the two seasons. Humic acid at 30 ml/L gave
the highest records in comparison to other activators, but the superiority in both
seasons was found due to the collecting between wounding and HA at 30 ml/L
treatment, which registered the utmost high means. This may indicate the role of HA
in providing the soil microbes with energy and improving nutrients retention in the soil
(Dorer and Peacock, 1997), beside containing NPK and some micronutrients that may
activating some enzymatic systems and root primordia to form new roots (Evans and
Li, 2003). Furthermore, wounding is considered a mechanical process that plays a vital
role in enhancing the formation of new roots.
Concerning the rooting efficiency index (REI%), as a real indicator for rooting
strength in offshoots of date palm cv. Haiany, data in Table (1) show that all activators
caused a significant increment in such parameter, with the mastery of HA treatment
(30 ml/L), which registered the highest percentages of REI in the two seasons. On the
other hand, unwounding treatment gave a higher percentage of REI with a significant
difference when compared to wounding treatment in the first season, but in the second
one, the opposite was the right, as unwounding treatment slightly decreased this
parameter with a non-significant difference comparing with wounding one. In general,
the prevalence in both seasons was for the interaction between HA at 30 ml/L and
either wounding or unwounding treatment, which recorded the highest values over
control and other treatments in both seasons. This may be attributed to that this
combination gave the longest roots over all other individual and combined treatments
as previously mentioned.
Improving rooting traits due to auxins mixture may explain the role of them in
encouragement of the cambium cells, besides some other meristematic cells, especially
parenchyma cells for division and enlargement near the base of offshoots to form
adventitious roots (Morini et al. 2003). These results are in accordance with those
183
attained by Vij et al. (1977) on date palm cv. Majdool, Singh et al. (2003) on date
palm cvs. Anbara, Barhi, Hilali, Khalas, Majdool, Succari and Sultana, and Al-Obeed
(2005) on date palm cvs. Segie and Khalas.
2- Vegetative and root growth:
Similar observations to those of rooting parameters were also obtained regarding
vegetative and root growth characters (Tables, 2 and 3), where the length of the first new
leaf (cm), No. new-formed leaves/offshoot, as well as fresh and dry weights (g) of the
first new leaf and roots were significantly increased in response to the different activators
and wounding treatments employed in this work in the two seasons. Among activators,
HA at 30 ml/L gave the highest averages, but the mastership in both seasons was for the
combined treatment between wounding and HA at 30 ml/L, which recorded the utmost
high means in all previous characters over these of control and other treatments. This
may be attributed to the synergistic effect of both wounding, as a mechanical process
activates root primordia to grow early and consequently absorb higher water and
nutrients, and humic acid as an organic fertilizer provides the new formed organs with
macro- and micro-nutrients necessary for the best and healthy growth.
Data were also exhibited that auxins mixture (3000 ppm) gave a good effect on the
length of the first new leaf and No. new-formed leaves/offshoot, while sulphur (10
g/offshoot) gave better fresh and dry weights of the first new leaf and roots. This may
be reasonable since auxins usually act as promotive agents for growth and
development, while sulphur improves soil fertility and increases nutrients availability.
Analogus results were also observed by Al-Mana et al. (1996) on date palm cvs.
Shishi and Shahl, Abdel-Galeil et al. (2010) on date palm cv. Zaghloul, El-Sayed et al.
(2010) on Spathiphyllum and Deshbhartar et al. (2010) on Cajanus cajan.
3- Chemical composition:
According to data presented in Table (4), it could be concluded that all activators
raised the leaf content of chlorophyll a, b and carotenoids (mg/g F.W.) with various
significance differences compared to control means in the two seasons. However, the
highest content of these pigments was found due to auxins mixture treatment at 3000
ppm in both seasons. This may interpret the role of auxins on promoting stroma
lamella formation and grana and chlorophyll appearance during normal leaf growth
184
(Morini et al., 2003). As for wounding treatment, it caused a significant increment in
chlorophyll a content in the first and second seasons, but for the content of
chlorophyll b and carotenoids, the augmentation was slight with non-significant
difference in comparison to unwounding treatment. However, the greatest content in
these pigments in both seasons resulted from the combined treatment between
wounding and 3000 ppm auxins mixture treatment. In addition, 10 g sulphur/offshoot
treatment induced a pronounced increment in the means of chlorophylls a and b, that
were closely near to those of auxins mixture treatment. In this regard, Ravi and
Channal (2010) stated that sulphur has a vital effect on chlorophylls biosynthesis,
although it is not include in their structure.
In the matter of total indoles and total phenols content (ppm), data in Table (5)
indicate that all activators significantly raised indoles content in the leaves of treated
plants, especially HA at 30 ml/L treatment, that elevated this constituent to 0.36 ppm
in both seasons, while wounding treatment does not affect of such parameter.
However, the highest indoles content in the two seasons was scored by the
interaction between 30 ml/L HA and wounding treatment. The opposite was the right
concerning phenols content, as it was not affected by either nitrobien or yeast
treatments, but it was slightly decreased by humic acid and sulphur treatments in the
two seasons. On the other hand, wounding treatment decreased this measurement to
the minimum value (0.06 ppm in both seasons) against 0.58 and 0.61 ppm for
unwounding treatment with highly significant differences in the first and second
seasons, respectively. This may be the main reason for the good formation of roots on
the bases of wounded offshoots rather than unwounded ones.
Data in Table (6) exhibit that treatment of 30 g nitrobien/offshoot significantly
augmented the percentages of both N and K in the leaves over control and all other
treatments, and followed by HA at 30 ml/L treatment in the two seasons. The opposite
was the right regarding P%, which reached the maximum by HA treatment, and
followed by nitrobien one in both seasons. This may indicate the beneficial effect of
nitrobien, as N-fixer biofertilizer, and humic acid, as a complete fertilizer supplies the
plants with various nutrients. Wounding treatment, on the other side slightly declined
N and K content in most cases of the two seasons, but significantly reduced P%
185
comparing with unwounding treatment in the first and second seasons. In general, the
excellence in both seasons was for the combined treatment between either nitrobien or
humic acid and wounding. The depression of nutrients content because of wounding
treatment may be attributed to the excessive growth in leaves and roots that usually
consume more nutrients.
The previous gains are in good agreement with those of El-Sayed et al. (2010) and
Abdel-Galeil et al (2010) on date palm cv. Zaghloul, Abdel-Fattah et al. (2009) on
Dracaena and Ruscus, and El-Sayed et al. (2010 a) on Spathiphyllum.
Due to the above results, it could be advised to wound the bases of Phoenix
dactylifera L. cv. Haiany aerial offshoots before wrapping them with polyethylene
bags and drenching the rooting medium with humic acid NPK (10:10:10) liquid
organic fertilizer at the rate of 30 ml/L four times from march to October every two
months intervals for the best rooting and growth (Photo, 1).
Acknowledgement
The authors wish to express their sincere gratitude and appreciation to Prof. Dr.
Sayed M. Shahin, Head Researchs, Botanical Gars. Res. Dept., Hort. Res. Inst., ARC
for his guidance and preparation of the manuscript.
186
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189
Season
Table (a): Some physical and chemical properties of the used sand during 2009 and 2010 seasons.
Cations (meq/L)
Particle size distribution (%)
S.P.
Coarse
sand
Fine
sand
Silt
clay
2009
89.03
2.05
0.40
8.52
2010
84.76
6.29
1.50
7.45
E.C.
pH
(ds/m)
Anions (meq/L)
Ca++
Mg++
Na+
K+
23.01
3.56 7.9 7.50
1.63
33.6 0.50
3.20
22.00 18.03
21.87
3.78 7.8 19.42
8.33
7.2
1.60
7.8 26.30
HCO3-
0.75
Table (b): Some physical and chemical properties of the used peatmoss in the two seasons.
Organic matter.…………
Ash.………………………
Density (vol. dry) .……..
90-95%
8-10%
80.90 mg/l.
K……………..…
Fe…………….…
Mn ……..………
1.77 %
421 ppm
27 ppm
pH value.………………...
3.4
Zn .......................
41 ppm
Water relation capacity...
Salinity………………......
N………………………....
P………………..
60-75%
0.3 g/l.
1.09 %
0.23%
Cu ………...….…
Mg ………………
8.8 ppm
3.3 ppm
Table (c): Chemical composition of the commercial yeast solution used in the two seasons.
Proteins
47.0%
Niacin
300-500 µ/g
Carbohydrates
33.0%
Pyrodoxin
28.0 µ/g
Minerals
8.0%
Pantathenate
70.0 µ/g
Nucleic acids
8.0%
Biotin
1.3 µ/g
Lipids
4.0%
Cholin
4000 µ/g
Thiamine
60-100µ/g
Folic acid
5.13 µ/g
Riboflavin
35-50 µ/g
Vit. B12
0.001 µ/g
Approximate composition of minerals (mg/g)
Na
0.12
Cu
8.0
Ca
0.75
Se
0.1
Fe
0.02
Mn
0.02
Mg
1.65
Cr
2.2
K
21.0
Ni
3.0
P
13.5
Va
0.04
S
3.9
Mo
0.4
Zn
0.17
Sn
3.0
Si
0.03
Li
0.17
190
Cl-
SO4--
Table (d): The different components of the liquid organic fertilizer (HA) used in
the two seasons.
Components
Humic acid (%)
Organic matter/total solid (%)
Total HA/total solid
Organic carbon (%)
C/N ratio
pH
Value
2.9
42.51
165.80
24.64
2.46
8.10
Components
EC(dS/m)
N (%)
P (%)
K (%)
Ca (%)
Mg (%)
Value
59.3
10.00
10.00
10.00
0.06
0.05
Components
B (mg/L.)
Fe (mg/L.)
Mn (mg/L.)
Zn (mg/L.)
Value
900.00
90.00
90.00
Table 1. Effect of activators, wounding and their interactions on rooting parameters
of Phoenix dactylifera L. cv. Haiany offshoots during 2009 and 2010 seasons.
Wounding
Activators
Rooting (%)
No. roots/offshoot
The longest root length
(cm)
Rooting efficiency index
(%)
UnUnUnUnWound
Wound
Wound
Wound
wounde Mean
wounde Mean
wounde Mean
wounde Mean
ed
ed
ed
ed
d
d
d
d
First season: 2009
Control
30.00g 30.00g 30.00E 28.33gh 21.93h 25.13F 18.33g 14.78h 16.56E 100.00g 100.00g 100.00D
Nitro.
(30g/offsh.)
55.00d 48.33e 51.67C 60.00d 42.33f 51.17D 31.67cd 28.10d 29.89C 172.78d 190.12c 181.45B
Yeast (500ml/L) 48.33e 41.33f 44.83D 43.50f 35.00g 39.25E 23.00e 19.56f 21.28D 125.48f 132.34e 128.91C
Auxins mix.
(3000ppm)
70.50c 63.46dc 66.98B 88.36b 70.46c 79.41B 33.39c 30.00dc 31.70B
182.16c
202.98b 192.57B
d
H.A. (30 ml/L) 96.67a 83.33b 90.00A 183.30a 91.58b 137.44A 41.50a 36.87b 39.19A 226.41a 249.46a 237.94A
Sulphur
(10g/offsh.)
Mean
66.67ed 64.00dc 65.33B 75.00cd 50.76e 62.88C 31.67cd 27.33de 29.50C 172.78d
184.91c 178.58B
d
C
61.20A 55.08B
176.64
A
79.75A 52.01B
29.93A 26.10B
163.27B
Second season: 2010
Control
Nitro.
(30g/offsh.)
30.50h 33.00h 31.75E 28.00g 23.16h 25.58E 17.67fe 15.38f 16.53E 100.00f 100.00f 100.00d
56.67e 50.33f 53.50C 53.76f 42.33f 48.00D 28.00cd 23.69d 25.25C 158.46d
154.03d
156.25C
e
Yeast (500ml/L) 46.71fg 43.63g 45.17D 58.00ed 38.90fg 48.45D 21.67ed 19.81e 20.74D 122.64e 128.81e 125.73D
Auxins mix.
(3000ppm)
68.47c 61.10d 64.79B 86.33bc 63.65d 74.99B 35.00b 25.67dc 30.34B 198.08b 166.91c 182.50B
H.A. (30 ml/L) 97.86a 80.00b 88.93A 147.00a 90.33b 118.67A 43.10a 41.00ab 42.05A 243.92a 266.58a 255.25A
Sulphur
(10g/offsh.)
Mean
66.23cd 60.50d 63.39B 71.33c 53.18e 62.26C 30.00c 24.67d 27.34C 169.78c 160.40d 165.09C
61.08 A 54.76B
74.07A 51.93B
29.24A 25.04B
165.48A 162.79A
- Nitro.= Nitrobien, offsh.= offshoot, mix.= mixture and H.A.= Humic acid
- Means within a column or row having the same letters are not significantly different according to
Duncan's Multiple Range Test (DMRT) at 5% level.
191
Table (2) Effect of activators, wounding and their interactions on the first new leaf length and number of
new formed leaves of Phoenix dactylifera L. cv. Haiany offshoots during 2009 and 2010 seasons.
Wounding
First new leaf length (cm)
Wounded
Activators
Unwounded
Mean
No. new formed leaves/offshoot
Wounded
Unwounded
Mean
First season: 2009
Control
80.00e
77.33e
78.67D
2.00c
1.67c
1.84C
Nitro. (30g/offsh.)
106.00de
85.50e
95.76C
3.00b
2.67bc
2.84B
Yeast (500 ml/L)
125.33c
112.70d
118.96B
3.00b
2.00c
2.50B
Auxins mix. (3000ppm)
141.70b
123.30c
132.50A
3.50ab
3.00b
3.25AB
H.A. (30 ml/L)
156.71a
126.69c
141.70A
4.00a
3.33ba
3.67A
Sulphur (10g/offsh.)
126.33c
83.70e
105.00BC
3.00b
2.33cb
2.67B
Mean
122.68A
101.54B
3.08A
2.50B
Second season: 2010
Control
83.67e
76.33f
80.00D
1.83c
1.33c
1.58C
Nitro. (30g/offsh.)
98.33d
80.67e
89.50C
2.67b
2.28cb
2.48AB
Yeast (500 ml/L)
131.50b
104.56c
118.03B
2.33bc
2.00cb
2.16B
Auxins mix. (3000ppm)
150.00a
128.31b
139.16A
3.00ba
2.33bc
2.67AB
H.A. (30 ml/L)
153.10a
136.70b
144.90A
3.67a
3.00ba
3.33A
Sulphur (10g/offsh.)
98.73d
83.50e
91.12C
2.67b
2.33bc
2.50AB
Mean
119.22A
101.68B
2.70A
2.21B
- Nitro.= Nitrobien, offsh.= offshoot, mix.= mixture and H.A.= Humic acid
- Means within a column or row having the same letters are not significantly different according to
Duncan's Multiple Range Test (DMRT) at 5% level.
192
Table 3. Effect of activators, wounding and their interactions on fresh and dry weights of the first new
leaf and roots of Phoenix dactylifera L. cv. Haiany offshoots during 2009 and 2010 seasons.
Fresh weight (g)
Wounding
Activators
The first new leaf
Dry weight (g)
Roots
The first new leaf
Roots
UnUnUnUnWound
Wound
Wound
Wound
wounde Mean
wounde Mean
wounde Mean
wounde Mean
ed
ed
ed
ed
d
d
d
d
First season: 2009
Control
186.33f 175.67f 181.00D 18.33f 17.00f 17.67E 77.63g 71.89gh 74.76E 11.25e 10.11f 10.68E
Nitro.
(30g/offsh.)
156.70g 154.70g 155.70E 32.67d 30.50d 31.59C 64.65h 57.73j 61.19F 13.21d 12.18de 12.70D
Yeast
(500ml/L)
232.00d
221.30e
11.39D
226.70C 26.78de 21.56e 24.17D 90.15f 88.71f 89.43D 11.80e 10.97fe
d
E
131.40d
Auxins mix. 243.50c
205.20e 224.30C 35.17dc 23.00e 29.09C
98.52e 114.96C 16.36c 13.81d 15.09C
d
e
(3000ppm)
H.A. (30 ml/L) 294.00a 251.36c 272.68A 73.00a 61.23d 67.12A 149.00c 135.31d 142.16B 34.46a 30.90ad 32.68A
Sulphur
(10g/offsh.)
Mean
270.10b
239.31d
254.71D 43.31c 38.21cd 40.76B 165.10a 158.39b 161.75A 21.50b 19.33bc 20.42B
c
230.44A 207.92B
38.21A 31.92B
112.99A101.76B
18.10A 16.22B
Second season: 2010
Control
183.13g 165.00h 174.07E 17.56e 15.33e 16.45E 78.90f 71.20g
75.05E
10.28ef 9.39f
D
9.84E
Nitro.
(30g/offsh.)
158.70h
151.50i 155.10F 33.10c 30.33cd 31.72C 73.48fg 63.50h 68.49E 12.63ed 11.33e 11.98D
i
Yeast
(500ml/L)
217.00e 205.33f 211.17D 28.56dc 23.27d
25.92D
10.92D
88.67ef 78.91f 83.79D 11.45e 10.39ef
E
E
221.10e
Auxins mix.
232.30d
226.70C 34.93c 21.88de 28.41D 112.63d 93.82e 103.23C 16.50c 14.00d 15.25C
d
(3000ppm)
H.A. (30 ml/L) 286.51a 245.36c 265.94A 67.00a 43.96d 55.48A 142.70a 133.40b 138.05A 21.80a 19.76ab 20.78A
Sulphur
(10g/offsh.)
Mean
261.18b 218.81e 240.00B 41.50b 37.44cd 39.42B 132.73b 121.33c 127.03B 18.96b 17.60bc 18.28B
223.14A 201.18B
37.11A 28.68B
104.85A 93.69B
15.27A 13.75B
- Nitro.= Nitrobien, offsh.= offshoot, mix.= mixture and H.A.= Humic acid
- Means within a column or row having the same letters are not significantly different according to
Duncan's Multiple Range Test (DMRT) at 5% level.
193
Table 4. Effect of activators, wounding and their interactions on pigments content in Phoenix dactylifera
L. cv. Haiany leaves during 2009 and 2010 seasons.
Wounding Chlorophyll a (mg/g F.W.)
Activators
Chlorophyll b (mg/g F.W.)
Carotenoids (mg/g F.W.)
Wounded Unwounded Mean Wounded Unwounded Mean Wounded Unwounded Mean
First season: 2009
1.35h
1.33h
1.34E
1.16d
1.13d
1.14C
0.06e
0.06e
0.06E
Nitro. (30g/offsh.) 1.78g
1.73g
1.76D
1.17cd
1.09d
1.13C
1.54c
1.50c
1.52C
Yeast (500ml/L)
3.27c
3.03d
3.15B
1.43b
1.34cd
1.39B
2.75b
2.70b
2.73B
Auxins mix.
(3000ppm)
4.88a
4.04b
4.46A
2.04a
1.90a
1.97A
3.98a
3.94a
3.96A
H.A. (30 ml/L)
2.53e
2.23f
2.38C
1.38b
1.37b
1.38B
0.44d
0.10e
0.27D
Sulphur
(10g/offsh.)
3.23c
3.11d
3.174B
1.99a
1.92a
1.96A
0.14e
0.14e
0.14E
Mean
2.84A
2.58B
1.53A
1.46A
1.49A
1.41A
Control
Second season: 2010
1.11j
1.28h
1.20E
1.15d
1.13d
1 .14D
0.06d
0.06d
0.06E
Nitro. (30g/offsh.) 1.77g
1.63g
1.70D
1.08d
1.03d
1.06D
1.51c
1.49c
1.50C
Yeast (500ml/L)
3.18c
3.00d
3.09B
1.42c
1.41c
1.42C
2.75b
2.57b
2.66B
Auxins mix.
(3000ppm)
4.71a
4.13b
4.42A
2.00a
2.00a
2.00A
3.95a
3.87a
3.91A
H.A. (30 ml/L)
2.51e
2.20f
2.36C
1.37c
1.33c
1.35C
0.50d
0.48d
0.49D
Sulphur
(10g/offsh.)
3.21c
3.19c
3.20B
1.95a
1.77b
1.86B
0.54d
0.13d
0.34DE
Mean
2.75A
2.57B
1.50A
1.45A
1.55D
1.43A
Control
- Nitro.= Nitrobien, offsh.= offshoot, mix.= mixture and H.A.= Humic acid
- Means within a column or row having the same letters are not significantly different according to
Duncan's Multiple Range Test (DMRT) at 5% level.
194
Table (5) Effect of activators, wounding and their interactions on indoles and phenols content in the
leaves of Phoenix dactylifera L. cv. Haiany offshoots during 2009 and 2010 seasons.
Indoles (ppm)
Wounding
Wounded
Activators
Unwounded
Phenols (ppm)
Mean
Wounded
Unwounded
Mean
First season: 2009
Control
0.12i
0.13hi
0.12E
0.05c
0.60ab
0.33AB
Nitro. (30g/offsh.)
0.17e
0.16ef
0.17D
0.06c
0.56b
0.31AB
Yeast (500 ml/L)
0.15fg
0.34bc
0.25B
0.08c
0.57b
0.32AB
Auxins mix. (3000ppm)
0.33c
0.14gh
0.24B
0.05c
0.76a
0.40A
H.A. (30 ml/L)
0.35ab
0.36a
0.36A
0.06c
0.52b
0.29B
Sulphur (10g/offsh.)
0.19d
0.20d
0.20C
0.07c
0.45b
0.26B
Mean
0.22A
0.22A
0.06B
0.58A
Second season: 2010
Control
0.12h
0.12h
0.12F
0.05d
0.59b
0.32AB
Nitro. (30g/offsh.)
0.17f
0.17f
0.17E
0.06d
0.57b
0.32AB
Yeast (500 ml/L)
0.14g
0.35b
0.25B
0.08d
0.55b
0.32AB
Auxins mix. (3000ppm)
0.30c
0.15g
0.22C
0.05d
0.77a
0.41A
H.A. (30 ml/L)
0.35b
0.36a
0.36A
0.06d
0.50c
0.28B
Sulphur (10g/offsh.)
0.19e
0.20d
0.20D
0.07d
0.45c
0.26B
Mean
0.21A
0.23A
0.06B
0.61A
- Nitro.= Nitrobien, offsh.= offshoot, mix.= mixture and H.A.= Humic acid
- Means within a column or row having the same letters are not significantly different according to
Duncan's Multiple Range Test (DMRT) at 5% level.
Control
HA + Wounding tr.
Photo (1) Effect of the interaction between wounding and HA at 30 ml/L on rooting compared to control
195
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196
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197
198
PP 06
Role of yeast as a new biofertilizer for improving yield and fruit
quality of both hayany and sewy date palm cultivars.
Gadalla, E. G1, Abo Rekab, Zeinab A. M2 and Nady, N .H2
Date Palm Research lab., Agricultural Research Center, Egypt 1.
Horticulture Research Institute, Agricultural Research Center, Egypt 2.
Abstract
This investigation was conducted for two successive seasons (2008 and 2009) to
assess the effect of active dry yeast as a foliar spray on yield and improving fruit
quality of Hayany and Sewy date cultivars. The active dry yeast was sprayed in three
concentrations 25gm/L, 50gm/L and 75gm/L per tree during fruit setting stage on May
(one spray) and during both fruit setting and fruit development stages on July (twice
spray). Results cleared that, spray of active dry yeast at 75gm/L,50gm/ L followed by
25 gm/L induced a striking improvement in fruit setting, fruit retention and decreased
fruit dropping percentages as well as fruit physical properties (fruit weight, flesh
weight, fruit length, fruit diameter, fruit volume).while fruit chemical properties are
increasing by increased of concentrations of active dry yeast (increasing total sugars,
reducing, non-reducing sugar and TSS percentages and decrease tannins, total acidity
and crude fibers percentages). Results also revealed that Sewy cultivar gave the
highest values of fruit weight, flesh weight, fruit setting, fruit retention, decreased fruit
dropping, total sugars and TSS percentages. Active dry yeast as foliar spray at 75gm/L
,50gm/L followed by 25 gm/L per tree for twice spray (during fruit set stage on May
and during fruit development on July) are recommended for improving yield and fruit
quality of Hayany and Sewy date palm cultivars.
Introduction
Date palm ( Phoenix dactylifera L. ) belongs to family : palmae ( Arecaceae ) is one
of the oldest trees in the world, because of its easy cultivation under variable soils and
climates . Date palm is one of the most important fruit crops grown in Egypt for its
unique nutritional value. The number of fruitful female palms in Egypt is 11.412.969
million planted on approximately 86.083 feddans producing 1.159.687 ton of dates
199
(according to the annual agricultural statistics of FAO (Food and Agriculture
Organization (2005). Hayany and Sewy are the best soft and semi-dry type date palm
cultivars, respectively. Yeast (Saccharomyces cervicisae ) is considered as a new
promising biofertilizer for many crops. The positive effects of applying yeast could be
due to one or more of its merits. Yeast aids in activating photosynthesis process
through enhancing the release of carbon dioxide Larson et al., (1962). Yeast contains
tryptophan which consider precursor of IAA, so it increases size of fruit (Moor, 1979).
Yeast contains cytokinins which enhancing the synthesis of protein and RNA Natio et
al., (1981). In addition, it contains some important nutrients as N, P, K and some
common amino acids (approximately 18 amino acids). (Abou-Zaid, 1984). Generally,
spraying with active yeast solution improving fruit quality, increasing both fruit
physical characteristic and fruit chemical characteristics (except tannins, total acidity
and crude fibers)these results were due to the contents of the active yeast which
consists
of
Protein
(41.2%),
Arginine
(2.6%),
Glycine
(2.6%),
Histidine(1.4%),Isolaysinel (2.9%), Lauicine (3.5%),Lysine(3.8%),Methionine(0.6%),
cystine(0.6%),
Phenyl
alanine(3%),
Tyrosine(2.14%),
Theronine
(2.6%),
Tryptophan(0.5%) and Vitamin B (2.9%), Ash (7.55%), Glycogen(6.54%),Fats
(2.9%)and Cellulose (4.92%),according to N.R.P(1977) and Ahmed et al.,(1998).
On the other hand, spraying with active yeast solution contains high amounts of
four vitamins, especially B which plays an important role in improving growth and
controlling the incidence of fungi diseases( Tarrow and Nakase (1975), laz et al
.,(2000), Abd El-hady (2001), El-sayed(2002) and Ahmed et al., (2003).
Active dry yeast at l0gm/L enhanced fruit set, yield and physical and chemical
properties (Aki et al., 1997). Dry yeast as a foliar spray increased TSS and yield,
lowered acidity and effectively enhanced the nutritional status and yield (Ahmed et al.,
1998). Fertilizing with active dry yeast increased fruit '' weight, diameter and length ''
and fruit set %, especially with 20gm/L yeast as application. This investigation aimed
to study the effect of active dry yeast as a foliar spray (25,50 and 75 gm/L) as a natural
stimulator, at two different times of the growing season on yield and fruit quality of
two date palm cultivars (Hayany and Sewy).
200
Materials and methods
This investigation was conducted through the two successive seasons of 2008 and
2009 on two date palm cultivars Hayany and Sewy (three female date palm trees for
each cultivar), grown at the orchard of Horticultures institute Research, Giza
governorate , where the soil is light clay. Selected trees were about 20 years old,
planted at 8 × 8 meters apart, similar as possible in growth vigor, height and fruiting
capacity in the preceding years, free from pathological and physiological and received
the common culture practices .Active dry yeast was dissolved in water before adding
to the date palm trees as a foliar Spray treatment in three concentrations 25,50 and 75
gm/L plus 500gm sucrose per tree :while the control was sprayed with distilled water
only .The treatments were carried out during the setting and fruiting season as follows:
three bunches were selected randomly in each tree and(6 spikes/ bunch were selected
randomly for each concentration as well as for control),hence,the experiment involved
the following treatments 1-active dry yeast solution at 25 gm/L . 2-active dry yeast
solution at 50 gm/L. 3-active dry yeast solution at 75gm/L 4- control (spray with
distilled water). Dates and numbers of spraying with active dry yeast solution were
recorded as follows:-1-once a season sprays (at fruit setting stage on May). 2-twice a
season spray (at fruit setting stage (on May as well as in1 fruit development stage on
July Number of fruit set, number of fruit retention, fruit drop percentage and the
average palm yield (kg) were recorded .The experimental palms were arranged in
Split.Split design. At the maturity stage each Season, samples of fruits (about 30) were
taken randomly from each bunch per treatment and the following data were recorded:
1- Fruit physical properties.
Fruit weight (gm), fruit length (cm), fruit diameter (cm), flesh weight, (gm) fruit volume
(Cm3) .and seed weight (gm).
Fruit chemical properties.
Total soluble solids (T.S.S) as percentage of fruit juice using Digital refractometer.
Percentage of total acidity as g. citric acid / 100 gm f.wt.as described by A. O. A. C.
(1980). Total tannins concentration was expressed as mg tannins / g f.wt. Fruit peel as
described by Resenabatt and pelluso (1941). Crude fibers were determined on 1 g fruit
flesh sample by adding glacial acetic acid and nitric acid (10: 1) as described by A. O.
201
A. C. (1985). Reducing and total sugars concentrations were determined according to
Lane and Eynon method, as described by A. O. A. C. (1985). The non-reducing sugar
concentration was calculated from the difference between total and reducing sugar.
Statistical analysis:
All data were subjected to statistical analysis according to the procedure reported by
snedecor and cochran (1980). L S D at 5% level of significance was used to compare
between means according to steel (1960).
Results and discussion
Data in Tables and figures (1, 2, 3 and 4) revealed that active dry yeast as foliar
spray with concentration of 25gm/L, 50gm/L and 75gm/L for twice spray caused
increasing in fruit set number, fruit retention number and yield , while induce
decreasing in fruit dropping percentage. Data showed significant variable response in
cultivars, whereas Sewy gained the highest value for fruit setting number and fruit
retention number, in the same time Sewy cultivar gave the lowest value of fruit
dropping percentage at 75gm/L,50gm/L and 25 gm/L of active dry yeast for twice
spray in both seasons of study. Also data in Table and figure (1) cleared that there was
no difference between once Spray and twice Spray in both seasons of study, while
there was no difference between once spray and twice spray in the second season of
study for fruit retention number (Table and figure 2). The improving effect of active
yeast solution might be attributed to their effect on stimulating growth and nutritional
status of the date palm tree. The present results are in accordance with those obtained
by Abd –El-wahab (1999), Ebrahim et al., (2000),Abd El-Moniem et al., (2002)and
Ahmed et al.,(2003).
present results are in accordance with those obtained by Abd –El-wahab (1999),
Ebrahim et al., (2000),Abd El-Moniem et al., (2002)and Ahmed et al.,(2003).
Fruit Physical Properties:
Positive effects attributed to spray with active dry yeast and different tested
concentrations of the active dry yeast were evident on physical characteristics of fruit
weight, flesh weight, seed weight, fruit length, fruit diameter and fruit volume (Tables
and figures 5, 6, 7, 8 ,9 and 10). The highest values of those parameters were detected
in both cultivars under study and in both seasons, active dry yeast is considered as a
202
source for IAA and cytokinin-like substances which encourage the uptake of various
nutrients (Moor, 1979 and Ferguson et al., 1987). Data presented in Tables and figures
(5, 6 and 7) obviously revealed that, the effect of 75 gm/L active yeast for twice spray
was more pronounced than the other treatments, followed by the concentration of 50
and 25 gm/L active yeast in both seasons of study. Referring to the effect of cultivars,
the tested cultivars showed variable response in this concern as Sewy cultivar gave the
highest value of fruit weight and flesh weight, while, Hayany cultivar gave the highest
value of seed weight in both seasons of study (there was no difference between once
spray and twice spray). These results are in agreement with Laz et al., (2000) and Abd
El-Wahab et al., (2008). Data in Tables ,Figures (8, 9 and 10)and photos concerning
fruit length, fruit diameter and fruit volume showed that the obtained results are nearly
similar, where, the concentration of 75 gm/L followed by 50 and 25 gm/L active yeast
(as foliar spray) for twice spray gave the highest value of fruit length fruit diameter
and fruit volume. Hayany cultivar gave the highest value of fruit length, fruit diameter
(except in the second season Hayany and Sewy cultivars gave the same value of fruit
diameter) and fruit volume. Concerning the interaction between the treatments and
cultivars, the results were obtained by Ahmed et al., (1998), Laz et al., (2000) and Abd
El-Wahab et al., (2008).
Fruit Chemical Properties:
It is clear from the data in Tables and figures (11, 12, 13, 14, 15, 16 and 17) that
treatments with active yeast caused a substantial and significant improvement in fruit
quality (total sugars, reducing and non-reducing sugar, T.S.S. percentage, tannins
percentage, total acidity and crude fibers) in both seasons of study. Data in Tables and
figures(11, 12, 13 and 14) revealed that, active yeast spray caused a substantial and
significant improvement in total sugars, reducing and non-reducing and T.S.S.
percentages. It is noteworthy to mention that 75gm/L active yeast spray for twice spray
was more pronounced that the other treatments in both studied seasons. The values of
total sugars and T.S.S. contents differed significantly between the two date palm
cultivar. The highest percentages was in Sewy, while, Hayany cultivar gave the
highest value for reducing sugar, also once spray gave the highest value for reducing
sugar. The increasing of total sugars percentage related to increasing photosynthesis
203
and synthesis and accumulation of sugars, so increased TSS (El-Mogy et al., 1998).
These results are in harmony with those found by Ahmed et al., (1998) and Laz et al.,
(2000). Data presented in Tables and figures(15, 16 and 17) revealed that, treatment
with active yeast induced better effect in decreasing tannins, total acidity and crude
fibers percentages. Moreover, it is clear that, concentration of 75gm/L for twice spray
was the most effective than the other treatments in both seasons and gave the lowest
values of tannins, total acidity and crude fibers percentages. Results also cleared that
Sewy gained the lowest values of tannins, total acidity and crude fibers. Concerning
the interaction between the treatments and cultivars were significant. These results are
in harmony with Ahmed et al., (1998) they found that active yeast as a foliar spray
caused lowered acidity.
On the other hand, spraying with active yeast solution contains high amounts of
four vitamins, especially B which plays an important role in improving growth and
controlling the incidence of fungi diseases. These results are in agreement with those
found by Tarrow and Nakase (1975), Laz et al., (2000), Abd El-hady (2001), El-sayed
(2002) and Ahmed et al., (2003).
Generally, spraying with active yeast solution improving fruit quality, increasing
both fruit physical characteristic and fruit chemical characteristics (except tannins,
total acidity and crude fibers)these results were due to the contents of the active yeast
which consists of protein (41.2%), arginine (2.6%), glycine (2.6%), histidine (1.4%),
isolaysinel (2.9%), lauicine (3.5%),lysine(3.8%),methionine(0.6%), cystine(0.6%),
phenyl alanine(3%), tyrosine(2.14%), theronine (2.6%), tryptophan(0.5%) and vitamin
B (2.9%), ash (7.55%), glycogen(6.54%),fats (2.9%)and Cellulose (4.92%),according
to N.R.P(1977) and Ahmed et al.,(1998).
204
References
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207
208
209
210
211
212
213
214
215
Table(17) - Effect of active dry yeast on crude fibers percentage (%) of Hayany and
Sewy cultivars during 2008 and 2009 Seasons.
Fig (1) - Effect of active dry yeast on Setting fruit number of Hayany and
Sewycultivars during 2008 and 2009 Seasons.
Fig (2) - Effect of active dry yeast on fruit retention number of Hayany and
Sewy cultivars during 2008 and 2009 Seasons.
216
Fig (3) - Effect of active dry yeast on fruit drop percentage of Hayany and
Sewy cultivars during 2006 and 2009 seasons.
Fig (4) - Effect of active dry yeast on yield (kg/tree) of Hayany and
Sewy cultivars during 2008 and 2009 Seasons.
Fig (5) - Effect of active dry yeast on flesh weight (gm) of Hayany and
Sewy cultivars during 2008 and 2009 Seasons.
217
Fig (6) - Effect of active dry yeast on fruit weight (gm) of Hayany and
Sewy cultivars during 2008 and 2009 Seasons.
Fig (7) - Effect of active dry yeast on seed weight (gm) of Hayany and
Sewy cultivars during 2008 and 2009 Seasons.
Fig (8) - Effect of active dry yeast on fruit length (cm) of Hayany and
Sewy cultivars during 2008 and 2009 Seasons.
218
Fig (9) - Effect of active dry yeast on fruit diameter (cm) of Hayany and
Sewy cultivars during 2008 and 2009 Seasons.
Fig (10) - Effect of active dry yeast on fruit volume (cm3) of Hayany and
Sewy cultivars during 2008 and 2009 Seasons.
Fig (11) - Effect of active dry yeast on total sugars percentage (%) of Hayany
and Sewy cultivars during 2008 and 2009 Seasons.
219
Fig (12) - Effect of active dry yeast on reducing sugars percentage (%) of
Hayany and Sewy cultivars during 2008 and 2009 Seasons.
Fig (13) - Effect of active dry yeast on non reducing sugars percentage (%) of
Hayany and Sewy cultivars during 2008 and 2009 Seasons.
Fig (14) - Effect of active dry yeast on total soluble solids percentage (%) of
Hayany and Sewy cultivars during 2008 and 2009 Seasons.
220
Fig (15) - Effect of active dry yeast on Tannins percentage (%) of Hayany
And Sewy cultivars during 2008 and 2009 Seasons.
Fig (16) - Effect of active dry yeast on total acidity percentage (%) of Hayany and
Sewy cultivars during 2008 and 2009 Seasons.
221
Fig (17) - Effect of active dry yeast on crude fibers percentage (%) of Hayany
and Sewy cultivars during 2008 and 2009 Seasons.
222
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223
224
PP 07
Impact of reducing soil in the area of date palm dike (Jura) and
the annual amount of irrigation water and fruits cover at date
palm with the conditions of organic farming
Rashed M. F.
Plant Pathology Research Institute – Agric. Res. Cent. – Egypt
Technical Manager - Alfakhra Farms For Date Palm – Alghat – Saudi Arabia –
dmfawzy@windowslive.com – d_mfawzyrashed@yahoo.com
Abstract
Rhizosphere area and irrigation water are the important factors for increase or
decrease in the crop of date palm. Organic farms and additional amount of compost
yearly causes the increase of surface inside or around the palm tree. Therefore
additional soil to elevate the dikes and creating new rizosphere area. Experiment was
conducted by two methods: 1. Removing the soil under the palm tree making it level to
the surface soil. 2. Removing the soil under the palm tree and dig 15 cm. underneath.
This work depended on 40% from water and minerals absorbed through 60 cm. deep
from the surface of soil (Zied, A, et al., 2005 ). The experiment was carried out two
time (2009, 2010). The result was increase in the crop and increase in the dry leaves in
first year only without affecting the crop of dates. Another experiment to reduce the
amount of irrigation water and increase the times of irrigation was conducted. The
result was decrease in the number of bunches and amount of crop and size of fruits but
the dry leaves were less in comparison to control .
Key words: date palm- soil- compost- rizosphere.
Introduction
The result of adding organic fertilizer per year for Date Palm in organic farms, the
palm Dike rise from the surface of the soil and emerge in this region, the roots of the
Palm spread in the base of palm Dike(Jura) is need additions of soil a year to
leveling the borders of the high Dike addition to the non-renewal or leveling the soil
and work for the continued presence of some pests and diseases that inhabit the soil
.These lesions are waiting for the existence of appropriate conditions to infect the plant
225
and then we started working experience in the removal of this rise and renew the soil
in two ways. First remove the soil around the base of the Palm and leveling the
ground surface and the second to remove the soil around the base of the Palm with the
deepening of the soil beneath the earth's surface for a distance of 15 centimeters with
the retention in both cases around the base of palm with filing up of soil .
Do not appear until the aerial roots and the lack of humidity and access to the trunk,
depending on the area to absorb the initial 40% of water and salts from the soil surface
is located to 60 cm (Zaid et al 2009).
The experiment was repeated during the two seasons farm 2009 and 2010 and the
results were good, especially as the crop was not affected as a result of the removal
and deepening the soil in addition to ease of service down the Palm and easily separate
the off-shoots, which became clear after the deepening of Jura as well as the ease of
tillage and serve the soil in a farm of palm without hindrance from the high dikes– On
the other hand resulted in reduction of 50% and 25% of irrigation water annual bee
with convergence times of irrigation according to an annual calendar led to the decline
in the number of bunches resulting in irrigation low-bunches resulting from the
comparison irrigation usual on farms and then a few annual crop of trees in trial The
size of the fruits, it has not been affected in the case of reduced irrigation 25%,but it
has been affected in size in case reduction of water 50%. In a test coverage of the fruit
with the beginning of Khelal stage (Dates or Besr) when you start coloring partial
results have been few infestation with Dust Mites and fungal fruit spots resulting from
the wounded fungus Thielaviopsis Paradoxa (Rashed MF (1998) and Nafea AMA
(1995)), as well as non-affected the sun that burned fruits(sunrise injury).
Material and Methods
Reduce of the soil in the palm Dike ( Jurua).
First, the selection of 12 palm trees, Khidry variety of old more than 10 years to carry
out 3 treatments each treatment 4 Palms (from10-2008 until 10-2010 ).Second, 75
tree Saqae var and 75 Khidry var. were selected to make treatments on each item on
each treatment to take place on 25 palm of every varieties (from 10-2009 until 102010 and the treatments were as follows:
226
First treatment of the leveling of Jura surface soil with the work of Dike on the Palm a
heightof15cm.
- Second treatment :leveling of soil around the palm and then dredging the bottom of
the soilsurface to 15 cm
-Third Treatment : left the Jura as they are for comparison
At harvest (month 9-2010 ) is the weight of each crop and calculate number of dry
leaves for each treatment .Reduce of irrigation with rapprochement the number of
irrigations of the (Sokary variety).
150 Palm was chosen for three transactions each transaction 50 Palm as follows:
The first : 50% reduction of the annual amount of water as in the following table:
IrrigationExperiment
Place of
No. of Trees
Variety
Age
Soil Type
50 trees
Sokari
Adult
Sandy
No. of
Irrigation/
Year
Amount of
Water per
Irrigation
period of
Irrigation
1
130 times
650 L
45 min.*
84.5 cu.m
2
130 times
975L
65 min.*
126.75 cu.m
Control
87
2000 L
120 min.*
172
Experiment
Gaa FARM
Means of Irrigation
TREATMINTS
Total Amount
of
Water/ Year/
Tree
• Note : can be change leaning on Bubbler flow
• No. Of Irrigation in Months through the Year
The second
comparison (total number in the comparison includes the weeks
between the months) Compared to a normal irrigation farms is as follows:
227
Treatment Month Jan Feb Mar
s
.
.
.
Apr.
May Jun Jul. Aug. Sep Oct. Nov Dec
.
.
.
.
.
Total
1
No.
7
8
10
12
15
15 15
15
12
10
6
5
130
2
NO.
7
9
11
13
14
14 15
14
12
10
6
5
130
Control
NO.
4
4
4
8
8
8
8
8
8
8
4
86
* Quantity of water/ irrigation= 2 cum
8
** No. of irrigation/ year= 86 times ***
Amount of water/year/tree= 172 cum
Method of calculating the weight of the crop to be compared.
Each time the crop harvest is weighed each tree (h).
Weighed each crop trees each one separately and combining the number of times (h)
Divides the weight of each (h) the number of trees in the treatment (x) to indicate the
rate of production per tree (s)
Weight of total yield for the treatment = x (h)
Rate of production per tree (s) = x (h) / x
How to calculate the size of fruits
Taken from each tree in the treatment of the fruit of 30 randomly measured the rate of
development of the fruit along the fruit lengthwise and measured and then divided the
result by the number and width is measured by putting next to each other horizontally
and then measured and divided the result by the number.
Bunchescover:
The fungus was isolated from fruit spots at the beginning of coloring (khilal stage) the isolation from samany var. in Egypt after the sterilization of fruit surface of cotton
with ethyl alcohol then put the fruit in the room moisture under the bell glass was
unarmed after the appearance of fungus developing on the spots were defined in the
Plant Pathology Research Institute- Department of fungi- Agricultural Research Center
- Egypt. A way the definition of fungus at the level of genus, according to the taxonomic
systems of Ellis (1971), Barnett and Hunter (1972) and was covered fruits of the season
following the injury to prevent infection resulting from insects and sand storms.
The same work has been with Vnkha var. and some ( no name) palms in the farm
in Saudi Arabia with the appearance of fruit spots and the work of the coverage,
228
leaving a group of palm trees of 50 palm trees under the experiment to observe the
infection arising from the lack of coverage and has registered cases spots and infested
with spider dust mites and , effecting of sun especially in June and July.The infection
were recorded and calculate trees infected regardless of the severity of the infection is
where the registration of any tree fruit symptoms appeared on the spots or strings of
the spider mite or the combustion of fruit from the sun and measure the percentage of
infected trees every symptom of infection on the unit
Results and discussion
First: modify the Dikes ( Jura) :
Fig. 1: Comparison: Jura form before the modification
Fig.2 The first Treatment: form of Jura after deeping 15 cm
from the soil surface
229
Fig.3 Second Treatment : Jura leveling with the level of the soil surface
and the work of an end to them not to get out of irrigation water
A – 1 DIKES EXPERIMENTS
Farm : 2nd In Fakhra farm No. of trees: 4 in each treatment - Variety : Khidri
Age of experiment : 2year
Deferent weight of fruits between experiment & control
No. of harvesting
times
Type of Treatment
1
Deep 15cm
Leveling of
surface soil
Control
Net weight KG
391.4
436.3
356.3
Net weight / tree KG
98.0
109.0
89.0
Dry leaves NO.
4
7
5
A – 2- DIKES EXPERIMENTS
Farm : 1st Gaa - No. of trees : 25 in each treatment - Variety : Khidri
Age of experiment : 1year
230
Deferent weight of fruits between experiment & control
No. of
Type of Treatment
harvesting times
1
Deep 15cm
Leveling of
Control
surface soil
Net weight
2230.9 kg
2252.3 kg
2205.2 kg
Net weight /
97.0
98.0
96.0
19
23
40
tree
Dry leaves
A– 3- DIKES EXPERIMENTS
Farm : 1st Gaa - No. of trees : 25 in each treatment - Variety :SAGAAY
Age of experiment : 1year
Deferent weight of fruits between experiment & control
No. of
Type of Treatment
harvesting times
1
Deep 15cm
Deep 15cm
Control
Net weight
1795.7 kg
1775.8 kg
1655.6 kg
Net weight /
71.8
71.0
69.0
145
206
80
tree
Dry leaves
The farm after the experiment:
It is noted in Figure 4 easily tilled the soil in the case of development of Jura on the
level of the soil surface in the form of Fig. 5 and Fig.6 the ease of service of
deepening the ground and free of weeds
231
Fig. 4 and 5 and 6 illustrate the case of the farm after the experiment
Fig.4,5,6
232
These results show that the reduction of the soil, either in the first treatment (15 cm
below the soil surface) , the second treatment (leveling of Jura with the surface of soil)
did not affect on the crop, but it increased in both coefficients, both the first year or
second year the dry leaves were increased in the first year only in order to cut the roots
in the surface layer of the variety Sqaay only either Khidri variety it is not affected by
cutting the roots, but the dry leaves in the experiment were lower than the comparison
in the second year that the treatments were close - this experiment reduces the
additions of soil, which raises the limits of Jura and is working on activating the root
zone and increase the activity of microbes beneficial to increase the fertility of the soil
(Hussein Fathi et al 1979) and the removal of soil beneath the plant each period of
work to get rid of pests and diseases that are dormant in the soil from one season to
another until the appropriate conditions for the infection - in addition to the case of
good farm and ease of service of soil after the experiment as shown figures 4, 5 and 6.
The results of the experiment of irrigation
Number of bunches after pollination and bearing fruits shows that the average number
of bunches in 50 tree in first treatment (50% reduction of irrigation water) were) 14)
and second treatment (25% reduction of irrigation water) were (16) and were
comparable normal Irrigation) was (18) and transfer the final results after harvest were
as follows:
– EXPERIMENT of IRRIGATION Reduction
No. of trees for 3 treatments : 50 - Variety : Sokary - Age of experiment : 2 Year
Deferent weight of fruits between experiment & control
233
Net
weight
Treatments
KG.
3350
Y
SOKARI
EXEPRIMEN
T
LONG/c
WIDTH/c
m
m
IRRIGATION
EX. 50%
3.2 cm
2.8 cm
3.6
3,2
3.6
3.2
DIKE- 15deep
4.2
2.1
DIKE-surface
4.3
2.3
DIKE – Cont.
4.1
2.2
DIKE- 15deep
4.5
2.4
DIKE-surface
4.6
2.3
DIKE – Cont.
4.7
2.3
DIKE- 15deep
4.5
2.5
DIKE-surface
4.5
2.5
4.6
2.7
Dry
weight leaves
per tree
3015.7 61.5
Reduction 50%
VARIET
Avg.
67
191
157
RE
reduction
IRRIGATION
EX 25%
IRRIGATION
CONT.
KHIDRI
level
KHIDRI
level
SAKAEY
level
DIKE – Cont.
REDUCTION 25%
3588.6 73.2
CONTROL
234
240
Experiment has shown two results the first decline in yield due to lower amount of
water received by the plant from the beginning the number of bunches and then
decreased final crop, but the convergence periods of irrigation in the experiment
reported in reducing the number of dry leaves, especially in the summer months, with
the number of dry leaves by 21% for comparison
SIZE OF FRUITS IN EXEPRIMENTS (irrigation & dike reduction )
Impact at the volume of fruits
The results of the size of fruits in the different experiment confirmation of the above
In the experiment of irrigation were fruit smaller than control in length and width in
the case 50% decrease while in case 25% decrease not affected by size only limited
quantity of the crop to the lack of bunches resulting when flowering but in the
experiment of dikes and reduce soil was the best size fruit in treatment of the leveling
of dike( Jura) with the surface of the soil in the second year in Kidri var. in the other
treatments, there was no significant differences between the sizes.
Experiment of bunches covering
Symptoms resulting from the lack of covering (Figs7, 8,9)
Fig. 7 Fruit rot (infection of thielaviopsis paradoxa at khilal stage )
235
Fig.8 Spider of dust mite: (natural infection)
Fig.9 Sun effecting (natural infection )
Fig.10 Covering of bunches
236
Results Coverage:
Infection
50 tree not covered
50 tree covered
Fruits spots
3%
0.0
Dust Mite
40%
2%
Sunburn
4%
0.0
Fig. 11 Date fruits covering : notes it is free from any infection (var. no name)
Fig. 12 The same tree the previous (no name), but the
bunches not covered on the same tree to compare
237
- notes infection with dust mites and fruit spots
The same tree the previous (no
name), but the bunches not covered on the same tree to compare
- notes infection with dust mites and fruit spots Conclusion
Results reflect the experiment of dikes that the removal of soil by tillage and others
to a distance of 15 Cm below the soil surface does not affect production and can be
done very two or three year whenever higher the tree dikes from soil surface to ease
agriculture operations below the trees. Water for irrigation is the main factor affecting
the production. Any reduction of irrigation water must be well thought out so well for
the quantity and quality of production.
The use of fruit covering technique and the
quality of the bag and time of coverage are very important in the control of pests and
diseases, especially in organic farming
238
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[3] Alnisf Yusuf (1997), Nkhltak – Al Safah – Kuwait.
[4] Barnett., H.L. and B.B. HUNTER, 1972. Illustrated Genera of Imperfect Fin
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[6] Hussein Fathi Ahmed, Alakjtani Mahmoud Said, Wali Youssef Amin, 1979,
Date palm
cultivation and production of dates in the Arab and Muslim
worlds - Press Ain Shams University - Cairo, Egypt.
[7] Ibrahim Atef, Khalif
Mohammed Nazif,( 1998), date palm cultivation and
stewardship, production in the Arab world – Dar El Maaref House
Alexandria - Egypt.
[8] Nafea, A.M.A. 1995. Pollution of dates by post-harvest pathogens. M.Sc.
Thesis, Fac. Agric. Ain Shams Univ. 166 pp.
[9] Rashed, M.F. (1998) Pathological Studies on Black Scorch disease of Date Palm
.Ph.D.Thesis,Fac.Agric.,CairoUniv.110pp.
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239
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240
PP 08
Influence of nutrients spray application on the yield traits of
"Zaghloul Cv." date palm
El Assar A. M.* and O. A. M. El Sehrawy **
*Agriculture Research Center, Horticulture Research Institute (Nubaria Horticulture
Research Station), Giza, Egypt.
** Sustainable Development of Environment Department, Environmental studies &
Research Institute (ESRI), Sadat Branch, Menofia University, Egypt.
a-assar@hotmail.com, oaelsehrawy@gmail.com
Abstract
This experiment was conducted in two successive growth years (2009 and 2010
years) on 30 years old "Zaghloul Cv." date palm growing in calcareous soil. Labeled
female uniform date palm trees were submitted to the usual farm management such as
irrigation, pollination, pruning and soil fertilization with both of mineral (N, P, K and
S) and organic (animal manure) to study the influence of spraying some macro and
micro- elements on the yield and both of their fruit physical and chemical quality
traits. Fruit bunches sprayed two times with N (250 & 500 ppm), K (500 & 1000
ppm), B (1000 & 2000 ppm) and Zn (250 & 500 ppm) at Kimri stage for the 1st time
and at Khalal stage for the 2nd time. The obtained results indicated that all the studied
treatments significantly improved the yield of "Zaghloul Cv." date palms however;
both of Boron and Nitrogen treatments were superior. Fruit weight and flesh/seed ratio
were significantly improved with particular effect own view for Zinc which may
increase the flesh weight .While, Potassium, Boron and Zinc treatments improved both
of the TSS (%) and total sugars (%) as well as decreased both of the fruit juice acidity
(%) and soluble tannins (%).
Introduction
Date palm “Phoenix dactylifera L.” (Nakhla in Arabic) is the tree of life and one of the
oldest fruit crops known around the world. It considers one of the most important fruits
not only in the Arab countries but also in other Middle Eastern/North African countries.
The date palm can grow well in hard environmental conditions, such as drought, salinity,
241
high temperature, and poor soils. Date palm is the most successful and important
subsistence crop in most of the hot arid desert regions [6] (Botes and Zaid, 1999).
Dates have been used as a major/main staple food for several thousands of years.
Their high energy value and good store-ability make dates a wise choice of crop in
places where they can be grown. Date palm starts to be the expend crop all over Egypt.
In Egypt, dates are important traditional crops. According to [14] FAO (2009), Egypt
is considered as the first country of the top ten date producers (1,130,000 tones).
Economically Zaghloul date is the most important soft cultivar grown in Egypt. It is
usually harvested and consumed at Khalal stage when fruits reach full maturity and are
crunchy and red in color. As Date palm is considered one of the suitable trees which
can be cultivated in the new reclaimed desert regions, most of the date palms produced
in Egypt are grown in sandy, loam and sandy loam or even in a carbonate rich soil.
These types of soils may become deficient in N, P, K, Mg and B [22] (Tisdale and
Nelson, 1978). It has also been reported that large amounts of macro and
microelements are depleted annually from the soil as a result of high crop yields and
pruning of leaves [13] (El-Shurafa, 1984). Several investigators have been studied the
fruit physical properties and chemical constituents of various date palm cultivars; and
the factors that affected their composition but little investigations searching the spray
application nutrients which consider a suitable method to overcome soil problems in
extend regions.
Nourish effect of macro and micro elements had important role in fruit set, retention
development and cause efficient yield and fruit quality improvement. While soil
application can supply enough nutrients to improve plant production, it also causes
world-wild anxiety about environmental contamination for nutrients leaching into
ground water [7] (Dinnes et al., 2002). Foliar fertilization has the advantage of using
low application rates, uniform distribution of fertilizer materials and quick responses
to applied nutrients.
Materials and Methods
This experiment was conducted in two successive growth seasons (2009 and 2010
years), at Nubaria Horticulture Research Station orchard under the calcareous soil
conditions on 30 years old date palm trees "Zaghloul Cv.". Labeled female uniform date
242
palm trees were submitted to the usual farm management such as irrigation, soil
fertilization pollination and pruning. The organic fertilizer (animal manure) was added at
winter (mid of December) as one dose while the mineral fertilizer (Ammonium sulphate
500 g + potassium sulphate 250 g) was added on two doses (the 1 st dose was mixed with
organic fertilizer at the winter and the 2nd dose was added at the end of May (during fruit
growth period). Thus, each palm received 1000 g rock phosphate and 500 g mineral
sulphur at the time of animal manure addition. The experimental treatments were as
follows (1) distilled water (control), (2) nitrogen (250 ppm), (3) nitrogen (500 ppm), (4)
potassium (500 ppm), (5) potassium (1000 ppm), (6) boron (1000 ppm), (7) boron (2000
ppm), (8) zinc (250 ppm) and (9) zinc (500 ppm). Nitrogen was added as Urea (46%),
potassium was added as potassium sulphate (K2SO4) (49%), boron was added as boric
acid (H3BO3) and zinc was added as zinc sulphate (ZnSO4). The used chemical solutions
were provided with wetter agent (Tween-20) to enhance distribution and absorption.
Selected palms were thinned to eight bunches after Hababouk stage. Fruit bunches were
sprayed twice, at Kimri stage for the 1st time and at Khalal stage for the 2nd time. Samples
were collected at harvest stage (1st week of October). The obtained data were tabulated
and analyzed using Completely Randomized Block Design (CRBD) with 9 treatments and
four replications according to [21] Steel and Torrie (1980). Yield of studied palms was
recorded in terms of kg per palm at harvest time in farm. In laboratory, physical fruit
characteristics such as fruit weight (g), length and diameter (cm) were measured. As well
as flesh weight (g), seed weight (g) and flesh / seed ratio were determined. Fruit quality
characteristics were determined as follows: total soluble solids percentage (TSS%) in fruit
juice was measured using hand refactometer, juice acidity percentage (as malic acid) was
titrated [2] (A.O.A.C., 1995), total sugars percentage was determined in dried fruit flesh
under 56 Co in an oven until constant weight [17] (Malik and Singh1980), total protein
(%) according to [15] Jackson (1967) and dry matter percentage was taken after drying
fruit flesh samples under 65 Co until constant weight.
Results and Discussion
Yield of palm (kg / palm):
Data of Table (1) indicated that yield of palm was statistically affected by
experimental treatments. Yield of control treatment was significant lowest in the two
243
studied seasons (78.3 and 79.1 kg/palm for 1st and 2nd seasons, respectively) as
compared with yield of all other treatments. Yield of boron at 2000 (ppm) treatment was
significantly superior but without significant difference as compared with yield of
nitrogen at 500 (ppm) treatment in the 1st and 2nd seasons (130 and 126 kg/palm for 1st
season; 133 and 133 kg/palm for the 2nd season, respectively). Yield of potassium
treatments at (500 and 1000 ppm) and zinc treatments (250 and 500 ppm) have no
significant differences in the 1st study season (99.2 and 99.8 kg/palm; 93.9 and 99.1
kg/palm, respectively). However, yield of boron 2000 (ppm) treatment was significantly
superior the yield of both zinc treatments in the two seasons of study, Table (1). These
results are in line with those of [11] El-Sayed El-Badawy and El-Dengawy (2001) on
Hayany cv. date palm, [16] Khayyat et al. (2007) on Shahany cv. date palm and [12] ElSehrawy (2008) on Anna apple trees. But, [23] Yogaratnam and Greenham (1982a) have
shown that urea spray on apple trees did not increase the yield
Fruit weight (g):
Table (1) cleared that, fruit weight values of control treatment was significantly
lower than fruit weight of all other studied treatments (20.3 and 21.3 g for the 1 st and
2nd seasons, respectively). In addition, fruit weight values of both nitrogen and boron
treatments were significantly superior all other studied treatments without significant
differences for the two studied seasons. As for the potassium and zinc treatments, fruit
weight values were significantly medium without significant differences. These results
are agreed with those of [11] El-Sayed El-Badawy and El-Dengawy on Hayany cv.
date palm (2001) and [19] Mengel (2002) on gases plants.
Seed weight (g):
Data tabulated in Table (1) indicated that differences among values of seed weight
were not significant. It means that seed weight trait was not significantly affected by
all experimental factors in the two seasons of study. Likewise, it means that the
differences were found among the fruit weights were due to flesh weight differences
not due to seed weight differences. These results are in harmony with those of [9] ElAssar and El-Kouny (2010) on Zaghloul cv. date palm.
244
Flesh/seed (ratio):
Data of Table (1) cleared that flesh/seed ratio under control treatment was
significantly the lowest in comparison with those of other treatments in the two studied
seasons (7.25 and 7.34 for 1st and 2nd seasons, respectively). It consider a logical result
because fruit weight of control treatment was significantly lowest, regardless the seed
weight trait. On the other hand, flesh/seed ratio for nitrogen (250 & 500 ppm), boron
(1000 & 2000 ppm) and zinc (250 & 500 ppm) treatments were significantly superior
both potassium (500 & 1000 ppm) treatments without significant differences in the
two seasons of study. These results are in line with those of [11] El-Sayed El-Badawy
and El-Dengawy (2001) on Hayany cv. date palm and [16] Khayyat et al (2007) on
Shahany cv. date palm.
Total soluble solids percentage (TSS %):
Total soluble solids percentage values were significantly affected by the
experimental treatments in the two studied seasons. Data in Table (2) showed that TSS
(%) values related with potassium (500 ppm), potassium (1000 ppm), boron (1000
ppm) and boron (2000 ppm) treatments were significantly superior all other TSS (%)
values in the 1st study season without significant differences (24.15, 24.75, 24.15 and
24.85 %, respectively). TSS (%) values of both zinc treatments (250 and 500 ppm)
came second without significant difference (23.00 and 23.10 %) followed by both
nitrogen treatments (250 and 500 ppm) without significant difference (20.55 and 20.75
%) while TSS (%) of control treatment was significantly the lowest value (18.20 %).
TSS (%) values of all treatments had taken the same significant places in the 2 nd
season (Table 2). These results are in line with those of [4] Basha and Shaheen (1986)
on Nebut Seif and Ruzeizi cvs. date palm, [11] El-Sayed El-Badawy and El-Dengawy
(2001) on Hayany cv. date palm and [12] El-Sehrawy (2008) on Anna apple trees.
While [16] Khayyat, et al. (2007) on Shahany cv. date palm reported that TSS (%) trait
not significantly affected by similar treatments.
Fruit juice acidity percentage (%):
Fruit juice acidity percentage values were significantly affected by all the studied
experimental treatments during the two seasons of study, Table (2). Data indicated
that both of zinc treatments (250 & 500 ppm) were significantly superior to all other
245
studied treatments without significant differences in the two studied seasons (1.41 and
1.38 (%) for the 1st season; 1.39 and 1.35 (%) for the 2nd season, respectively). Values
of studied criterion related with nitrogen (250 & 500 ppm), and boron (1000 & 2000
ppm) treatments were significantly came second without significant differences
between the two levels of each factor (1.48, 1.45, 1.50 and 1.48 (%) for the 1st season,
respectively and 1.48, 1.44, 1.51 and 1.48 (%) for the 2nd season, respectively).
While, fruit juice acidity (%) which related with both of potassium treatments (500 &
1000 ppm) were significantly came third without significant difference, (Table 2).
Always, control treatment leads to absolute highest value of fruit juice acidity (1.80
and 1.78 (%) for the 1st and the 2nd seasons, respectively). [3] Bacha and Abo-Hassan
(1983) on Khudari cv. date palm, [10] El-Kouny et al. (2004) on Zaghloul cv. date
palm and [8] El-Assar (2005) on Zaghloul cv. date palm studied the mineral and
organic fertilization and found similar results. Also, these results agree with those of
[12] El-Sehrawy (2008) who studied the influence of mineral, bio and organic
fertilization on Anna apple trees. [5] Bacha et al. (1995) reported similar results on
grape with zinc foliar application.
Total sugars percentage (%):
Data of Table (2) cleared that this parameter had significantly affected by the
studied factors. Boron (1000 & 2000 ppm) and nitrogen (500 ppm) treatments
significantly caused the high total sugars (%) values in comparison with all other
studied treatments and levels in the two seasons of study without significant
differences (80.45, 80.45 and 80.35 (%), respectively for the 1st season; and 80.75,
80.65 and 80.75 (%), respectively for the 2nd season). Data tabulated in Table (2)
illustrated that no significant difference was found between related trait values of
either two boron levels or two zinc levels in the two studied seasons. Low significant
value of total sugar (%) was related with control treatment in the two seasons of study
(70.25 and 70.15 (%) for the 1st and 2nd season, respectively). [18] Marzouk and
Kassem (2011) found similar results when studied the application of organic and/or
mineral fertilizers to improving fruit quality, nutritional value and yield of "Zaghloul"
dates. On the other side, [12] El-Sehrawy (2008) found that both of the used treatments
246
of organic and bio fertilization significantly increased the total sugar (%) of Anna
apple fruits as compared with those of mineral.
Soluble tannins percentage (%):
Results reported in Table (2) showed that soluble tannins (%) values significantly
affected by the studied treatments in the 1st season. Whereas, all studied factors and
their levels have absolute low soluble tannins (%) values without significant
differences as compared with control treatment's value. While, no significant
differences have noticed among values related with all the studied factors, levels and
control treatments in the 2nd season. These results are in line with those of [11] ElSayed El-Badawy and El-Dengawy (2001) on Hayany cv. date palm and those of [9]
El Assar and El Kouny (2010) on Zaghloul cv. date palm.
Total protein (%):
Table (2) illustrates significant impact of studied treatments on total protein
percentage, whereas N (500 ppm) significantly superior those of all other treatments in
the two studied seasons (2.10 and 2.12% for the 1st and 2nd seasons, respectively).
Followed by N (250 ppm) which have significant difference as compared with all other
treatments in the two seasons of study (1.99 and 2.00% for the 1st and 2nd seasons,
respectively). Control treatment had significantly the lowest value without significant
difference in comparison with K (500 and 1000 ppm) and B (100 ppm) in the two
studied seasons. Moreover, date of Table (2) indicated that Zn (500 ppm) lead to the
high protein percentage as compared with control, K (500 and 1000 ppm) and B (1000
and 2000 ppm) in the two seasons of study beside Zn (250 ppm) at the 1st season. These
results are in line with those of [1] Abd El-Zaher (2008) on Barhee cv. date palm and
[20] Soliman and Al-Obeed (2011) on Khalas and Nabut-Sif date palm cultivars.
247
References
[1] Abd El-Zaher M. H. 2008. Studies on Seedless Fruits (Stone less) of Date Palm cv.
Barhee: Effect of Spraying with Some Natural Materials on Pollinated Fruits,
Unpollinated Fruits and Properties of the Fruit Set. Journal of Applied Sciences
Research, 4(7): 906 - 916.
[2] Association of Official Agricultural Chemists. 1995. "Official Methods of
Analysis" 15th ed. Published by A.O.A.C. Washington, D.C. (U.S.A.).
[3] Bacha M. A. and A. A. Abo-Hassan. 1983. Effect of soil fertilization on yield, fruit
quality and mineral content of "Khudari" date palm variety. The 1st Symposium on
the date palm in Saudi Arabia, King Saud University, Riyadh, Saudi Arabia: 174-180.
[4] Basha M. A. and M. A. Shaheen. 1986. The effect of different leaf/bunch ratios on
yield and fruit quality of Nebut Seif and Ruzeizi date palm cultivars. Arab Gulf J.
Sci. Res. 4 (1): 341 - 347.
[5] Bacha M. A., S. M. Sabbah and M. A. El-Hamady. 1995. Effect of foliar
application of Iron, Zinc and Manganese on yield composition of Thompson
seedless and Roumy Red grape cultivars. Alex. J. Agric. Res. 40 (3): 315 - 331.
[6] Botes A. and A. Zaid. 1999. The economic importance of date production and
international trade. In: Zaid, A., Arias, E.J. (Eds.), Date Palm Cultivation, pp. 45–
57 (FAO plant production and protection paper no. 156).
[7] Dinnes D. L., D. L. Karlen, D. B. Jaynes, T. K. Kaspar, J. L. Hatfield, T. S. Colvin
and C. A. Cambardella. 2002. Nitrogen management strategies to reduce nitrate
leaching in tile-drained Midwestern soils. Agron. J., 94: 153 - 171.
[8] El-Assar A. M. 2005. Response of "Zaghloul" date palm yield to various organic
and inorganic fertilization types as well as fruit-thinning models in a rich carbonate
soil. J. Agric. Sci. Mansoura Univ., 30 (5): 2795 - 2814.
[9] El Assar A. M. and H. M. El Kouny. 2010. Reaction of multifarious nitrogen
fertilizer resources on the yield traits of "Zaghloul Cv." date palm and the
calcareous soil fertility. J. of Plant Production, vol. 1(3): 347- 365.
[10] El Kouny H. M., A. M. El Assar and S. G. Mohamed. 2004 Effectiveness of natural
organic amendments, biologically activated compost, and mineral fertilization
248
sources of potassium in improved soil properties and productivity of “Zaghloul
Cv.” date palm in a calcareous soil. Assiut J. Agric. Sci., 35 (2): 195 - 214.
[11] El-Sayed El-Badawy T. E. and E. F. A. El-Dengawy. 2001. Effect of calcium and
zinc sprays on fruit dropping nature of Hayany date cultivar. I - Yield and fruit
quality. Second International Conference on Date Palms (Al-Ain, UAE, March
25-27, 2001).
[12] El-Sehrawy O. A. 2008. Influence of Bio and Organic Fertilization on Growth of
Anna Apple in the Reclaimed Land. Ph.D. Thesis, Dept. of Hort., Fac. Agric.
Menofia University, Egypt.
[13] El-Shurafa M. 1984. Annual loss of minerals from date palm. Date Palm J., 3:
278 – 290.
[14] FAO. 2009. Food and Agriculture Organization of the United Nations. Fuleki T.,
Francis F. J., 1968. Quantitative methods for anthocyanins. 1. Extraction and
determination of total anthocyanin in cranberries. J. Food Sci., 33: 72 –77.
[15] Jackson M. I. 1967. Soil Chemical Analysis. Prentice Hall, Inc. N J, California, USA.
[16] Khayyat M., E. Tafazoli, S. Eshghi and S. Rajaee. 2007. Effect of nitrogen,
boron, potassium and zinc sprays on yield and fruit quality of date palm.
American-Eurasian J. Agric. & Environ. Sci., 2 (3): 289 - 296.
[17] Malik C. P. and M. B. Singh. 1980. Plant enzymology and histoenzymology. A
Text Manual. Kalyani Publishers, New Delhi.
[18] Marzouk H. A. and H. A. Kassem. 2011. Improving fruit quality, nutritional
value and yield of Zaghloul dates by the application of organic and/or mineral
fertilizers. Scientia Horticulturae, Vol. 127, 3: 249 – 254.
[19] Mengel K. 2002. Alternative or complementary role of foliar supply in mineral
nutrition. Acta Hortic. 594: 33 - 47.
[20] Soliman S. S. and R. S. Al-Obeed. 2011. Effect of Boron and Sugar Spray on
Fruit Retention and Quality of Date Palm. American-Eurasian J. Agric. &
Environ. Sci., 10 (3): 404 - 409.
[21] Steel R. G. D. and T. H. Torrie. 1980. Principles and procedures of statistics. NY.
2nd ed. McGraw Hill, NY, USA. (220).
249
[22] Tisdale S. L. and W. L. Nelson. 1978. Soil Fertility and Fertilizers. Macmillan
Publishing Co. Inc., New York, USA, pp.: 67–70.
[23] Yogaratnam N. and W. P. Greenham. 1982. The application of foliar sprays
containing nitrogen, magnesium, zinc and boron to apple trees. 1. Effects on fruit
set and cropping. J. Hortic. Sci., 57: 151 - 158.
250
Table (1): The effect of experimental treatments on the yield, fruit weight and
flesh/seed ratio traits of "Zaghloul" date palm cultivar in 2009 and 2010 years.
Studied
treatments
Yield (kg/palm)
Fruit weight (g)
Seed weight (g)
Flesh/seed (ratio)
2009
2010
2009
2010
2009
2010
2009
2010
Control
78.3
79.1
20.3
21.3
2.8
2.9
7.25
7.34
N (250 ppm)
115
119
29.6
29.8
2.9
2.9
10.21
10.28
N (500 ppm)
126
133
30.1
31.4
3.0
3.1
10.03
10.13
K (500 ppm)
99.2
98.9
25.4
25.2
2.9
2.8
8.76
9.00
K (1000 ppm)
99.8
102
25.7
26.4
2.8
2.9
9.18
9.10
B (1000 ppm)
121
129
28.8
30.5
2.7
2.8
10.67
10.89
B (2000 ppm)
130
133
30.2
32.1
2.8
2.9
10.79
11.07
Zn (250 ppm)
93.9
92.8
25.5
25.6
2.4
2.5
10.63
10.24
Zn (500 ppm)
99.1
96.9
27.1
26.6
2.5
2.5
10.84
10.64
LSD (0.05)
6.5
5.9
1.9
1.7
N.S
N.S
0.85
0.95
Table (2): The effect of experimental treatments on fruit quality traits of
"Zaghloul" date palm cultivar in 2009 and 2010 years.
Studied
treatments
TSS (%)
Fruit juice
acidity (%)
Total sugars
(%)
Soluble
tannins (%)
2009
2010
2009
2010
2009
2010
2009
2010
Control
18.20
17.95
1.80
1.78
70.25
70.15
0.26
0.23
1.68
1.72
N (250 ppm)
20.55
20.65
1.48
1.48
79.50
80.10
0.20
0.20
1.99
2.00
N (500 ppm)
20.75
20.95
1.45
1.44
80.35
80.75
0.20
0.19
2.10
2.12
K (500 ppm)
24.15
24.25
1.61
1.60
79.85
79.75
0.20
0.19
1.68
1.69
K (1000 ppm)
24.75
25.15
1.58
1.56
78.75
78.65
0.21
0.20
1.72
1.72
B (1000 ppm)
24.15
24.55
1.50
1.51
80.45
80.75
0.20
0.22
1.68
1.69
B (2000 ppm)
24.85
24.95
1.48
1.48
80.45
80.65
0.21
0.19
1.75
1.76
Zn (250 ppm)
23.00
23.05
1.41
1.39
79.85
79.85
0.19
0.18
1.88
1.90
Zn (500 ppm)
23.10
23.05
1.38
1.35
79.70
79.75
0.19
0.19
1.92
1.92
LSD (0.05)
0.85
0.75
0.03
0.04
0.15
0.15
0.03
N.S
0.07
0.06
251
2009
2010
Total protein
(%)
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252
Agricultural Biotechnology
253
254
OP 10
Date palm biotechnology: current status and prospective-an overview
S. Mohan Jain,
Department of Agricultural Sciences, University of Helsinki,
PL-27, Helsinki, Finland. Email: mohan.jain@helsinki.fi
Abstract
The date palm is one of the most ancient plants, grows in the regions of Middle
East, North Africa, South Sahel, East and South Africa; its sexually propagation
hampers propagation of true-to-type genotypes due to heterozygosis. The vegetative
propagation is carried out with the off shoots, produced from axillary buds situated at
the base of the trunk during the juvenile life of palm tree. Offshoot production is slow;
their numbers are limited, laborious and can’t meet the rapidly growing demand of
varieties. To speed up the date palm genetic improvement, in vitro culture techniques
could be handy; however, genotype influence limits the effective use. Bioreactor is
being used for large-scale production of somatic embryos. Somaclonal variation is
common among in vitro-derived date palm plants. However, it could broaden genetic
variability together with mutagenesis; molecular markers AFLP used to identify
variability and to select useful variants. Dwarf date palm hybrid was developed by
embryo rescue by site-specific hybridization of Phoenix dactylifera and P. Pusilla. In
vitro germplasm conservation is done by cryopreservation for long-term storage.
Alternatively, in vitro shoot cultures and plantlets are stored at 40C for short termstorage. Micro-calli is produced from date palm protoplasts; Agrobacterium-mediated
transformation succeeded in GUS gene expression in callus. Date palm genomics can
distinguish multiple varieties and a specific region of the genome linked to gender.
Introduction
The unique characteristics of date palm can be truly called 'tree of life' and is
considered as one of the most ancient plant, and is distributed throughout the Middle
east, North Africa, South Sahel, areas of East and South Africa, and even certain parts
of Europe and USA. It makes a significant contribution towards the creation of
equable microclimates within oasis ecosystems and thus enabling sustainable
agricultural development in saline and drought affected areas. The rich fruit plays an
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important role in the nutrition of human population, and also several products are
made that generate employment and thus influence socio economic aspect of people.
Therefore it is widely acknowledged sustainability value in social, economical and
ecological terms. Moreover, this crop has a great potential as a source of renewable
energy, an alternate source to the fossil energy, by producing bio-fuel since its fruits
high carbohydrates, 44-88% total sugars.
Sexual propagation is widely used for date palm propagation. However this method
can't be used commercially for propagating the cultivars of interest in a true-to-type
manner. Interspecific hybridization between the date palm (Phoenix dactylifera) and
the dwarf date palm (P. pusilla) has been successfully carried out, aimed at the
development of short hybrid date palms (Sudhersan et al. 2009) Heterozygosity in
date palm is related to the dioecious nature of date palm. Half of the date palm
progeny is generally male and they don't produce fruits, and also large variation can
occur in the progeny. There is no known method for sexing date palm at an early stage
of tree development and that makes hard to eliminate non-productive male trees in the
nursery before planting in the field. Another drawback of seed propagation is that the
growth and maturation of seedlings is extremely slow. A date palm seedling may take
8-10 years or more before fruiting occurs. It is not surprising that little work has been
done on date palm genetic improvement for developing new cultivars by traditional
approaches. Therefore to speed up the date palm breeding programmes, particularly
the areas where date palm is threatened by red weevil, devastating diseases like
Bayoud and Brittle Leaf; as source of bio-fuel, biotechnology would of great help in
overcoming these problems (Jain et al 2011).
Problems facing date palm genetic improvement
The date palm cultivation encounters several constraints mainly due to its
development under harsh desert conditions, e.g. water shortages, high temperature and
irregular supply of amendments.
Date palm also faces many biotic constraints,
especially bayoud disease caused by Fusarium oxysporum f. sp. albedinis (Carpenter
and Klotz 1966; Djerbi 1988). This disease is the most devastating to the date palm
cultivation and was first described in southern Moroccan groves.
Currently, it
continues to spread across North African countries, especially in Morocco and Algeria
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where more than 12 million date palm trees have been destroyed so far. No effective
means is known to control this disease and only a few cultivars with poor-quality
fruits, unfortunately, are known to be resistant to bayoud (El Hadrami et al. 1998).
Therefore, proper date palm cultivation requires, disease resistant cultivars, pruning,
pollination, fruit thinning, bunch removal and fruit harvesting, are highly essential for
good quality fruit production. The cost of date production increases when the trees
grow taller, due to the high labour cost in many producing countries. Mechanization is
also expensive and unjustifiable in the case of small growers. Frequent climbing for
fruit harvesting is highly dangerous in the case of taller old trees. Tree height is one of
the major constraints to good quality date production. In order to reduce tree height
and to develop dwarf date palms, a related dwarf palm species, Phoenix pusilla, was
crossed with selected female date palm cultivars (Sudhersan et al. 2009).
Red palm weevil (RPW) is a major pest in date palm growing countries in the Near
East including the United Arab Emirates (UAE), Iran, Egypt and others (Jain et al
2011). It appeared for the first time in the Middle East in 1985. It is a great cause of
concern to the date palm growers in these countries. The control of RPW is mainly
done by applying chemical insecticides through direct injection into the trunk of the
date palm tree or by fumigation. Pheromone traps are also commonly used to control
RPW, which still requires more refinement for more effectiveness to control this pest.
Baculoviruses could be another way to control RPW, especially genetically engineered
ones inserted with a set of genes dealing with neuro toxin, light-emission (fire fly
gene), and heat tolerance. Another approach would be to express Bacillus
thuringiensis (Bt) crystal insecticidal protein genes, to address problems related to
insect pests (Sharma et al. 2002) and chitinase (to address problems related to basal
stem rot).
Date palm propagation methods
Available techniques of rapid multiplication of date palm have contributed
immensely to meet the increased demand of date palm fruits worldwide (Jain et al
2011). Traditionally, date palm is propagated by both sexually through seeds and
vegetatively by off shoots that produced from axillary buds situated on the base of the
trunk during the juvenile phase in date palm tree. It is quite slow for off shoots to
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develop and that hampers vegetative propagation of date palm trees. So far, there is no
available technique to speed up in increasing the off shoot numbers as well as reduce
the time in developing them. The use of off shoots preserve true-to-type character of
multiplied genotypes. Moreover, sexual propagation of date palm is unsuitable for
commercial production/propagation of true-to-type value-added genotypes. It is due to
heterozygous nature of date palm seedlings and their dioecious nature (Jain, 2007a). In
addition, half of this progeny will be composed of male trees which aren’t
distinguished before flowering stage. The female plants will produce variable fruits
and generally of inferior quality (Eke et al 2005). Furthermore, seed propagation
method has another limitation that the growth and maturation of seedlings is extremely
low, and therefore, date palm seedling may begin to fruit after 8-10 years of plantation.
Although offshoot propagation is a true-to-type technique, it is not commercially
practical for the following reasons:
- Offshoot production is limited to a relatively short vegetative phase of about 10
to 15 years;
- Only a limited number of offshoots are produced during this phase (20 to 30
offshoots, depending on variety);
- Some varieties produce more offshoots than others (some do not produce
offshoots at all);
- Offshoot survival rate is low;
- The use of offshoots enhances the spread of date palm diseases and pests;
Offshoot propagation is difficult, laborious, and therefore expensive.
In vitro propagation of date palm
The use of in vitro techniques such as somatic embryogenesis and organogenesis is
highly suitable for large-scale plant multiplication of vegetatively propagated crops.
The success of these techniques is highly genotypic dependent, however, have
successfully been applied for plant propagation in wide ranging crops including date
palm (Jain, 2007a). Micropropagation via direct organogenesis is widely used for rapid
clonal propagation of elite genetic material of date palm (Khierallah and Bader 2007).
The performance of micropropagated date palm seems to be better than conventionally
grown plants in terms of yield, early flowering time, and quite uniform in fruit quality
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and physical properties. Aaouine (2003) reported plant regeneration from 30 genotypes
of date palm via direct shoot organogenesis. The major concern with this approach is
somaclonal variation that is dependent on various factors including genotype, explants,
plant growth regulators (Jain, 2001). Moreover, it is highly desirable to maintain
genetic fidelity of regenerated plants, which can be studied by various molecular
markers Micropropagation has an advantage of using low concentrations of plant
growth regulators, consequently callus phase is avoided. Direct regeneration of
vegetative buds minimizes the risk of somaclonal variation among regenerants. The
duration of culture period is limited by frequent subcultures for maintaining and
providing shoot cultures for plantlet production. However, the highest number of
subcultures must be determined before starting the fresh cultures from the mother
plants. This is done to prevent or reduce somaclonal variation. Currently, only a few
laboratories use this technique to produce date palm in vitro-plants at commercial level,
mainly in Morocco, Saudi Arabia and United Arab Emirates. Micropropagation
technique has been used commercially in selected date palm cultivars (Jain, 2006)
described advantages and limitations of date palm micropropagation; major advantages
are year round availability of plants, quality control, rapid production of plants of elite
cultivars, and cold storage of elite genetic material
Embryo rescue
It involves the removal of a zygotic embryo from the seed and planting it in a sterile
nutrient medium. Embryo culture has several potential applications in agricultural
crop improvement research programs. It is used to save embryos that fail to develop
naturally in interspecific or intergeneric hybridization where defective endosperms are
common (Hodel 1977). Embryo culture may also be used to reduce lengthy dormancy
periods or with seeds difficult to germinate due to physical or physiological factors.
Excised embryos cultured in vitro, under suitable basal nutrient culture media, usually
germinate immediately. Embryo culture also can be useful in seedling developmental
studies. Sudhersan (et al. 2009) were successful in reducing the date palm height by
embryo rescue of a cross between a dwarf palm species Phoenix pusilla and cultivated
selected P. dactylifera cultivars (Sudhersan et al. 2009). This is the first report on
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reducing the plant height in date palm by embryo rescue, and opens the way to
genetically improve date palm in a short time.
Protoplasts
Date palm biotechnology is routinely used in tissue, organ and cell culture for largescale plant production and multiplication. Protoplast technique is yet to reach a stage
of being used routinely in date palm genetic improvement, especially for somatic cell
hybridization. The protoplast, consisting of the cell cytoplasm bounded by the plasma
membrane and free from cell wall. The availability of commercial enzymes enables
the production of large numbers of uniform protoplasts. Regeneration of fertile plants
from isolated protoplasts was reported in tobacco (Nicotiana tabacum) for the first
time by Nagata and Takebe (1971) and Takebe et al. (1971). Presently, a protoplastto-plant system has been reported for more than 400 species, of which the family
Solanaceae is predominantly represented, followed by Leguminosae, Gramineae,
Compositae, Cruciferae, Umbelliferae and Rosaceae (Davey et al. 2005).
So far, to the best of our knowledge there are only two reports on date palm
protoplasts. Chabane et al. (2007) reported callus formation from protoplasts in cvs.
Deglet Noor and Takerboucht. Similarly, Rizkalla et al. (2007) succeeded in inducing
callus from protoplasts in Barhee and Zaghloul cvs. Critically important steps toward
plant regeneration from recalcitrant date palm protoplasts have been achieved in the
recent past. Callus regeneration was achieved in commercial cvs. Deglet Noor,
Takerboucht, Barhee and Zaghloul. The use of feeder layer was the main factor for
inducing cell divisions as well as subsequent microcallus and callus formation. One of
the most reliable tools to produce resistant plants with good date fruit quality is genetic
improvement. This approach would enable (1) the selection of resistant cultivars and
cultivars with excellent fruit quality through field trials, (2) and then combining both
traits in one cultivar through conventional (crossbreeding) or somatic hybridization.
Also resistance genes can be taken from a cultivar or species with high resistance level
to a particular disease through asymmetric somatic cell hybridization, partial genome
transfer from donor to the recipient parent. By this approach, virus resistant plants
have been produced by fusing protoplasts of Solanum brevidens and S. tuberosum
(Valkonen et al 1994).
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Somatic embryogenesis
Somatic embryogenesis has tremendous potential for rapid large-scale plant
production. In date palm, this technology can be used for the large-scale propagation,
thereby opening the way for the production of artificial seeds. Somatic embryogenesis
of date palm has been quite successful in plant regeneration (Fki et al 2003; Al-Khayri
2005). The most frequently used explants of date palm are apical shoot tips and lateral
buds for successful plant regeneration (Jain 2007a). However, it should be noted that
factors controlling callus induction are so numerous that’s why other optimizations are
still to be done to improve the quality of the embryogenic calli and to increase the
frequency of callus induction from diverse explants. Both abnormal somatic embryos
differentiation and somaclonal variation were associated with the utilization of high
concentrations of 2,4-D. Reducing its concentration significantly had minimized the
number of abnormal somatic embryos and somaclonal variants (Fki 2005). Smith and
Aynsley (1995) studied field performance of tissue culture derived date palm clonally
produced by somatic embryogenesis, and the results demonstrated that these plants
started bearing fruits within 4 years from field planting of small plants with leaf length
100 cm and 1.5 cm diameter at the base. The main advantages of somatic
embryogenesis are ideal for cryopreservation, cost effective for large-scale
propagation, and embryo production in a bioreactor (Table 1).
In addition, further studies are still to be done to find other biochemical and new
molecular markers of embryogenesis in date palm. Most of the methods used to assess
somaclonal variations have limitations: cytogenetically analysis cannot reveal
alteration in specific genes, isozyme markers are subject to ontogenic variation, and
molecular markers investigate only a small part of the genome. Hence, field
performance analyses remain the most reliable strategy to assess genetic integrity in
date palm. Studies related to the cryopreservation of date palm embryogenic cultures
are scarce that’s why developing innovative procedures will be beneficial for date
palm genetic resources preservation and a fabulous support for commercial
propagation laboratories. The preliminary studies revealed that embryogenic cultures
constitute an adequate plant material for further experiments on mutagenesis induction
for useful mutants’ selection, transfer of genes and isolation of regenerable protoplasts.
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Finally, there are still a number of problems such as abnormal somatic embryos
differentiation, endophytic bacteria proliferation in in vitro culture and somaclonal
variation, needing further extensive research to be totally solved. Concerning the
endophytic bacterial contamination, only juvenile explants could be used to establish
clean in vitro tissue culture since antibiotics such as cefotaxim have only a
bacteriostatic effect. Immaturity of vascular tissue in these explants may explain the
absence of this kind of contaminants in such explants (Fki 2005).
Genetic diversity conservation
Plant genetic diversity is highly essential for the genetic improvement of crops for
sustainable agriculture and its gradual loss is as a consequence to rapid human
population growth, industrialization, deforestation, and natural calamities (Jain,
2010ab; 2011ab). In the future, the impact of climate change may have an adverse
impact on sustainable date palm productions as well other crops. The conservation,
distribution and proper utilization of plant genetic diversity/resources become
necessary for the development and improvement of date palm cultivars for sustainable
crop production. The conservation, distribution, and utilization of natural and induced
genetic diversity have become essential by the establishment of gene/germplasm bank
both at the national and international levels. The Gene bank should encourage
researchers to survey and monitor the genetic diversity of natural populations and
landraces on farmer’s fields. In vitro conservation techniques, cryopreservation or
cryo-storage and cold storage, are excellent system for genetic resources conservation
of forest trees and horticultural crops. Cold-storage approach has disadvantage of
frequent subculture and that may run into a risk of contamination and somaclonal
variation. Cryo-storage has an advantage of long-term storage without going through
frequent subcultures and somaclonal variation. For this, in vitro cultures are suitable,
e.g. somatic embryos/ cell suspension, callus, and should be able to regenerate plants
with minimal somaclonal variation. In date palm, the most common in vitro culture
approach has been somatic embryogenesis, which is very much dependent on genotype
and culture medium for plant multiplication, even though there is a risk of genetic
variability among regenerated plants For the first time, cryo-storage of date palm
somatic embryos was done in Tunisia, FAO/IAEA project, and plant regeneration is
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yet to be accomplished. In Asia, National Bureau of Plant Genetic Resources
(NBPGRI, India) is the biggest germplasm bank, and conserves mainly local
germplasm seed and vegetative propagated crops and introduces new crops as well.
In vitro conservation and cryopreservation of germplasm
The purpose of date palm genetic material conservation is to protect from
deforestation, man-made environmental pollution, and natural calamities such as
hurricane, floods, drought, fire etc. In Grenada, hurricane Ivan and Emily in 2004 and
2005 damaged 90% nutmeg and other spice trees, and resulted in loss of agriculture
production, elite germplasm, and exports. The basic requirement of in vitro
conservation and cryopreservation of genetic resources is the reliable plant
regeneration from in vitro explants and large-scale disease-free plant multiplication. In
failing to plant regeneration, this technique may is useless to storing in vitro cultures.
Most common in vitro cultures are being used such as shoot tips, callus, cell
suspension, microspore, and somatic embryos. At low temperature, 0-5 OC, growth of
stored shoot cultures is slowed down and that reduces the number of subcultures on the
fresh culture media without influencing the genetic stability of cultures. It allows store
cultures for several years as long as over 10 years depending on plant type. However,
rooted shoots enhances storage time much longer, e.g. in strawberry shoot cultures that
developed excellent roots could be stored for three years without change of culture
medium under low light intensity and 40C (S. M. Jain personnel communication). The
growth rate can also be reduced by increasing sucrose concentration or addition of
mannitol or sorbitol in the culture medium. Bekheet et al (2001) were successful in the
conservation of in vitro tissues including shoot buds and callus cultures of date palm
var. Zaghloul by slow growth method for 12 months at 5oC in the darkness. In vitro
conservation has many advantages: disease-free planting material, high plant
multiplication rate, all year round plant supply to the growers, potential of producing
low cost planting material, and maintain the genetic fidelity verified with molecular
markers. The major disadvantages of in vitro conservation are: loss of genetic material
by contamination due to bacteria, fungi, virus and mites; subcultures on the fresh
culture medium; labour intensive; destruction of stored genetic material due to fire or
earth quake; and power supply interruptions. Therefore, utmost precaution should be
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taken to use healthy plant tissues for storage, and also test for virus-free material
especially for example in cassava, strawberry and so on before initiating in vitro
cultures for storage.
Cryopreservation
Cryo-storage or cryopreservation is widely used for long-term storage of in vitro
cultures of genetic material under ultra low temperatures, usually at -196 oC in the
liquid nitrogen (Subaith et al, 2007; Bekheet et al 2007). This method preserves
contamination-free material and prevents somaclonal variation. Since date palm in
vitro culture has been worked out for plant regeneration, several groups have been
engaged in cryo-storage of date palm tissues such as shoot tips, nodular cultures,
callus, and somatic embryogenic cultures (Bekheet et al 2007). Cryoprotectant
treatment is given before plunging the tissue in the liquid nitrogen for preventing ice
crystal formation in the tissue in order to avoid any damage to the tissue that may
adversely affect plant regeneration upon thawing of cryo-stored material. The common
cryoprotects are polyehthylglycol (PEG), glucose, and dimethylsulfoxide (DMSO). In
date palm, somatic embryo growth remains normal when treated with cryo-protectant
mixture of glycerol and sucrose. The growth rate or germination rate of somatic
embryos should remain normal after the cryopreservation and that would reflect any
adverse impact of various treatments during the following the protocol.
Cryo-therapy for virus elimination
Cryopreservation has application for the elimination of viruses, which is also
termed as cryo-therapy. Several viruses have been eliminated from various plants
such as cucumber mosaic virus and banana streak virus from banana (Helliot et al
2002), grape virus A (GVA) in vitro-grown shoot tips of Vitis vinifera L. (Wang et al
2003), potato leafroll virus (PLRV) and potato virus Y (PVY) from potato shoot tips
(Wang et al 2006). The cryopreservation method allows only the survival of small
areas of cells located in the meristematic dome and at the base of the primordial
(Helliot et al 2002). Therefore, cryo-therapy would be an alternative efficient
procedure to eliminate viruses to producing virus-free plant material and
simultaneously long-term storage of genetic material.
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Mutation breeding
The exploitation of genetic variability is essential for the development of new
cultivars. Genetic variability can be induced by chemical and physical mutagens, TDNA insertional mutagenesis, and tissue culture-derived variation or somaclonal
variation. The most common physical mutagen used is gamma radiation. In this
chapter, we will stick to physical mutagens only. Induced mutations are random
changes in the nuclear DNA or cytoplasmic organ, resulting in chromosomal or
genomic mutations that enable plant breeders to select useful mutants such as disease
resistant, high yield etc. First of all, gamma irradiation breaks DNA into small
fragments and secondly DNA starts repair mechanism. During this 2nd step, new
variations develop or mutations occur. In date palm, there is hardly any work done on
mutation induction, except that of FAO/IAEA Coordinated Research Project on
development of Bayoud disease resistant date palm mutant varieties in North Africa
(Jain 2005, 2006). Mutation induction in date palm is feasible now due to a reliable
plant regeneration system via somatic embryogenesis and organogenesis. Somatic
embryogenesis system is more preferable approach due to single cell origin of somatic
embryos and that prevents or reduces the occurrence of chimeras. Moreover, mutant
somatic embryos are germinated into direct plantlets in a single step, avoiding
laborious rooting step. The irradiation of multicellular structures, e.g. seed, meristem
tissue or offshoots, may result in chimeras in regenerated plants, and that would
require a lot of extra work to dissociate chimeras by plant multiplication up to M1V4
generation (Jain 2007ab).
Mutant isolation
Mutant isolation can be done in two ways either in a single step or stepwise
selection. In the first approach, irradiated cells are put under very high selection
pressure for the isolation of mutant cell clumps/lines. The initial selection pressure
should be as high as high LD75. Remove isolated mutant cells and transfer them on the
fresh culture medium with reduced selection pressure allowing them to recover from
the initial selection pressure for about one week. The selected lines are put for shoot
and root differentiation. Before selected mutant lines are put for shoot differentiation,
they should be grown for 2 generations devoid of selection pressure and put them back
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again to the selection pressure. This step is done to make sure that the selected mutant
lines are stable due to genetic changes rather than due to epigenetic changes. In the
second approach, the selection pressure is reduced stepwise, from high to low
concentration. All other steps are more or less similar to the first approach.
In vitro selection of mutants, normally type of the selection pressure varies, e.g. salt
concentration, fungal toxin, polyethyl glycol (PEG), herbicide etc. For appropriate
selection pressure, it is better to determine LD50 dose.
The third option is to select mutants at the whole plantlet level, e.g. by spraying
herbicide or water withholding for drought tolerant selection, fungal toxin spraying or
injection. In date palm, bayoud disease resistant mutant plants were selected in the
greenhouse by treating them with isolated toxin from Fusarium oxysporum f. sp.
albedinis fungus causal agent (Jain 2006). These plants are already in the field for the
last four years. So far, they are doing just fine.
Somaclonal variation
Somaclonal variation is an essential component of date palm breeding in which
variation regenerated from somatic cells can be used for the introduction of new
agronomic, tolerance or quality traits (El Hadrami and El Hadrami 2009; Jain 2001);
has a real advantage in widening the genetic basis of this species, relying more or less
solely on vegetative propagation. Variation in the somaclones has often been
associated with changes in chromosome numbers and/or structure, punctual mutations
or DNA methylation or other epigenetic events (Jain et al 1998; Brar and Jain 1998).
Somaclonal variation is undesirable from an industrial production stand point of view
but may provide an enrichment of the genes pool. Its frequency depends, among
others, on the genotype and the length of the proliferation process.
Jain (2006) reported that rapid shoot proliferation can be achieved from various
parts of the plant including shoot tips, stem cuttings, auxiliary buds and roots. He also
pointed out that the selection of the genotype and the number of sub-culture cycles
help limit the appearance of somaclones after the step of plant regeneration. Many offtype plants and abnormal dwarf phenotypes with low fruit sets may still be observed
among the in vitro-propagated populations with high frequencies. These phenotypes
are not always detectable at seedling stages and often become apparent a few years
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after planting. However, the technological advances and the development of molecular
markers have made it possible, in recent years, to early and accurately detect these
variants and eliminate them for the mass production (Saker et al. 2000). These offtypes and somaclones can be further investigated to enrich the genetic pool.
In vitro-selection represents useful biotechnology tools in date palm breeding for
tolerance to biotic and abiotic stresses i.e., drought, salinity, and diseases and pests
(Jain et al 2011). These techniques also offer an improvement of the value-added of
the new genotypes with traits such as an increase in the number and/or size of fruits or
their texture or taste, or a modification in flower structure (Witjaksono 2003). By
applying specific selective agents or providing particular conditions to in vitropropagated tissues, somaclones with desired traits can be produced at a high
frequency. Causes of somaclonal variation during the multiplication are diverse and
tightly dependent upon the genotype, its level of ploidy, the growth conditions and
duration of selection (Maluszynski and Kasha 2002). Studies of the determinants of
such a variation revealed that it can be due to changes at the gene level through genetic
events such as duplication, translocation, mutation by insertion or deletion of
transposable elements, or methylation. It can also occur at the chromosome level
through instability, inversion, and transient or permanent ploidy changes (Kumar and
Mathur 2004). These phenomena often lead to irreversible pleiotropic and epigenetic
events and the production of variants called chimera.
Genetic transformation
The global population growth rate is alarming and the situation demands to enhance
food production to feed new mouths by developing new tools for plant breeders. Since
date palm is more or less like a food crop and feeds people and serves as nutrition
security, genetically engineered date palm would be able to generate disease and pest
resistant plants by over expression of bio pesticide and antifungal. Growing of such
palms will significantly reduce the hundreds of tons of pesticide applied yearly risking
human health and degradation of the ecosystem. Genetic engineering would assist in
reducing time scale in developing new cultivars, only when precisely single trait
genes to be expressed without altering the remaining genetic makeup. However,
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genetically modified (GM) crops have yet to win the confidence of the consumer
worldwide even though growing area of GM crops is expanding.
A large number of plant species have subsequently been genetically transformed,
primarily using two different strategies for DNA delivery into totipotent cells; T-DNA
delivery with Agrobacterium tumefaciens (Horsch et al. 1984) and direct gene transfer
with particle bombardment.
Generally, Agrobacterium-mediated transformation has
several advantages over particle bombardment method e.g. integration of a welldefined DNA sequence, typically low copy number and preferential integration into
actively transcribed chromosomal regions (Gheysen et al. 1998). Many approaches
have been pursued in order to improve the efficiency of Agrobacterium-mediated
transformation in recalcitrant monocot plant species, e.g. use of hypervirulent
Agrobacterium strains, use of particular combinations of Agrobacterium and plasmids,
optimization of co-culture media and conditions that increase the interaction of
Agrobacterium with the plant cell (Cheng et al. 2004; Kumlehn et al. 2006). For date
palm, Agrobacterium-mediated transformation used GUS (β-glucuronidase) as a
reporter gene, which is easy to assay. So far, no conclusive report is available on the
expression of economically-important genes in date palm to the present. The first report
on successful infection of date palm embryogenic callus with Agrobacterium, and that
led to the development of its gene transfer system (Saker et al. 2009). It involves callus
production from shoot tip explants on callus induction medium (CIM) containing MS
salts, B5 vitamins, 30 g/l sucrose, 10 mg/l 2, 4-D, 3 mg/l 2ip, 170 mg/l KH2PO4 and 3
g/l activated charcoal, followed by mass propagation of the proliferated microcalli on
MS medium supplemented with 0.4 mg/l NAA and 0.1 mg/l 2ip. Factors influencing
transient expression of the GUS gene were evaluated following the infection of
embryogenic callus; results indicated that high bacterial density (OD600 1-1.5) and
prolonged infection (2 hrs) gave the highest percentage of GUS-expressing calli
concluding date palm gene transfer achievable. Alternatively, direct gene transfer in date
palm cells was optimized by particle bombardment method (Habashi et al. 2008; Saker
et al. (2007) A construct harbouring a cholesterol oxidase gene, which renders plants
resistance to insect attack, was introduced into embryogenic date palm callus using
PDS1000/He particle bombardment system. Three calli colonies out of 200 putative
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transformants microcalli colonies, which have been bombarded with DNA-coated
particles, gave positive GUS expression. The successful integration of GUS gene in
GUS positive clones was verified by PCR.
The reported system involves the
establishment of embryogenic callus cultures from shoot tip explants, followed by
shooting of the embryogenic callus with DNA coated particles under optimized
physical conditions The most effective physical factors influencing gene delivery
using a bio-listic gun were flight distance of micro-projectiles and their size and
applied pressure, cell and tissue type dependent (Iida et al. 1990).
Molecular markers
Molecular markers are an increasingly important resource for all crops.
DNA
markers, especially those based on simple sequence repeats and single nucleotide
polymorphisms, are playing an increasingly important role in plant variety
identification, germplasm resource collection and breeding activities. The major types of
DNA markers are described and the resources available to the date palm community are
identified. In general, the molecular marker resources for date palm are somewhat
limited. However, most of the available DNA marker types have been used on some
material, mostly to cluster date palm varieties into related groups. The most profound
effect on the development of the DNA marker resources for date palm is the newly
available shotgun sequence. Mining this sequence database and the steady lowering of
the costs of high throughput sequencing will increase rapidly the molecular marker
resources and their application to date palm over the next few years
It is clear that the date palm genome is structured similarly to that of other
characterized plants. Therefore all the tools that have been developed for using DNA
markers are available. Preliminary studies have demonstrated that population
structures and lineage relationships can be identified with the current crop of DNA
markers. As noted, the availability of the complete genome sequence will facilitate
the development of a suite of different marker types to be applied appropriately. The
development of a series of sequenced tagged sites (probably based in SSRs) will
supply resources needed for the screening of collections to reduce the number of
samples kept in germplasm banks. They will also add impetus to identifying markers
linked to the various disease-resistant genes. With the steady increase in the
269
sequencing resources, SNPs will also become more useful but the relative costs of
SNP and SSR analyses may well determine which of the two-marker systems
becomes most widely used. It is undoubted that the collection of many high
polymorphism information content SSR primer pairs and validated SNPs will provide
the tools for phylogenetic analyses as well as germplasm conservation. However,
once genomic regions associated with important characteristics such as disease
resistance, taste and post-harvest stability, the sequencing of these regions and the
identification of the actual bases for these characteristics can be incorporated into the
breeding and improvement programs. The identification of off-types arising in tissue
culture propagation and the complete genome sequencing of normal and off-type
individuals will lead to the identification of both markers for assessing off-type
individuals in the regenerated plants as well as the ‘mutations’ responsible for these
off phenotypes. Therefore these molecular markers and the tools developed through
their use will facilitate the improvements in available germplasm for increasing the
area under date palm cultivation as well as for the overall improvement of the plant
material available to growers.
Traditional and modern genetic improvement in date palm need extended time
periods and considerable funds. Therefore, they can be assisted by molecular markers
that give better and more efficient research strategies. Data based on molecular
markers such RAPDs, have been developed to molecularly characterize date-palm
genotypes of cultivars and to examine their phylogenetic relationships (Trifi et al.
2000). Earlier results showed the use of molecular markers as tools to evaluate genetic
diversity and genotyping of date-palm cultivars (Jain et al 2011). Based on statistical
analysis, Sedra (2007c) reported certain informative molecular markers which are
associated with specific phonological characters in date palm. Previous study of datepalm mitochondrial DNA gave evidence of two plasmid-like DNAs that seem to be
linked to bayoud-disease resistance (Benslimane et al. 1996) but these markers cannot
distinguish both cultivars studied (Trifi 2001). Each marker corresponds to one part of
date palm DNA and the genome has the size estimated to 1.7 pg and it is constituted of
more than ١٠١٢ nucleic bases. These data seem to suggest that the higher the number of
markers used the greater the probability to achieve more precise results. Our research
270
using several hundred RAPD and ISSR primers allowed identifying several markers as
candidates which can distinguish partially or totally between resistant and susceptible
cultivars of date palm. The difficulty and relatively weak efficiency were probably due
to the nature of the genetic status of resistance. These preliminary research results
open new doors to explore the use of molecular technologies in the development of a
breeding program of date palm in order to rapidly select new cultivars desired by
farmers, and fruit more in demand by different markets. They also may provide an area
of research and a construction program of the date palm genetic map.
Genomics
Genomics is carried out to study the whole genome of an organism, which is the
sum total of DNA molecules harbouring all genes of an organism. This type of work is
performed to study all the genes of a given cell, tissue and organism; DNA (genome)
as well as RNA (transcriptome), and protein (proteome) in the context of a regulatory
network as well across taxa (evolution). The field includes intensive efforts to
determine the entire DNA sequence of various organisms and to construct a genetic
map, using large-scale sequencing technology, to generate massive, adequate and
high-quality data, by using bioinformatics tools for assembly, annotation and in-depth
analysis. A major branch of genomics is still focused on sequencing the genomes of
various species, but the knowledge of full genomes has created the possibility for the
field of transciptomics, proteomics, bioinformatics, function genomics, metagenomics
and system biology.
A team from Weil Cornell Medical College in Qatar tried to sequence the entire date
palm genome using Solexa (illumine) sequencer based on a shotgun method. They
announced the finished draft map in 2009 and released the sequence data subsequently:
(http://qatar-weill.cornell.edu/research/datepalmGenome/index.html). According to
their analyses, the genome assembly has a predicted genome size of ~550Mbp. The
following are genome parameters of their draft sequence assembly:
• 45,000 scaffolds greater than 2kb
• Scaffold N50 is 4250bp
• 850,000 novel high quality SNPs between parental alleles
• GC content of the nuclear genome is 37%
271
• 302Mb of assembled sequence with 18.5Mb of ordered gaps
• Unique sequence is 292Mb at the 24-mer level
The date palm genomic project (DPGP) is being carried out at the King Abdulaziz
City for Science and Technology (KACST) jointly with the Beijing Institute of
Genomics, Chinese Academy of Science (BIG/CAS). The objectives are
bioinformatics, genetics, biochemistry, transcriptomes and post-genomics. Data have
been generated by using second-generation sequencers and sequence assembling has
been working on most likely in a complex process where different types of data are
integrated to ensure both quality and contiguity.
The first phase of the DPGP is focused on genomics and bioinformatics that pave
the way for genetic and biochemical studies.
• The specific aims of the DPGP are: a working draft with sequence coverage; 10x
from 454 and 50x from SOLiD; a complete map will be built with end-sequences
from BACs and Fosmids; a genome diversity map built with shotgun sequencing of
30 cultivars; each with 30x of SOLiD reads; the date palm transcriptomes: fulllength cDNA, over 30,000 unigenes; and expression profiles for leaves, roots, and
flowers (~50 tissue samples).
• They have already preliminary data on genome sequencing and assembly,
chloroplast genome sequencing and transcriptomics.
Conclusions and prospects
Date palm is life-line of people living in Sahara and sub-Sahara regions and also an
important source of income in Near Eastern countries. Most of the date palm trees are
very old, as old as 70-100 years and perhaps are becoming more vulnerable to various
diseases and pests. One of the reasons could be due to global warming or global
climatic changes. An increase in global temperature would bring new pests and disease
and get rid of some existing types. Since date palm has a long life cycle, it could
become more vulnerable to the global warming, and that is why it is highly desirable
to pay more attention to the genetic improvement of date palm varieties that could with
stand natural calamities without compromising the yield and quality. The use of
chemical insecticide and pesticides is very common to control diseases and pests of
date palm. These practices could become deadly health hazard to human health and
272
that may also curtail their export market. Innovative techniques are needed to apply for
the control of disease and pests, and that is where genetic modifications of organisms
would be of highly effective. Genetic engineering of baculoviruses may be of great
help in controlling the RPW by inserting a set of genes including neuro toxin (gene
from scorpion or snake), light-emitting (fire-fly), and heat tolerance (bacterial gene).
The engineered baculoviruses would multiply inside the insects and kill them
instaneously. One could monitor the rate of viral multiplication inside the insect by
light meter. Insertion of Bt gene in date palm won't be the right approach due to long
life cycle of date palm and it would be rather difficult to predict the behaviour of
transgene in the long run. Moreover, food safety regulations don't permit to insert Bt
gene in food crops.
The progress of in vitro culture techniques has enabled date palm micro propagation
more as a routine technique for large-scale plant production in many countries. The
influence of genotype has handicapped micro propagation of different commercially
valuable date palm varieties. This area needs serious attention by modifying the
culture medium well suited for several date palm cultivars. This type of work perhaps
may require more empirical work in order to modify the composition of the culture
medium. Now the question arises how well molecular approach would assist plant
tissue culturists to modify the culture medium and growing conditions or the selection
of appropriate explants or pre-conditioning of explants. To answer these questions,
plenty of work is foreseen and in other words this area of research is 'virgin'.
The date palm shoot multiplication rate could be improved by using liquid culture
system or 'bioreactor'. Few groups have started working on liquid culture for in vitro
propagation of date palm. RITA bioreactor, based on temporary immersion system,
should be tried in date palm shoot multiplication and somatic embryo production.
Micro propagation via organogenesis or direct shoot formation is extensive labouroriented. Somatic embryogenesis may reduce labour cost and also asset in developing
automated somatic embryo production. However, genetic fidelity of micro propagated
plants should be maintained with minimal somaclonal variation, otherwise there will
be severe economic loses to the growers. Molecular marker analysis would be an ideal
approach to identify genetic variability at the early stage of plant development. It
273
would be difficult to identify point mutations or any genetic change at the early stage
of plant development because it may not express phenotypically and may express at
the later stage of plant development. This scenario occurred in oil palm tissue culturederived plants in Malaysia and the oil palm industry lost millions of US dollars.
Haploid production in date palm has not yet been accomplished. Inflorescence
culture will be one way to induce haploid somatic embryo production. Fki et al (2003)
induced callus from immature inflorescence of date palm var. Deglet Nour, and the
calli originated from the proliferation of floral primordia showed embryogenic
potential. The capacity of inflorescence to form callus was much higher than cultured
leaves. They did not determine the ploidy level of callus and regenerated plants from
inflorescence-derived callus. In the future, the success of this type of work would
revolutionise date palm genetic improvement program as well as molecular genetics
for useful gene identification.
Somatic embryogenic cell suspension is an excellent system for mutation induction
and isolates useful mutants of date palm. Direct mutant somatic embryos can be
produced and germinated into mutant somatic seedlings. These mutant seedlings can
further be micro propagated for large-scale production. The utmost care should be
taken while handling somatic embryogenic cultures, and in failing to do, the chances
getting somaclonal variation becomes very high. This approach is an excellent
example of combining mutagenesis and biotechnology for date palm improvement.
Transgenic date palm is long way to go before consumers accept to consume them and
consequently export market will also be lost. Therefore, transgenic approach to modify
date palm should be followed with a great cautious, even though it has a great potential
to overcome several of its problems.
274
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279
Table 1. Advantages and disadvantages of somatic embryogenesis (Jain, 2007b)
Advantages
Disadvantages
Cost effective clonal propagation
Low number of field-plant able
plantlets
Both shoot and root meristem
Highly genotypic dependent
development in the same step of the
process
Quick and easy to scale-up in liquid
Inability to produce somatic embryos
cultures, e.g. bioreactors
from mature seeds in many plant
species
Long-term storage via
Gradual fluctuation and eventual
cryopreservation
decline in embryogenic culture
productivity
Establishment of gene bank
Somatic embryogenic cultures from
seeds or seedlings have unproven
genetic value
Production of somatic seeds by
Long life cycle may show genetic
encapsulation of mature somatic
variability or new mutations at the
embryos
later stage of development
Somatic seedlings may be rejuvenated
Genetic transformation
Automation of somatic embryo
production
Somatic seedlings are virus-free
Mutation induction
280
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OP 11
Influence of shoot-tip callus induction medium on in vitro
morphogenesis of date palm inflorescence explants
Adel Ahmed Abul-Soad
Date Palm Research Institute, Shah Abdul Latif Univ., Khairpur, Pakistan
adelaboelsoaud@gmail.com
Abstract
Among individual groups of PGRs Auxins, particularly 2,4-D is the most routinely
used agent to mediate the transition from somatic to embryonic cells. The 2,4-D-rich
medium which has widely been used for callus induction from the shoot-tip explants
was utilized to induce the direct somatic embryos from the inflorescence explants of
date palm (Phoenix dactylifera L.). The appropriate growth stage of initial explants
among different cultivars and the period of incubation on a high auxin medium which
is required to switch on the somatic cells into embryonic cells were investigated. The
inflorescence explants were excised from seven Pakistani varieties at different growth
stages has been exposed to high-content 2,4-D medium for different periods of time
(3,6 and 24 weeks) then transferred to a lower 2,4-D medium. Cultures of those
periods named batches I, II and III. A comparison among these three batches of
cultures was carried out. The obtained results revealed that spikelets of different
varieties with same length are not necessarily to be of the same age. The length varied
according to variety, time of excision, location of the inflorescence on a tree’s head
and within same inflorescence. Accordingly, the response could differ from a variety
to variety. Only batch-II explants were able to produce direct green shoots after 8
subcultures (24 weeks). On the other hand, the explants of batch I and III failed to
induce organs even after pursuing to 14 sub-cultures and the overwhelming majority of
explants went into blackish.
Key words: Date palm, inflorescence, micropropagation, Phoenix dactylifera L.,
somatic embryogenesis.
Introduction
Date palm (Phoenix dactylifera L.) is an economic crop in Middle East and similar
regions in the world. It is traditionally propagated by offshoots (axillary buds in leaf
283
axils). Limited number of offshoots particularly from elite varieties and expansion in
land reclamation made a problem. The reasonable way to overcome this problem was
the production of palms by tissue culture technique. Tissue culture technique in date
palm emerged since three decades depending mainly on the offshoot explants [17]. The
explants derived from offshoots and usage of a high-auxin medium is causing many
technical problems such as endogenous bacterial contamination, browning, somaclonal
variations and long-term duration of production. Therefore, the micropropagation by
shoot tip of the offshoots takes about three years, if the protocol is well-known [5].
Moreover, most of the commercial laboratories all over the world are using somatic
embryogenesis [19; 10; 9]. Thus, a viable alternative explant is needed.
The inflorescence explants usage avoided all the above mentioned obstacles. Since
1973, several workers attempted to culture palm inflorescences. Inflorescences of
several species have been micropropagated in vitro [1; 7]. Subsequently the high
possibilities of inflorescence explants to produce direct [6] and in-direct shoot
formation of date palm [11; 7; 18] were investigated with variable success.
Date palm explants derived of shoot tip used to re-subculture onto that medium up
to friable callus formation. After then, friable callus was transferred onto a free plant
growth regulators medium to differentiate into somatic embryos. This cycle takes
about 1-2 years with risk of getting somaclonal variation because of prolonged subculturing the explants on a high-auxin medium. A novel idea which applied in this
study was by using the high auxin medium for a short period of time in the beginning
only to trigger the pathway of embryogenic cell program, then shifting to a lowerauxin medium to allow the growth and development of induced embryos. Arisen
question which wanted to be answered by this study is for how long the date palm
explants need to start the embryogenic pathway. This study aimed to investigate first:
the role of initial pulse by auxin to trigger the pathway of somatic embryogenesis for
the explants of date palm inflorescence. The famous and frequently used nutrient
medium for somatic embryogenesis of date palm is MS [16] basal nutrient medium
supplemented with 100 mg l-1 2,4 D + 3 mg l-1 2iP + 3 g l-1 Activated Charcoal [19].
This medium was utilized for 3, 6 or 24 weeks after then the explants transferred onto
lower-auxin medium to allow further growth and development of somatic embryos.
284
The second is to study the impact of variety and age of the explants on somatic
embryos induction.
Research Methodology
This work was carried out in the Biotechnology Lab. of Date Palm Research
Institute, Shah Abdul Latif University, Khairpur, Sindh, Pakistan in 2009-11.
Plant Materials:
An immature inflorescence was excised from a single female palm of 5 Pakistani
varieties Gajar, Kachoo Wari, Khar, Karbline, and Khormo were growing in a major
area of date palm in Pakistan, Khairpur district. However, trees of other two varieties
Dedhi and Aseel were subjected for inflorescence excision for more few times (3 and
4, respectively) at different time intervals and named Dedhi (1 to 3) and Aseel (1 to 4).
The inflorescence excision was done during the period from January 19 th – February
7th of 2009. The excised inflorescence was kept in a clean plastic bag and carried
carefully to the lab.
Surface sterilization:
The whole inflorescence was washed under the running tap water for few minutes
and then surface sterilized by immersing in 1 % sodium hypochlorite solution with few
drops of Tween-20 for a minute under aseptic conditions. Thereafter, the intact
inflorescence was washed gently with sterilized distilled water 3 times. In the next
step, the outer cover of the inflorescence was entirely removed (Fig. 1) and the spikelet
explants were cultured individually onto different treatments. Each treatment consisted
of 72 tubes, each involved an explant.
Media composition and treatments:
The basal nutrient medium which employed in current study contained Murashige
& Skoog basal salts [16] supplemented with (in mg l-1): 100.0 myo-inositol; 0.5
nicotinic acid; 0.5 pyridoxine-HCl; 0.4 thiamine-HCl; 2.0 glycine; 2100.0 agar (Agar
Technical, Oxoid, Inc.); 1300.0 Gel (Gellan Gum, Caisson Laboratories, Inc.) and
30000.0 sucrose. Regarding the plant growth regulators and Activated Charcoal (AC),
this basal nutrient medium was supplemented with 100 mg l -1 2,4-D + 3 mg l-1 2iP and
3 g l-1 AC at zero sub-culture (initial culture) and continued sub-culturing up to 24
weeks. Those cultures named batch-I. After 3 weeks, a portion of those cultures
285
(batch-I) was sub-cultured for more 3 weeks onto a lower-auxin medium with 10 mg l1
2,4-D + 3 mg l-1 2iP + 1.5 g l-1 AC and named batch-II, however, the rest of batch-I
was sub-cultured on the same high-auxin medium. After 6 weeks, a third portion was
taken from batch-1 and sub-cultured onto the lower-auxin medium and named batchIII, while the cultures of other two batches were sub-cultured on same media
composition. All three batches’ cultures (three treatments) were subjected to
subculture process every 3 weeks up to 24 weeks initially (8 subcultures).
After preparation of the medium the pH was adjusted to 5.7 ± 0.1 and was
dispensed into small culture tubes (25 × 150 mm) in aliquots of 15 ml per tube and
tubes were capped with aluminum foil. Media were then autoclaved for 20 minutes at
1.11 Kg/Cm2 at 121°C.
Incubation conditions:
In vitro explants were incubated under darkness in a temperature-controlled
chamber at 25 ± 2 °C. Some observations have been taken and the following data were
recorded every 3 weeks:
1. Length of spikelets (age) and spathes excision date from mother tree, Table 1.
2. Browning, swelling and somatic embryo formation [it is expressed as scores and
presented as +, ++, +++, - represent poor, moderate, high and no response,
respectively, according the method described by [9;15], Table 2.
Results and Discussion
Based on evaluation of 84 date palm varieties are growing in Khairpur district,
Sindh, Pakistan [14], 7 predominant varieties were selected to be micropropagated by
tissue culture using inflorescence explants. It is noticed that the targeted response to
induce somatic embryos varied according to the variety and age of the explants.
It is shown in Table 1 that the length of intact spikelets in terms of explant age was
in a wide range of 5 – 55 mm. This range was recorded for all inflorescences before
and during flowering season from 19th January – 7th February, 2009. It is reported that
the physiological age of the explant is necessary to meet the targeted response as
friable or unfriable embryogenic callus formation [2; 3].
Regarding the appropriate age of the spikelet explants, the obtained data indicated
that the early excised inflorescence of Aseel-1 cv. was less responded than the elder
286
explants of late excision date. Since, the early-age explants were short around 15 mm
and the most of explants became brown in color and their initial florets which lied on
the spikelet were tiny. On the other hand, the explants of other elder ages produced
globular structures arose from the florets. It is important to note that, the spikelets of a
same length of different varieties are not necessarily to be of a same age. The length
varied according to a variety, different time of excision, location on the tree at same
time and even inside same inflorescence (Fig. 1). Furthermore, this range is increased
within different varieties. For example, the length of Gajar and Khar Cvs., where the
Gajar’s length of early excised inflorescence was 25-30 mm while late excised Khar
cv. was 22-25 mm. Same trend was found between Kashoo Wari and Karbline Cvs.
Accordingly, the response could differ from variety to variety on same nutrient
medium composition. Thus, the determination of the appropriate age in terms of length
is routinely needed to be investigated to each variety within a particular area. This will
give only an idea or figure about the appropriate time to excise the explant from a
mother tree which may differ but not widely to the surrounded conditions such as
climatic conditions and nutritional state of the date palm tree after harvesting of last
fruit season. Thereafter, there are other factors need to be fixed to allow further
response inside test tubes, for instance plant growth regulators, basal salts and sucrose
concentration [8].
For Aseel and Dedhi, length of spikelets increased by delaying the time of excision
from the mother tree. The length range of Aseel 1-4 was 5-15; 15-20; 25-30; and 40-45
mm, respectively. However, length may also differ in next year due to the other factors
mentioned above especially the location of inflorescence on a tree’s head which was
constant in this study for all 4 trees of Aseel cv. The opposite was found in the case of
Dedhi-2 and Dedhi-3 which excised at same day Feb 6 and the length of inflorescence
was 22-26 and 19-21 mm, respectively. This result is assuring the value of
inflorescence location.
It is reported that not all the inflorescences have same age and inflorescences are
emerging nearly in three groups appearing one after each other. The earlier and first
set of inflorescences are emerging from middle level, then the lower and finally the
upper level on a tree’s head [6].
287
The spikelet explants of Dedhi variety after 24 weeks in culture, three
inflorescences have been excised from the mother trees of Dedhi variety at three
different times (Dedhi1-3). Data in Table 1 and observations indicated that the
intermediate time was relatively suitable for culturing. But the spikelet explants of
Dedhi-1 resulted in shrank and brownish florets. Although, some of these explants
produced initial signs of callus formation and others remained without observable
change and became entirely brown.
This was the role of explant age and variety but, the initial medium absolutely
integrating with those two factors to trigger the induction of somatic embryos of
inflorescence explants.
Among individual groups of PGRs auxins are the most routinely used agents to
mediate the transition from somatic to embryonic cells. In more than 80% of 124
recently published protocols, induction of somatic embryogenesis required the
presence of auxins alone, or in combination with cytokinins [13]. A high auxin level
was speculated to be necessary to disrupt normal development. This has subsequently
been confirmed in date palm [12]. Nevertheless, the need to avoid using strong
stimulator of somaclonal variation 2,4-D and using other alternatives like NAA and
IAA were also performed [3].
Current study aimed at avoiding prolonged cultures on a high 2,4-D medium, then
investigating the proper initial period of time which is necessary for a high 2,4-D medium
and finally to expose the impact of two lower (10 mg l-1) and higher (100 mg l-1) 2,4-D
media on the embryogenic callus induction from the initial inflorescence explants.
In order to compare the response of different varieties, observations on initial
response of the explants after 3 weeks in culture were significantly varied among
different varieties. Since, the response of Khar cv. explants was much better than other
varieties. All the spikelet explants induced distinct globular structures instead of the
florets lied on the spikelet axis. The globular structures were larger in this variety than
other varieties and bright. As well as, the explants swelled and remain white in color.
In general, these results indicated the genetic potential (genotype) of this variety to
form such structures compared to other varieties.
288
Observations after a couple of sub-cultures (after 6 weeks) indicated a very little
callus formation in the cultures of batch-I compared to cultures of batch-II.
Nevertheless, this weak stimulated effect of the high-auxin medium was exceptional
and not permanent as no more callus formation occurred from the cultures of batch-I
now or later. The produced callus morphology of different varieties varied. But in
general, for example in the case of Karbline cv. explants, the white friable callus was
formed on the responded explants only and the ordinary appearance of the florets
nearly disappeared to be replaced by aggregates of such white callus (Fig 2).
On contrary for the silent (un-responded) spikelet explants, the most of swelled
florets’ color was bright creamy or pale yellowish and the florets appeared filled with
water and mostly have retained such original shape for the subsequent subcultures
until went into brownish balls or to shrink. Rarely, these brownish balls after so many
subcultures found maintained clusters of globular structures (pro-embryos). These
results are in harmony with same findings by [7] when reported that these structures
supposed to give globular pro-embryos when maturation will take place by reculturing onto the same medium for many sub-cultures.
The same response of silent explants has been obtained when long-spikelet explants
(highly organized tissues) were used. This may show the interaction effect between
media and explant age. Since, the florets swelled and traces of callus tissue were
localized in the center of closed florets top as in the explants of Aseel-4 cv.
During sub-culturing process, not many explants have given embryogenic callus.
The majority of responded explants belonged to batch-II. Most of these explants were
for 4 trees of Dedhi-1 and -2, Khar, Karbline and Aseel-3 Cvs. All explant were with
minimal browning and callus formation partially increased by sub-culturing (Table 2).
Using a 2,4-D media caused burning for the terminal parts of the spikelet explants
and became dark brown. Although the phenomenon of burnt terminal parts of spikelet
explants was observed in the majority of explants among studied varieties, the
responded ones (produced embryogenic callus) showed less brownish tips. However,
some varieties showed little burnt terminal parts phenomenon. It seems that because of
mainly the genetic background of a variety then the developmental stage and at the end
the medium composition.
289
As it is mentioned before that the developmental stage of initial explants is different
in terms of the length of a spikelet which may differ within a single inflorescence. In
this concern, measurement for the length of all spikelets within a single inflorescence
has been performed (Fig. 1). It was found that the longest spikelets presented in the
middle of the inflorescence, surrounded by the outer shorter spikelets in the four
directions, as they exist in a pyramid construction.
Moreover, the spikelets of a single inflorescence could not be able to expose same
response for all of them on the same medium composition. It could be the content of
endogenous hormone which may be different. Furthermore, rarely all florets on a
single spikelet explant have responded.
The accurate origin of produced callus was not clear. However, it was clear that the
callus emerged from the core of a floret. It seems that the callus originated from newly
growing meristem which is supposed to give the different parts of the flower in the
future. Sometimes callus has been emerged from the basal part of the floret and the
brown remained-floret’s parts floated on the callus.
The interaction effect between the variety and media was detected after 24 weeks in
culture. Few explants of Kashoo Wari cv. which belonged to Batch-II surprisingly
showed high differentiation ability. Most of the explants produced embryogenic callus
(nodular callus or friable callus) white and loose. Sometimes it was mixed with the
differentiated somatic embryos and roots (Fig. 3). These cultures were transferred to a
free-auxin medium under low light conditions [4] to pursue differentiation into the
intact plantlets (Fig. 4).
On other hand, the overwhelming majority of batch-1 and –III cultures produced
silent explants. These were having vitrified and swelled florets (Fig. 2). It is worth to
mention here that such shape was dominant on the high-auxin medium where no
progress in growth was taken place after 24 weeks from initial culture done.
Although, Tisserat’s media [19] have proved viable callus formation media for the
shoot tip explant but, current study have proved those media inaccurate media formula
can be used for the inflorescence explants. Nevertheless, a pulse of the modified highauxin medium which used in this study for 3 weeks was relatively enough to trigger
the embryogenic callus formation of inflorescence explants.
290
In can be concluded that a difference was found among spikelet explants of
different varieties at the same time of inflorescence excision, location of those
inflorescences on same palm palm’s head and spikelets within a single inflorescence.
Thus, the appropriate age in terms of length of spikelet explants of a variety in a
particular area should be investigated before micropropagation. For instance, the best
period of time to excise an inflorescence of Aseel variety was Jan. 24 and Dedhi was
Feb. 6 in Khairpur district, Pakistan.
Using a primary pulse of a high-auxin medium for 3 weeks only in the beginning of
establishment stage resulted in somatic embryos formation from the inflorescence
explants (cultures of batch-II). Continuous sub-culturing on the high-auxin media
failed to induce direct somatic embryos (cultures of batch-I). Although a little
unfriable callus formation occurred in the initial subcultures. However, a low-auxin
medium was needed for embryogenic cells to proceed into mature somatic embryos
(Batch II). Furthermore, exposure of initial inflorescence explants to a high-auxin
medium for long time had bad impact on the explants (Batch III). New composition of
nutrient medium is still needed to get better results from the inflorescence explants.
The late growth stage of Aseel-4 cv. explants often produced swelled white-callused
carpel. Phenomenon of burnt terminal parts of spikelet explants was recorded for the
majority of studied varieties after 6 weeks in culture. Also, vitrified and swelled florets
were observed on those explants.
291
References
[1] Abul-Soad, A. A. 2003. Biotechnological studies of date palm: Micropropagation
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(Phoenix dactylifera L.) micropropagation. The 4th Symposium on Date Palm in
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inflorescence of date palm (Phoenix dactylifera L. cv. Zaghloul). The 2nd International
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dactylifera L. cv. Zaghloul) propagation using somatic embryogenesis of female
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date palm (Phoenix dactylifera L.) Zaghloul cv. Egyptian Journal of Agricultural
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[10] Al-Kaayri, J. M. (2001) Optimization of biotin and thiamine requirements for
somatic embryogenesis of date palm (Phoenix dactylifera L.). In Vitro Cell. Dev.
Biol., Plant. Pp. 37453 - 37456.
[11] Drira, N. and A. Al-Sha’ary (1993) Analysis of date palm female floral initials
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University, Al-Hassa, Saudi Arabia. Pp. 161 - 170.
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[13] Gaj, M. D. (2004) Factors influencing somatic embryogenesis induction and plant
regeneration with particular reference to Arabidopsis thaliana (L.) Heynh. Plant
Growth Regul. Vol. 43. Pp. 27 – 47.
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dates. The 4th Symposium on Date Palm in Saudi Arabia. 5 - 8 May 2007, King
Faisal University Date Palm Research Center Hofuf, Kingdom of Saudi Arabia.
(Under publication)
[15] Mujib, A., S. Banrjee and P. D. Ghosh (2005) Origin, development and structure
of somatic embryos in selected Bulbous ornamentals: BAP as inducer. In: A.
Mujib and J. Samaj (eds.) Somatic Embryogenesis. Springer-Verlag Berlin
Heidelberg. 15 – 24.
[16] Murashige T. and F.A. Skoog (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiological Plantarum 15:473-479.
[17] Reuveni, O., Y. Adato and H. L. Kipins. 1972. A study of new and rapid methods
for the vegetative propagation of date palms. Date Grower’s Institute, 49th
Annual report. Vol.49. Pp. 17 - 24.
[18] Sidky, R. A., Z. E. Zaid, and A. A. Abul-Soad (2007) Direct somatic
embryogenesis of date palm (Phoenix dactylifera L.) by osmotic stress. Egyptian
Journal of Agricultural Research, 85(1B). Pp. 573- 582.
[19] Tisserat, B. 1979. Propagation of date palm (Phoenix dactylifera L.) in vitro. J. of
Exp. Bot.. No.30. Pp. 1275 - 1283.
293
Table 1. Inflorescence length (age), and excision date from mother
tree of different Pakistani varieties of Khairpur District, 2009.
Variety name
Length (mm)
Excision date (d-m-y)
Aseel-1
5 – 15
19 – 1 – 2009
Aseel-2
15 – 20
24 – 1 – 2009
Aseel-3
25 – 30
27 – 1 – 2009
Aseel-4
40 – 45
06 – 2 – 2009
Dedhi-1
9 – 12
27 – 1 – 2009
Dedhi-2
22 – 26
06 – 2 – 2009
Dedhi-3
19 – 21
06 – 2 – 2009
Gajar
25 – 30
22 – 1 – 2009
Kachoo Wari
40 – 50
23 – 1 – 2009
Khar
22 – 25
27 – 1 – 2009
Karbline
25 – 30
03 – 2 – 2009
Khormo
50 – 55
07 – 2 – 2009
Table 2. Effect of initial-medium supplemented with 2,4-D for 3, 6 and 24
weeks (Batches I-III, respectively) on browning, swelling and somatic
embryo formation of female spikelet explants of date palm for 7
Pakistani varieties, after 24 weeks in culture.
Duration on initial
Browning
Swelling
medium
Somatic embryo
formation
Batch I
+++
+++
-
Batch II
+
++
++
Batch III
++
+
-
Each treatment had 6 replicates (72tubes). +, ++, +++, - represent poor, moderate, high and no response, respectively.
Fig. 1. Initial spikelet explants of a single female inflorescence in a pyramid
structure, no. 1 is the longest spikelets, no. 2 is the middle and no. 3 is the shortest.
294
Fig. 2. The vitrified and swelled spikelet explants on the
high-auxin medium (100 mg l-1 2,4-D) after 24 weeks.
Fig. 3. Differentiated shoots and roots of batch-II cultures
of Kashoo Wari variety after 24 weeks in culture.
Fig. 4. Produced ex vitro plantlets from female inflorescence
explants of 7 Pakistani varieties after acclimatization in greenhouse.
295
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OP 12
Effect of number of subcultures and cultivar on in vitro multiplication
through direct organogenesis of date palm (Phoenix dactylifera L.)
Abd EL-Baky M. A., A. Seaf El-Dean and H. A. A. Metwally.
Central Laboratory for Date Palm Research and Development.
Agricultural Research Centre, Giza, Egypt.
Abstract
This study was carried out during 2009 – 2010 at private tissue culture laboratory in
Saudi Arabia to study the effect of number of subcultures and cultivar on
multiplication of date palm (Phoenix dactylifera L.). Four date palm cultivars
(Madjoul, Sukkary, Barhee and Ajwa) were investigated. Shoot tip explants were
cultured Murashigae and Skoog (1962) (MS) salts and vitamins supplemented with
40.0 g/L sucrose, 6.0 mg/L Naphthalen Acetic Acid (NAA), 1.0 mg/L 2ip, 1.0 mg/L
BA and 1.0 mg/ L Kin. Resulted buds have been obtained after 24 weeks. The resulted
buds were cultured on (MS) salts and vitamins media supplemented with 40.0 g/L
sucrose,
170.0 mg/L KH2PO4-2H2O, 200.0 mg/L glutamine, 100.0 mg/L Myo-
inositol, 30.0 mg/L Ca-pantothianate and 100.0 mg/L P.V.P. In addition, five
treatments (in mg/L), i.e., M1- 0.1 NAA + 0.05 BA, M2- 0.5 BA, M3- 0.1 NAA + 1.0
BA, M4- 0.1 IBA + 1.0 BA + 1.0 Kin and M5- 0.1 IBA + 2.5 2ip were tested for
stimulating shoot development through direct organogenesis. The developed shoots
which separated into individual plantlets were cultured on rooting medium contained
(MS) salts and vitamins with: 30.0 g/L sucrose + 7.0 g/L agar + 1.0 mg/L NAA + 3.0
mg/L tyrosine + 3.0 mg/L Ca-pantothianate, without activated charcoal (A C).
Survival percentage, number of roots per plant, root length (cm) and plantlet length
were recorded.
Key word: Date palm- Phoenix dactylifera.- Direct Organogenesis- In-vitro .
Introduction
Date Palm (Phoenix dactylifera L.) is one of the oldest fruits trees to be cultivated
in the Arabian Gulf region possibly southern Iraq (AL-Bakr 1972). Generally the
cultivated species spread from the Middle East to the Nile valley, where its greatest
297
numbers are located. In Saudi Arabia, date palm consider the very important
horticulture tree, where most of the population depend for their nutrition on the dates
and this is linked to Islamic customs and the correlation between the Arabs and
Muslims with this blessed tree, which stated in the holy books, especially in the holy
Koran, also, the correlation between the palm tree and dates with the Prophet
Muhammad. Commercial cultivation expansion of superior cultivars of date palm has
been restricted by slow propagation through offshoots. Production of offshoots by the
palm is naturally limited and limits its spread and propagation (AL-Bakr, 1972).
Propagation by seeds is also unsatisfactory because date palm is heterozygous and
about half of the progeny will be males and half will be females which are not true to
type (Mater, 1986). Many investigations have been made to employ tissue culture
techniques for date palm propagation (Ammar and Benbadis 1977; Euewens and Blake
1977; Poulain et al., 1979; Reynolds and Murashige 1979; Rhiss et al., 1979). Arab
countries, especially, Saudi Arabia, United Arab Emirates, the Kingdom of Morocco
and the Arab Republic of Egypt has taken care to establish many of the tissue culture
laboratories for in-vitro micro-propagation of date palm cultivars, especially, that have
superior date fruit characteristics. Some success has been made based on
organogenesis. Organogenesis in date palm has a low efficiency due to the low number
of explants that respond in-vitro, the long time required for the initiation phase, the
low multiplication rate, and the strong influence of the variety (Poulain et al., 1979
and Beauchesne, 1982). So that, we are interest in this investigation, to propagate
some of superior date palm cultivars such as Madjoul, Sukkary, Barhee and Ajwa
through direct organogenesis technique.
Materials and method
This study was carried out through successive period (2009 – 2010). The
experimental work was performed at private tissue culture laboratory in Saudi Arabia
to study the effect of number of subcultures and cultivars on multiplication of some of
superior date palm cultivars i.e., Madjoul, Sukkary, Barhee and Ajwa via direct
organogenesis technique.
Offshoots weighing about 10-15 kg and with height up to 1.0 m and diameter up to
15-20 cm. were chosen for this study. Before the separation process, chosen offshoots
298
have been treated by using disinfectants fungal. Also, periodic spraying by using
Cupper-Oxychloro for 3 months, then, the chosen offshoots were separated and the
following operations was carried out: four date palm cultivars offshoots (Madjoul,
Sukkary, Barhee and Ajwa) were carefully transferred to the laboratory. Fibrous
sheath and leaves were removed by using sharp knife. Removing leaves were
continued acropetally towards the apical meristem with 4-6 leaf primordia. The apical
meristem and leaf primordia were used as explant material. All vegetative explants
were rinsed immediately in running tap water for one hour, then, soaked in sterile antioxidant solution of 100.0 mg/L ascorbic acid and 150.0 mg/L citric acid overnight.
After that, explants were surface sterilized under aseptic conditions by using 70%
ethyl alcohol for 1 minutes, then, explants were basic sterilized by rinsed one-time
with sterile distilled water and transferred to double surface sterilization by sodium
hypochlorite (NaOCl2, Clorox 5.25% active ingredient) supplemented with two drops
of Tween-20 per 100 ml solution, the first one by 50% Clorox for 10 minutes and
thoroughly washed with sterilized distilled water for one time and the second one by
25% Clorox for 20 minutes, then, washed with sterilized distilled water for three times.
Before culturing and under aseptic conditions, explants were rinsed three times by
sterilized distilled water and the outer soft leaves were removed to obtain the meristem
tip which lengthen 2 – 2.5 cm and composed of the apical meristem and 4 leaf
primordia. Meristem tip and leaf primordial explants were cultured by using direct
organogenesis technique. The culture medium contained inorganic salts of MS basal
nutrient medium (Murashige and Skoog, 1962) supplemented with (in mg/L): 2.0
adenine Iso-pentenil + 200.0 glutamine + 170.0 KH2PO4-2H2O + 6.0 NAA + 1.0 2ip +
1.0 BA + 1.0 kin. The pH was adjusted to 5.7 ± 0.1 before the addition of 7.0 g/L agar
with or without 3 g/L activated charcoal (A C). The nutrient medium was dispensed
into small jars (150ml). All culture jars were maintained under darkness conditions at
27 ± 1˚C. Meristem tip and leaf primordial explants were cultured on the previous
medium for 8 weeks and were sub-cultured every 8 weeks till buds resulted upon
cultured explants after 24 weeks.
The developed buds cultured as 5 jars for each cultivar, each jar contains 5 buds which
were cultured for 8 weeks and were sub-cultured every 8 weeks to stimulating shoot
299
development for 24 weeks on Murashigae and Skoog (1962) (MS) salts and vitamins
supplemented with 40.0 g/L sucrose, 170.0 mg/L KH2PO4-2H2O, 200.0 mg/L glutamine,
100.0 mg/L Myo-inositol, 30.0 mg/L Ca-pantothianate and 100.0 mg/L P.V.P. In
addition, five treatments (in mg/L), i.e., M1- 0.1 NAA + 0.05 BA, M2- 0.5 BA, M3- 0.1
NAA + 1.0 BA, M4- 0.1 IBA + 1.0 BA + 1.0 Kin and M5- 0.1 IBA + 2.5 2ip.
Number of resulted buds in each jar was recorded, the average of buds number per jar
was calculated, in addition, the multiplication rate was determinate through this formula:
The developed buds
The primary buds
------------------------------------------------The primary buds
After we have obtained the suitable number of developed buds, then, the developed
buds were cultured on Murashigae and Skoog (1962) (MS) salts and vitamins
supplemented with (in mg/L): 40000.0 sucrose + 7000.0 agar + 1.0 kin + 3.0 tyrosine,
without activated charcoal (A C) (Beauchesne, 1982).
The developed shoots were separated into individual plantlets, then, cultured on
rooting medium that contained Murashigae and Skoog (1962) (MS) salts and vitamins
supplemented with 30.0 g/L sucrose + 7.0 g/L agar + 1.0 mg/L NAA + 3.0 mg/L
tyrosine + 3.0 mg/L Ca-pantothianate, without activated charcoal (A C). Survival
percentage, number of roots per plant, root length (cm) and plantlet length were
recorded.
Results and Discussions
Data presented in Table (1) showed the effect of media and number of sub-cultures
on multiplication value of some date palm cultivars, i.e., Madjoul, Barhee, Sukkary
and Ajwa. Data declared that, Barhee Cv. recorded the highest multiplication value
(120.69) compared with other cultivars, while, Ajwa Cv. gave the lowest value
(51.22). Referring to media effect, Fig. (1) showed that, medium no. 5 gave the
greatest value of multiplication (94.81), followed by medium no. 3 (72.36), medium
no. 4 (65.58), medium no. 2 (65.03), while, medium no. 1 gave the lowest value
(63.25). On the other hand, there was a gradually increase in multiplication and reach
the maximum significant value (203.93) in sub-culture 3 (Fig. 2). As shown in Table
(1), Madjoul, Barhee, Sukkary and Ajwa Cvs. gave the greatest value of multiplication
300
when cultured on medium no. 5 (132.65, 128.55, 59.25 and 58.80, respectively).
Regarding to the interaction between cultivar and subculture, through subculture 1,
Barhee Cv. gave the greatest value of multiplication (22.30), while, Ajwa Cv. gave the
lowest value (15.52). Concerning subculture 2, Barhee Cv. recorded the highest value
of multiplication (88.40), while, Sukkary Cv. recorded the lowest multiplication value
(46.80). In subculture 3, Barhee Cv. recorded the highest significant value of
multiplication (367.04), followed by Madjoul, Sukkary and Ajwa Cvs., (172.68,
138.60 and 137.40, respectively). Concerning the interaction between cultivar, media
and subculture, Madjoul Cv. cultured on Medium no. 5 in the third subculture recorded
the greatest significant value of multiplication (401.60), while, it recorded the lowest
value of multiplication (100.40) on medium no. 1& 4. Whereas, Sukkary Cv. cultured
on medium no. 1 gave the lowest value of multiplication (12.00).
Data presented in Table (2) stated the multiplication rate of four date palm cultivars
i.e., Madjoul, Barhee, Sukkary and Ajwa. Data declared that, Barhee Cv. recorded the
highest multiplication rate (72.41) followed by Madjoul Cv. and Sukkary Cv. (33.54
and 26.72, respectively), while, Ajwa Cv. gave the lowest rate (26.48). Referring to
media effect, Fig. (1) Showed that, medium no. 5 gave the greatest rate of
multiplication (54.45), followed by medium no. 3 (38.99), medium no. 4 (35.65),
medium no. 1 (35.01), while, medium no. 2 gave the lowest rate (34.83). On the other
hand, there was a gradually decrease in multiplication from sub-culture 1 (2.10) and
reach the minimum rate of multiplication (1.95) in sub-culture 3 (Fig. 3).
Data presented in Table (2) demonstrated that, Madjoul and Ajwa Cvs. showed a
gradually decrease in multiplication rate from subculture 1 reaching the minimum
significant rate in sub-culture 3 (1.46 and 1.95, respectively). Whereas, Barhee,
Sukkary showed different trend and showed a slight decrease in multiplication rate
from subculture 1 to subculture 2 then they appeared a slight increase in multiplication
in sub-culture 3.
As shown in Table (2), Madjoul, Sukkary and Ajwa Cvs. gave the greatest rate of
multiplication when cultured on medium no. 5 (79.32, 30.80 and 30.56, respectively),
whereas, Barhee Cv. gave the greatest rate when cultured on medium no. 1 (77.40). On
the other hand, Madjoul, Sukkary and Ajwa Cvs. recorded the lowest rate of
301
multiplication when cultured on medium no. 1 (19.08, 22.00 and 21.56, respectively),
while, Barhee Cv. gave the lowest rate when cultured on medium no. 4 (63.96).
Regarding to the interaction between cultivar and subculture, within subculture 1,
Barhee Cv. gave the highest multiplication rate (3.46), while, Ajwa Cv. gave the
lowest rate (2.10). Concerning subculture 2, Barhee Cv. recorded the greatest
multiplication rate (2.96), while, Sukkary Cv. gave the lowest multiplication rate
(1.97). In subculture 3, Barhee Cv. recorded the highest multiplication rate (3.16),
followed by Sukkary and Ajwa (1.98 and 1.95, respectively), while, Madjoul Cvs.
gave the lowest multiplication rate (1.46). Concerning the interaction between cultivar,
media and subculture, Madjoul Cv. cultured on Medium no. 1 within the subculture 1
recorded the highest multiplication rate (3.80), while, it recorded the lowest
multiplication rate (1.00) within subculture 3 on medium no. 3.
Table (3) showed the survival percentage of some date palm cultivars plants after 3
subcultures. The presented data reviled that, Barhee Cv. gave the greatest plants
survival percentage (99.14 %), followed by Sukkary Cv. (97.66 %), Ajwa Cv. (95.74
%); whereas, Madjoul Cv. showed the lowest plants survival percentage (82.22 %). In
addition, medium no. 2 recorded the highest plants survival percentage (96.49 %),
followed by medium 5 (94.72 %), medium 1 (94.03 %), medium 4 (91.97 %), while,
medium no. 3 recorded the lowest survival percentage (91.25 %). Regarding to the
interaction between cultivar and media, Barhee, Sukkary, Ajwa and Madjoul Cvs.
recorded the highest survival percentage (99.40 %, 98.75 %, 96.80 % and 92.30 %,
respectively) by using Media no. (3&5, 2, 5 and 2, respectively), whereas, they
showed the lowest survival percentage (98.70 %, 96.82 %, 93.80 % and 73.10 %,
respectively) by using Medium no. 1 for Barhee and Sukkary Cvs. and Medium no. 3
for Ajwa and Madjoul Cvs.
Data presented in Table (4) declared the formation of roots after 10, 20 and 30 days
for four date palm cultivars. As shown, there was a continuous increment in the
number of roots after 15, 30 and 45 days from culturing on rooting medium.
After 15 days, Madjoul Cv. gave the highest significant roots number (1.00)
followed by Sukkary Cv. (0.50), Barhee Cv. (0.30); whereas, Ajwa Cv. couldn't gave
any roots (0.00). After 30 days, the ranking of date palm cultivars was changed,
302
Madjoul Cv. gave the highest significant roots number (2.50) followed by Barhee,
Sukkary and Ajwa Cvs. (1.70, 1.50 and 1.10, respectively). After 45 days, Madjoul
Cv. recorded the greatest significant roots number (12.60) followed by Barhee, Ajwa
and Sukkary Cvs. (4.80, 4.50 and 2.70, respectively).
Data presented in Table (4) stated the root length through the study period 30 days,
it's clear that, there was a continuous increase in root length till 30 days. After 45 days
from culturing on rooting medium, Barhee Cv. gave the tallest significant roots (4.30
cm), followed by Sukkary Cv. (4.20 cm), Ajwa Cv. ranked third with significant
difference (3.40 cm), while, Madjoul Cv. plants gave the shortest roots (2.35 cm). As
shown in Table (4), there was a significant difference in the increasing of plant height
through the period of study (30 days). Plants of Madjoul Cv. showed the highest value
of plant height after 30 days (6.20 cm), followed by Barhee Cv. plants (4.60 cm) with
significant difference. Sukkary Cv. ranked third (2.90 cm), whereas, Aiwa Cv. gave
the lowest significant value of increasing in plant height (1.70 cm).
These results are in agreement with Faiza Masmodi - All ouche et al, (2010) which
reported that the in-vitro reversion of a shoot meristem from vegetative to reproductive
state was achieved within 1 to 5 months depending on the variety. Besides, Ammar et
al., (1984) reported that, on the sexual induction of young seedlings obtained from the
germination of Deglet Nour seeds. The present study, also, shows the potential gain
obtained by the application of new methods of sex induction in date palm. This
application of sex induction couldn't be achieved on all plant species, such as Murraya
paniculata (L.) as reported by Jumin and Ahmad (1999), Long (1987), Mechelle J.Mc
Cubbin and A.Zaid (2006) who reported that, using plant growth regulators in low
concentrations during the initiation process may help to reduce the possible variation.
In addition, Hussam S. M. Khierallah and Saleh M. Bader (2006) determined the
best combination of plant growth regulators and other conditions in order to achieve
organogenesis and multiplication directly from shoot tips of date palm without callus
formation by using MS modified medium supplemented with 2.0 mg/L 2ip, 1.0 mg/L
BA and 1.0 mg/L NAA was the best combination for bud formation from shoot tip
after 16 weeks. On the other hand, Zaid (2003) showed that, individual embryos
germinate directly to shoot and root without producing secondary embryos, while
303
repeat and multiply somatic embryos were used in multiplication stage of date palm to
produce more secondary somatic embryos which differentiated from callus on its
body. This process may be useful in date palm micro-propagation. Whereas, George
(1993) stated that multiply embryos to cluster from 3 – 4 embryos can occur on the
base of the original embryos. On the other hand, Tisserat (1984a) recorded that
production of plantlets from shoot tips and buds by organogenesis process is less well
developed technique compared with embryogenesis. But, producing plantlets of date
palm through organogenesis process should be clonal and less risky of genetic
variation than callus derived plantlets.
Belal et al., (1993) applied shoot multiplication starting from shoot tip explants,
isolated from the mother plants of two Egyptian date palm cultivars (Zaghloul and
Samani) using MS medium containing high cytokinin level (3.0 mg/L 2ip) and low
auxin level (0.1 mg/L NAA). Also, Belal and El-Deeb (1997) recorded that, using
medium containing 0.5 mg/L IAA, 0.5 mg/L NAA, 10.0 mg/L BAP and 5.0 mg/L 2ip
enhanced auxillary shoot formation.
304
References
[1] AL-Bakr, J.A. 1972. The Date Palm: Past, Present and Future. 2nd edn., AlWattenpress. Baghdad. Iraq. (In Arabic), 850 pp.
[2] Ammar, S. and A. Ben.badis. 1977. Multiplecation vegetative du pallmier
dattier (Phoenix dactylifera L.) par la culture de jeunes plants de semis. C. R.
Acad. Sc. Paris, series D.284: 1789-1792.
[3] Ammar. S, Benbadis. A and Tripathi. B. K. (1987). Floral induction in date
palm seedling (phoenix dactylifera L.Var. Deglet Nour) cultured in-vitro. Can J
Bot 65: (137-142).
[4] Beauchesne, G. 1982. The vegetative propagation of date palm through tissue
culture. In: 1st Symp. Date Palm, King Faisal Univ., Al-Hassa, Saudi Arabia,
March 1982, pp. 698–700.
[5] Belal, A. H. and El Deeb, M. D. (1997). Direct Organogenesis of date palm
(phoenix dactylifera L.). Assiut Journal of Agriculture Science, 28 (2): 1997.
[6] Belal, A. H.; Sabour, A. M.; Ibrahim, I. A. and El-Barkouky, M. H. (1993).
Shoot tip differentiation in-vitro for (phoenix dactylifera L.). The third Symp.
On date palm, King Faisal Univ. Al-Hassa, Saudi Arabia, Jan.17-20, 1993.
[7] Euewens, C. and J. Blake. 1977. Culture of coconut and date palm tissue with a
view of vegetative propagation. Acta Hortic. (78): 277–286.
[8] Faiza Masmoudi - All Oyshe, Besma Mejiou, Walid Krioa, Radhia Gargouri,
Bouzid and Drira, N. (2010). J. plant growth regulators (2010) 2g: 35-43.
[9] George, F. F. (1993). Plant growth regulators, plant propagation by tissue culture.
Printed in Great Britain by Butter and Tanner Lid. Krome, Somerset. pp. 425.
[10] Hussam S. M. Khierallah and Saleh M. Bader (2006). Micro-propagation of date
palm (Phoenix dactylifera. L. Var. Maktoom) through direct organogenesis. Proc
111rd on Date palm (213-225)
[11] Jumin, H. B. and Ahmad, M. (1999). High - frequency in-vitro flowering of
murraya (L.) Jack. Plant Cell Reb. 18: 764-768.
[12] Lang, A. (1987). Nicotiana.A. M. :Halevw A. H. (ed) Hand book of flowering
Vi.CRC press, Boca Raton, Fl, pp: 427-483.
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[13] Mater, A.A. 1986. In-vitro propagation of (Phoenix dactylifera L.). Date Palm J.,
4 (2): 137-152.
[14] Mechelle J. Mc Cubbin and Zaid, A. (2006). Would a combination of
Organogenesis and Embryogenesis techniques in date palm micro-propagation be
the Answer? Proc.111rd icon date palm (255-260)
[15] Murashige, T. and F.A. Skoog. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Plant Physiol., 15: 473-479.
[16] Poulain, C., A. Raiss, and G. Beauchesne. 1979. Multiplecation vegetative en
culture in-vitro du palmier-dattier (Ponenix dactylifera L.). C.R. Seances Acad.
Agric Fr., (11): 1151-1154.
[17] Reynolds, J.F. and T. Murashige. 1979. Asexual embryogenesis in callus culture
of plants. In-vitro. 15: 383-387.
[18] Rhiss, A., C. Poulain, and G. Beauchesne. 1979. La culture in-vitro appliquee a
la multiplication vegetative du palmier dattier (Ponenix dactylifera L.). Fruits,
34: 551–554.
[19] Tisserat, B. (1984a). Propagation of date palm by shoot tip cultures. Hort.
Science. Jorn., 19 (2): 230-231.
[20] Zaid Zeinab, E (2003). Comparative studies on the production of date palm
cultivars via tissue culture technique. Ph. D. Thesis. Cairo University. pp: 102-115.
306
Table (1): The effect of media and number of subcultures on multiplication
value of some date palm Cultivars (Madjoul, Barhee, Sukkary and Ajwa).
Cultivar
Media
Subcultures
Mean
0
1
2
3
1
5.0
15.00
47.00
100.40
41.85
2
5.0
20.20
64.80
131.00
55.25
3
5.0
20.80
65.00
130.00
55.20
4
5.0
15.00
47.00
100.40
41.85
5
5.0
24.00
100.00
401.60
132.65
Mean
5.0
19.00
64.76
172.68
65.36
1
5.0
22.40
87.20
392.00
126.65
2
5.0
21.60
81.60
345.60
113.45
3
5.0
21.20
86.00
382.20
123.60
4
5.0
23.20
91.80
324.80
111.20
5
5.0
23.20
95.40
390.60
128.55
Mean
5.0
22.32
88.40
367.04
120.69
1
5.0
12.00
37.00
115.00
42.25
2
5.0
16.00
41.00
121.00
45.75
3
5.0
17.00
56.00
144.00
55.50
4
5.0
15.00
46.00
154.00
55.00
5
5.0
19.00
54.00
159.00
59.25
Mean
5.0
15.80
46.80
138.60
51.55
1
5.0
14.00
37.20
112.80
42.25
2
5.0
15.20
43.40
119.00
45.65
3
5.0
15.40
56.60
143.60
55.15
4
5.0
14.00
44.20
153.80
54.25
5
5.0
19.00
53.40
157.80
58.80
Mean
5.0
15.52
46.96
137.40
51.22
5.0
18.16
61.73
203.93
72.21
Madjoul
Barhee
Sukkary
Ajwa
General Mean
LSD at 0.05:
Media (A)
4.92
Cultivar (B)
4.40
AXB
9.84
Subculture (C)
4.40
AXC
9.84
BXC
8.80
AXBXC
19.67
307
Table (2): The effect of media and number of subcultures on multiplication
rate of some date palm Cultivars (Madjoul, Barhee, Sukkary and Ajwa).
Cultivar
Madjoul
Barhee
Sukkary
Ajwa
Media
Subcultures
Mean
0
1
2
3
1
0.0
2.00
2.13
1.14
19.08
2
0.0
3.04
2.21
1.02
25.20
3
0.0
3.16
2.13
1.00
25.00
4
0.0
2.00
2.13
1.14
19.08
5
0.0
3.80
3.17
3.02
79.32
Mean
0.00
2.80
2.35
1.46
33.54
1
0.0
3.48
2.89
3.50
77.40
2
0.0
3.32
2.78
3.24
68.12
3
0.0
3.24
3.06
3.44
75.44
4
0.0
3.64
2.96
2.54
63.96
5
0.0
3.64
3.11
3.09
77.12
Mean
0.00
3.46
2.96
3.16
72.41
1
0.0
1.40
2.08
2.11
22.00
2
0.0
2.20
1.56
1.95
23.20
3
0.0
2.40
2.29
1.57
27.80
4
0.0
2.00
2.07
2.35
29.80
5
0.0
2.80
1.84
1.94
30.80
Mean
0.00
2.16
1.97
1.98
26.72
1
0.0
1.80
1.66
2.03
21.56
2
0.0
2.04
1.86
1.74
22.80
3
0.0
2.08
2.68
1.54
27.72
4
0.0
1.80
2.16
2.48
29.76
5
0.0
2.80
1.81
1.96
30.56
Mean
0.00
2.10
2.03
1.95
26.48
0.00
2.63
2.33
2.14
39.79
General Mean
308
Table (3): Survival of some date palm cultivars plants (Madjoul, Barhee,
Sukkary and Ajwa) after 3 Subcultures.
Cultivar
Madjoul
Barhee
Sukkary
Ajwa
Media
No. of plants
Survived plants
Died plants
Survival %
1
100
85
15
85.00
2
130
120
10
92.30
3
130
95
45
73.10
4
400
300
100
75.00
5
350
300
50
85.70
Mean
222.00
180.00
44.00
82.22
1
392
387
5
98.70
2
346
343
3
99.10
3
384
382
2
99.40
4
328
325
3
99.10
5
392
390
2
99.40
Mean
368.40
365.40
3.00
99.14
1
157
152
5
96.82
2
160
158
2
98.75
3
155
153
2
98.71
4
170
165
5
97.06
5
165
160
5
96.97
Mean
161.40
157.60
3.80
97.66
1
115
110
5
95.6
2
121
116
5
95.8
3
144
141
3
93.8
4
154
149
5
96.7
5
159
154
5
96.8
Mean
138.60
134.00
4.60
95.74
309
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310
OP 13
Protoplast culture before and after fusion between two different
cultivars in improvement of date palm ( Phoenix dactylifera L. ).
Chabane D1, Assani2 A and Bouguedoura N1. University of Houari Boumediene. Po
box 81 Avenue Ali Khodja El Biar Algiers. Algeria. email: chabanedj@yahoo.fr.
2
Department of Molecular and Cellular Biology, University of Guelph, Guelph,
Ontario, N1G 2W1. *Email: aassani@bell.net.
Abstract
Date palm, is an economically important plant in Saharan agriculture, where it
offers dates as staple food and ecological conditions for other cultivated plants. A
fungal disease called Bayoud threatens the date palm plantations in North Africa.
Bayoud, the fungal vascular wilt of date palm, is caused by Fusarium oxysporum fsp
albedinis. Genetic improvement is the most important and efficient tool to produce
resistant plant material with good date quality. Deglet nour and Takerboucht, which
are sensitive and resistant respectively to Fusariose; however, successful protoplast
regeneration is a prerequisite. In the present study, we report callus formation from
protoplasts in date palm. Protoplasts were isolated from embryogenic calli in Deglet
nour and Takerboucht. The protoplast yield depended on genotype, donor plant
material, mixture of enzyme solution and incubation time. Protoplasts isolated were
cultivated before and after fusion between two cultivars with Poly ethylene glycol
were induced very white and friable callus formation. Embryogenic cell from calli is
very powerful material for obtaining young plantlets
Key word: Date palm, protoplasts, microcallus,
protoplast fusion, somatic
embryogenesis, plant regeneration..
Introduction
Date palm (Phoenix dactylifera L.) is a monocotyledonous, dioecious and
diploid (2n = 2x = 36) plant of the family Arecaceae, cultivated in North Africa
and the Middle East, and in the other parts of the world as USA, spain and Italy
(Heselmans 1997). It offers dates as staple food and ecological conditions for
other cultivated plants.
311
There are two common methods to propagate palm trees, sexually by seeds and
asexually using offshoots. Generally, seed propagation gives half of the progeny male
and other half female.
The traditional vegetative propagation, with a very limited number of offshoots
(about 10-15 per tree) is less efficient comparing to in vitro micropropagation
procedures (Bouguedoura et al. 1990). Actually, date palm biotechnology is used in,
organ, tissue and cell culture for large-scale plant production and multiplication.
Protoplast technique is used in, date palm genetic improvement for somatic cell
hybridization. Presently, a protoplast-to-plant system has been reported for more than
400 species, of which the family Solanaceae is predominantly represented, followed
by Leguminosae, Gramineae, Compositae, Cruciferae, Umbelliferae and Rosaceae
(Davey et al. 2005). The totipotency of isolated protoplasts is fundamental for genetic
manipulation approaches involving somatic hybridization and genetic transformation.
Electrofusion and chemical treatments of protoplasts with polyethylene glycol (PEG)
techniques are commonly used. Somatic hybrids are routinely produced in citrus,
brassica, potato and other members of the Solanaceae (Grosser et al. 2001; Möllers et
al. 1994; Sacristan et al. 1989). However, success in monocots, which includes date
palm, is very limited.
To the best of our knowledge there are only two reports on date palm protoplasts.
Chabane et al. (2007) reported callus formation from protoplasts in cvs. Deglet Noor
and Takerboucht. Similarly, Rizkalla et al. (2007) succeeded in inducing callus from
protoplasts in Barhee and Zaghloul cvs. One of the most reliable tools to produce
resistant plants with good date fruit quality is genetic improvement. This approach
would enable (1) the selection of resistant cultivars and cultivars with excellent fruit
quality through field trials, (2) and then combining both traits in one cultivar through
conventional (crossbreeding) or somatic hybridization. Also resistance genes can be
taken from a cultivar or species with high resistance level to a particular disease
through genetic engineering method. These genes can be inserted into date palm
through genetic transformation approach. Once an elite tree is developed, it can be
micropropagated through in vitro culture. A successful production of a resistant elite
cultivar would reduce the use of chemicals for pest and disease control in date palm
312
plantations. Protoplasts can be isolated from various parts of the plant including
leaves, cotyledons, shoots, roots and flowers. In monocot species, the ideal material
for protoplast isolation is embryogenic cell suspension and prolific growing callus
cultures. However, embryogenic callus cultures are most suitable for protoplast
isolation in date palm (Chabane et al. 2007). This paper describes the current status of
date palm cell suspension and protoplast culture before and after fusion with PEG.
Research methodology
1. Cell suspension culture
1.1. Plant material
Two different cultivars were tested, Deglet nour from the southeast and
Takerboucht from Adrar in the south west of Algeria. Deglet nour has a good fruit
quality but sensitive to Fusariose and Takerboucht has a lower fruit quality but very
resistant to this pathogen. The offshoots used were taken from adult female plants.
1.2. Callus Induction
Offshoots were excised with a sharp knife, the shoot apical tip from them were used
for this investigation and then sterilized under aseptic conditions. They were sterilized
with 0.3% benlate (methyl [1-(butylcarbamoyl)benzoimidazol-2-yl] aminoformate)
(DuPont, France) for 30 min followed by transferring to 30% aqueous solution of
5.4% (v/v) sodium hypochlorite in water with two drops of tween 20 per 500 ml for 45
min. The tip was then rinsed three times of 10 min each with distilled water and
excised in small pieces (5 mm) and cultured on solid medium M 1 (Table 1, Chabane
1995) containing MS salts (Murashige., Skoog, 1962) and supplemented with 9.0 µM
2.4-D, 14.76 µM IPA, Morel vitamins (Morel., Wetmore, 1951), 87 mM sucrose, and
7gl-1 agar (Sigma, USA). The pH was adjusted to 5.7-5.8 before autoclaving (20 min,
120°C, 1 bar). The cultures were kept for 3-6months at 27°C in the dark. The explants
were subcultured at 4 weeks interval on the same medium composition. The calli were
separated and chopped with a scalpel blade and transferred onto the same fresh callus
induction medium.
1.3. Establishment of Cell Suspension Culture
Calli were first chopped thoroughly with a scalpel blade in a plastic Petri dish
containing 5 ml of suspension culture medium as described by Teixeira et al. (1995).
313
The small pieces obtained were transferred aseptically in suspension culture medium.
Chopped calli about 500 mg to 1000 mg were transferred aseptically into 100 ml
Erlenmeyer flasks containing 20 ml liquid medium of the same composition as the
callus induction medium, but without agar. The cultures were then placed on a rotary
shaker 90rpm at 28oC under a 12h/12 h (light/dark) photoperiod and a 50 µmol m-2 s-1
photon flux, provided by cool fluorescent lamps. The suspension was subcultured
every 2weeks. After about 2months of culture, small proembryonic masses (PEMs)
were observed.
1.5. Histological Analysis
For histological examination, a plant material was fixed in a mixture containing 4 ml
25 % glutaraldehyde solution, 50 ml phosphate buffer, pH 7.2, 20 ml of 10 %
paraformaldehyde solution, 1 g caffeine and the total volume was raised to 100 ml by
adding 26 ml distilled water. After fixation, this was followed by progressive dehydration
with ethanol and then embedded in paraffin or epoxy resin. Thin serial sections, 3.5-7 μm
thick, were usually made, using a microtome; stained with Periodic Acid Schiff (PAS)
combined with Naphthol Blue Black (NBB), as described by Fisher (1968).
2. Protoplast culture
2.1. Protoplasts Isolation
About 1g fresh weight of cell suspension of each cultivar (Deglet nour and
Takerboucht ) was used separately for protoplast isolation. The callus was cut into
small pieces, put into 15 ml enzyme solution in Petri dishes (9.5 cm diameter), and
placed in the dark at 27◦°C for 12-20 h. The enzyme solution EC (pH 5.6) was used
(Chabane et al. 2007), this enzyme solution contained 1.5(%, w/v) Cellulase RS
(Yakult Pharmaceutical Ind. Co., Ltd, Japan), 0.15(%, w/v) Pectolyase (Kyowa
Chemical Products Co, Ltd, Osaka Japan), 0.2(%, w/v) Hemicellulase (Sigma, USA),
3(%, w/v) KCl and 0.5(%, w/v) CaCl2. This enzyme solution was sterilized using a 0.2μm Millipore filter (Millipore, Billerica, MA, USA). The enzyme solution/pieces of
cell suspension mixture was incubated overnight at 27◦°C for 12-20 h in the dark.
Before the purification step, the protoplast suspension was transferred to a gyratory
shaker to increase the number of protoplasts released.
314
2.2. Purification of protoplasts
The protoplasts were purified as described by Assani et al (2006).The digestion
mixture was filtered through 100/25 µm metallic mesh combination to remove debris
and large cell colonies. Protoplasts were washed three times through centrifugation (65
g for 5 min) with a washing solution containing 204 mM KCl, 67 mM CaCl2 with pH
5.7. The protoplast suspension were rinsed again with 0.5 M mannitol and 67 mM
CaCl2 (centrifugation 65 g for 5 min) to minimize the salt content. The protoplast
viability was determined by fluorescein diacetate (FDA)(Widholm, 1972).
2.3. Protoplast fusion using PEG (polyethylene glycol 4000)
It is the chemical fusion with PEG. PEG (HOCH 2 (CH 2- O-CH 2)n CH 2 OH) is
a highly water soluble with a molecular weight ranging from 1500 to 6000, the ether
bond makes the molecule slightly electronegative allowing the formation of a film of
coating the periphery of the protoplast and hide the electrical charges. After
purification, mix 5ml of protoplast Takerboucht genotype with 5ml of protoplast
Deglet nour genotype (with same density equal to 105protoplasts/ml) intimately
mixed in a solution containing 0.5M mannitol and 0.5mM CaCl2. This mixture was
made in a plastic tube.
Place 300µl of the mixture of protoplasts, and wait for the protoplasts settle to the
bottom of the Petri dish. Meanwhile, miw 10ml of 50% PEG with 10ml of sterile
solution composed by 0.5M mannitol and 67mM CaCl2. Add 300µl of this solution of
PEG obtained and then drop it drop by drop around the protoplast solution. Conduct
this operation with an observation under an inverted microscope.
2.4. Culture of protoplasts isolated and fused
The isolated and fused protoplasts were cultured separately at a density of 10 6 on
nurse culture system, embryogenic offshoot-tip-derived calli of Deglet nour was used
as nurse cells for feeder layer. The nurse culture was prepared the same day the
protoplasts were isolated. The PCM liquid medium (double strength), with double
strength of MS salts, 9.0 µM of 2.4D, vitamins of Morel, 2.8 mM glucose, 278 mM
maltose, 170 mM sucrose and 2.5 mM Myo inositol (pH 5.7).
Callus suspension was made by small pieces (0.2mm) of friable callus and 100 ml of
PCM culture medium to obtain final cell concentration of 2% in PCM/ agarose mixture.
315
Agarose sea plaque 1.2 g (Sigma, USA) was dissolved separately in 100 ml of distilled
water with pH 5.7 and then autoclaved. When the temperature of the agarose solution
decreased to 30°C-35°C, it was carefully mixed with 100 ml PCM medium containing
nurse cells. Aliquot of 10 to 12 ml of this mixture were poured into small Petri dishes
(5.5 cm diameter). After solidification, the medium was covered with sterilized nitro
cellulose filter (AA type millipores), and 1ml of protoplast suspension in M5 medium
(see above). All cultures were maintained at 27°C in the dark.
2.5. Protoplasts development
Cell wall regeneration of protoplasts was observed with calcofluor white brightener
stain (Nagata et takebe, 1970). The microcalli formed were transferred onto callus
induction medium containing MS salts and supplemented with 13.5 µM 2.4 D and
14.76 µM IPA; Morel vitamins and 3 g gelrite. The calli were transferred to
regeneration medium, which consisted of MS salts supplemented with the same level
of IPA of the callus induction medium (see above) and 1.4 µM 2.4 D. The cultures
were kept in the dark at 27°C.
2.6. Data Collection and statistics
Viable protoplasts were counted, the protoplast yield was estimated with a Nageotte
hematocytometer. The protoplast yield was calculated using the following formula: Y=
Qx104/W (Y is the protoplasts number per milliliter, Q is the quantity of protoplasts in
average 5x1mm2, W is the fresh weight of plant material. Results were expressed as
yield per g. f. wt. of calli. The experiments were performed at least three times.
Results
3.1. Production of cell suspension and protoplasts
Embryogenic friable callus white or yellow (Fig. 1A) is the material of choice for
suspension establishment and protoplast isolation (Figs. 1B and 1C) in date palm. A
histological study revealed the presence of a very heterogeneous structure with cells at
different developmental and differentiation stages. It consists of (a) single
embryogenic cells with cytoplasm containing starch and lipoproteic storage grains,
with pink and thick polysaccharide walls, (b) dividing embryogenic cells, (c) cell
aggregates of several cells, (d) proembryonic clusters. Around them, we were noted
cells with less-dense cytoplasm with fragmentation zones between cell non316
embryogenic long single cells with less-dense cytoplasm.In this study, the protoplast
viability reached 80% in Deglet nour and 60% in Takerboucht calli. Concerning the
protoplast culture on feeder layer, the cell wall regeneration began after 2days with
shape transformation in oval form. The frequency of cell wall regeneration of cell
suspension-derived isolated protoplasts was around 50% either in Deglet nour or in
Takerboucht and 25% in protoplasts fused between two cultivars (Table 2).
In this study, it demonstrates that nurse culture was effective for mitotic activity of
date palm protoplasts isolated and fused, we were obtained a large number of cell
division. The dividing cells continued to grow and developed into microcalli on feeder
layer 8 weeks after protoplast plating (Fig. 1D). The number of microcalli was 20 000
per Petri dish in Deglet nour and 12 000 per Petri dish in Takerboucht. However, the
number of microcalli obtained after fusion by PEG was average 5000, whose inner
parts are comprised of small meristematic cells with a dense cytoplasm that is rich in
soluble protein which stained blue by NBB (Naphthol Blue Black) which could be a
credible indicator of the development towards somatic embryogenesis. The outer part
of those microcalli displaying cells with less-dense cytoplasm that is rich in starch and
lipoprotein storage grains.
4. Discussion
In the present investigation, we have established a protocol allowed the microcalli
formation from protoplasts of two cultivars isolated and fused in date palm.
The induction of highly prolific and embryogenic callus in date palm is done to
initiate a stable cell suspension culture, which is a very slow process requiring 6-12
months, calli are white, display a granular appearance and are friable. A histological
study revealed the presence of several globular proembryogenic clusters.
In terms of donor material, Shoot tips have been commonly used for callus
induction (Chabane et al. 2007; Zouine et al. 2005). Fki et al ( 2003) have showed that
inflorescences can form much higher quantity of callus that of leaves
The frequency of explants that produce callus can vary respectively in Takerboucht
and Deglet nour from 65-90 % depending on genotype.MS medium (Murashige and
Skoog 1962), supplemented with plant growth regulators (PGRs) such as 2,4-D, 2iP
(6-(γ-γ-dimethylallylamino) purine), is most frequently used for callus induction ); 9
317
µM 2,4-D and 14.8 µM 2iP in cv. Deglet Nour (Chabane 1995). In terms of vitamin
supplements, Morel and Wetmore vitamins (Morel and Wetmore 1951) are preferred.
In other cultivars of date palm as, Boufeggous, a high percentage of prolific calli
has been accomplished in MS medium containing 45 µM 2,4-D (Othmani et al. 2009);
450 µM 2,4-D in cv. Barhee (Bhaskaran and Smith 1992) , 9 µM 2,4-D in cv.
Amsekshi (Sané et al. 2006).
4.1. Cell Suspension Culture
The establishment of stable cell suspension culture required about 6 months from. A
fine chopping of calli is crucial for a successful initiation of embryogenic cell
suspension culture. In earlier studies, Fki et al. (2003), Sané et al. 2006) have showed
that chopping and sieving are necessary to establish cell suspension. Bhaskaran and
Smith (1992) have revealed that the embryogenic capability in suspension cultures
could be maintained for more than 3 years in date palm. Concerning plant regeneration
from Cell Suspension Culture, it appears that the chopping of callus increases the
number of proembryos, which develop into somatic embryos in about 6 months after
establishment of the suspension cultures. Zouine et al (2005) have noted the
development of somatic embryos from cell suspension in date palm.
4.2. Protoplast culture
The best response is achieved during the exponential phase of growth of the donor
tissues or cells (Ochatt et al. 1989). Nevertheless, in monocots, embryogenic cell
suspension cultures are most frequently used as a protoplast source as in banana
(Assani et al. 2001) and other species such as in rice (Jain et al., 1995], maize (Prioli
et al., 1989) and barley (Funatsuki et al., 1992).In contrast, in dicotyledonous species,
leaf tissue has been the most frequently used donor material for protoplast isolation. In
addition, the growth conditions for the donor plant play a core role for protoplast
isolation. In order to ensure the continuous supply of sterile leaf material, in vitro
shoot cultures are preferentially employed for protoplast isolation in various species
(Collonier et al. 2001; Möllers and Wenzel 1992).
The developing of protoplast to plant regeneration protocol in this study provides
the prerequisite for creating novel genotypes from Deglet nour with a good quality of
dates and Takerboucht. Unfortunately, all experiments realised till today on calli
318
organogenesis failed to give a high percentage of young plantlets.Takerboucht is
known to be resistant against bayoud disease. Calli production and organogenesis
induction from the combination of the two genomes by protoplast fusion may
contribute to produce a superior date palm variety. Pan et al (2004) has reported that
cell colonies were observed after 4 weeks of culture, and the protoplast-derived
colonies formed calluses when transferred onto 0.25% gellan gum-solidified MS
medium supplemented with 1.0 µmol l–1 BA and 2.0-µmol l–1 IBA. Shoot
organogenesis from protoplast-derived callus was induced on MS medium
supplemented with 5.0 µmol l–1 BA and 2.0 µmol l–1 IBA.
This study showed that nurse culture technique is a good tool to induce regeneration
in protoplasts. The number of microcalli obtained was important in protoplasts isolated
than in protoplasts fused, the possible explanation for decreasing the number of
microcalli in fused protoplasts could be explained by explosion of some of protoplasts
related to the negative interaction by addition of PEG. In contrast, only microcalli
obtained from fused protoplasts were showed regeneration of somatic embryos with
white young leaves, but the number of them was less (3to 5%). The results show that
somatic embryos could be induced from protoplasts fused by PEG. PEG may increase
the embryos formation capacity by varying the sucrose concentration in cells.
In conclusion, this study has demonstrated the possibility of inducing microcalli
formation from protoplasts isolated and fused of two Algerian cultivars of date palm
Deglet nour and Takerboucht by adding PEG at 5%, therefore, further study the
improvement of process of somatic embryo formation seems imperative. The study
may examine plant regeneration from protoplasts, it is necessary to search a quite
growth regulators composition to induce calluses transformation in complete plantlets
with a high number.
319
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322
Table 1. Medium composition and explants cultured for callus induction of date palm.
Medium
composition
(mg L-1)
Callus induction
Callus multiplication
Shoot
tips
Calli
MS salts
4,568
Morel vitamins
2
Fe-EDTA
65
Sucrose
30,000
Myo-inositol
100
Glutamine
100
NaH2PO4
170
Adenine
30
Biotine
0.01
Thiamine-HCl
0.1
Nicotinic acid
0.1
Pyridoxine-HCl
0.1
Ca-pentothenate
0.1
2,4-D
9 µM
4,568
2
65
30,000
100
100
170
30
0.01
0.1
0.1
0.1
0.1
9 µM
14.7µM
7,000
2iP
14.7µM
Agar
7,000
Explant:
323
Table 2: Influence of genotype, donor material on frequency of cell viability cell wall
regeneration 4 days after protoplast (isolated and fused) culture on feeder layer.
% of cell viability
Deglet nour
% of cell wall
regeneration
callus
65±2.0b
54±3.6 b
Cell
suspen
sion
75±2.0 a
55±1.0ab
Takerboucht
callus
57±2.6 b
38±3.6 b
Deglet nour
and Takerbo
ucht fused
callus
27±2.0 c
23±3.6 c
Deglet nour
Cell
and
suspen
Takerboucht
sion
fused
35±2.0 b
25±3.6 c
Data are means of three independent melatonin treatments. Differences between means were assessed
using Student-Newman-Keuls Test; Means with the same letter are not significantly different.
324
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326
OP 14
Effect of somaclonal variation , subculture number and explant type on
in vitro micropropagation of date palm cvs. Sewi,Zaghloul and Samani.
Essam Abdel-Rahman Madboly 1.; Amina Hamed Gomaa2 and Mohamed Ahmed Eisaa2
1 The Central Laboratory for Date Palm Researches, Agriculture Research Center,
Giza, Egypt..E-mail:esammadboly@yahoo.com
2 Department of Pomology, Faculty of Agriculture, Cairo University, Giza, Egypt.
Abstract
Protocol for in vitro micropropagation of plantlets from two types of explants were
excised, the first explant type was shoot tip explants (ST) and the second explant type
was leaf primordial explants (Lp) via tissue culture techniques of date palm(Phoenix
dactylifera L.) cvs. Sewi, Zaghloul and Samani were established in this study .
Increasing the subculture number from 2 to 5 decreased the normal somatic embryos
percentage developed from shoot tip explants and leaf primordia explants of Sewi,
Zaghloul and Samani cvs. . Shoot tip explants of Sewi, Zaghloul and Samani cvs. were
more effective than leaf primordia explants for callus induction ,normal somatic
embryo percentage and number of normal individual somatic embryos. Plantlets
regenerated from shoot tip explants of Sewi, Zaghloul and Samani cvs. showed higher
shoot length(cm) and root number compared with those obtained from leaf primordia
explants .Sewi cv. showed the best callus induction ,normal somatic embryo
percentage and number of individual somatic embryos compared with those obtained
from Zaghloul and Samani cvs. Identification through RAPD-PCR technique cleared
that plantlets regenerated from normal somatic embryo developed from embryogenic
callus obtained from subculture number 3 and 6 of Sewi, Zaghloul and Samani cvs.
Showed genetic variation comparing with mother plants of these cvs.. Genetic
variation occurred between plantlets regenerated through tissue culture techniques and
mother plants, this variation increased by increasing the subculture number and this
phenomenon called somaclonal variation.
Key Words: Date Palm (Phoenix dactylifera L.), In vitro, Tissue culture,
Micropropagation, Shoot tip explants, Leaf primordial explants,
Somatic embryogenesis, Somaclonal variation
327
Introduction
Date palm has been called the “tree of life “because of its indispensable utilization
in the economy and domestic life of inhabitants of palm – growing countries. Date
palm is considered one of the most important commercial crops in the Middle East and
Arab World. The entire tree of date palm is utilized to provide food, shelter, fiber,
furniture and many other products .Moreover, the date palm tree successfully tolerates
extremely adverse environmental conditions, including drought, high temperature and
salinity, which are the peculiar criteria of desert lands (Barreveld, 1993).
Date palm is a monocotyledonous and dioecious fruit tree with a high degree of
heterozygosity. Conventional vegetative propagation, made through offshoots, is very
slow and laborious. (Munier, 1973). The development of micropropagation protocols
is very important for this based on organogenesis and somatic embryogenesis.
Organogenesis in date palm has a low efficiency due to the low number of explants
that respond in vitro, the long time required for the imitation phase, the low
multiplication rate, and the strong influence of the variety (Beauchesne, 1982).
Propagation by both offshoots and tissue culture generally results in true-to-type
plants, but some off-type with abnormal phenotypes have developed in tissue culture
plants. These abnormalities may be due to somaclonal variation (Nasser et al., 2007).
Various molecular techniques are being practiced for fingerprinting such as random
amplified polymorphic DNA (RAPD). (Fukuoka et al., 1992). Optimized conditions
for regenerating random amplified polymorphic DNA (RAPD) markers using the
polymerase chain reaction (PCR) for date palm. They discussed the importance of
technique optimization to obtain reproducible and applicable RAPD patterns.
(Aitchitt et al., 1993) .
Somaclonal variation refers to the variation arising in cell cultures, regenerated
plants and their progenies, and this general term was given by Larkin and Scowroft
(1981). However, other types of variation arise by specific culture of cells or tissues,
which
include
culture:
protoplasts
(protoclonal);
anthers
and
microspores
(gametoclonal); callus (Calliclonal); apical meristem (mericlonal); leaf, stem, root or
other somatic tissues (somaclonal). (Jain et al., 2001).
328
The major objectives of the present study were to the effect of subculture number
and explants type on in vitro micropropagation of date palm cvs. Sewi, Zaghloul and
Samani and identification of somaclonal variation of date palm through RAPD-PCR
technique by comparing with effect of subculture number of plantlets produced from
in vitro micropropagation and mother plant.
Materials and methods
This study was carried out during of 2002-2006 in the Laboratory of the Central
Laboratory for Date Palm Researches and Development (CLDRD), Agriculture
Research Center (ARC), Giza, Egypt.
A . Plant Material
In this experiment healthy offshoots of date palm (Phoenix dactylifera L.) cultivars
Sewi(semi-dry cultivar) grown at Al-Wahaat El-Baharia in Giza, Zaghloul and Samani
(soft cultivars) grown at Rashid in Bihara were selected from mother trees. The young
offshoots were of about 3-5 years; ranging in weight from 5 - 7 kg and about 50 - 80
cm in length.
B . Preparation of explants and surface sterilization
The adventitious roots, fibrous sheath and leaves of selected young offshoot were
removed acropetally with the help of pruning scissors and serrated knife. The shoot tip
was cleaned by a brush, then soaked in 1: 1000 v/v benlate fungicide solution for 60
min and then rinsed by tap water.
The shoot tip was handled very carefully for removing soft inner leaves from the
shoot terminal part after peeling away all the mature leaves to obtain a shoot apex
which was 2 – 4 cm in length and about 1 – 2 cm in diameter as shown in Fig. (1).
The shoot apex was immediately washed with running tap water for 1 – 2 hours. All
excised shoot apexes were soaked temporarily in an anti-oxidant solution (150 mg/l
ascorbic acid and 100 mg/l citric acid) for 30 min. prior to surface sterilization.
Under aseptic conditions, shoot apex’s were soaked in 70% ethanol alcohol solution
for 30 seconds, followed by immersion in (1.0 g/l) of mercuric chloride for 5 min and
thoroughly washed with sterilized distilled water for one-time. After that additional
leaf primordial were removed from sterilized explants and then transferred to double
surface sterilization by commercial Clorox (5.25 % sodium hypochlorite NaOCl) plus
329
2 drop of Tween 20 per 100 ml solution ,the first one by 40% Clorox for 20 min with
rotary agitation, rinsed one times with sterilized distilled water and the second one by
60% Clorox for 20 min with rotary agitation, rinsed three times with sterilized distilled
water. Under aseptic conditions, outer soft leaves were removed to obtain a terminal
portion of the shoot. Two type of explants were excised in this experiment. The first
one was shoot tip explant (S T), shoot tip 5 – 10 mm in length (composed of apical
meristem, sub apical tissue) sliced longitudinally into 4 pieces, and the second explant
was leaf primordial explant (Lp) 5 – 10 mm in length(composed of several leaf
primordial) sliced longitudinally into 6-8 pieces .
C . Media preparation
The basal medium used in this experiment was formula of Murashige and Skoog
(1962) basal nutrient medium with the following modification in mg/l: 170 NaH 2PO4,
2H2O; 200 glutamine; 40 adenine sulfate; 0.4 thiamine-HCl. The basal nutrient
medium was supplemented with mg/l: 3000 activated charcoal; 30000 sucrose; and
6000 agar as described by Tisserat (1984).
The sterilized explants of each explant type (ST and Lp) were cultured on nutrient
medium which consists of the previous modified MS basal nutrient medium
supplemented with 100 mg/l 2, 4-D + 3 mg/l 2iP as described by Matar (1986).
The pH of all culture media was adjusted to 5.8 ± 0.1 prior to the addition of agar,
and then 35 ml of medium was dispensed into small jars (150ml). The culture jars
were sealed with caps of polyvenylpropelin. The jars were autoclaved at 121 °C, and
1.1 kg/cm2 for 20min.
One sterilized explant was cultured individually in each culture jar. The explant was
cultured horizontally with a good contact with the surface of the culture medium .
Each treatment consists of 3 replicates , each replicate consists of 6 culture jars , and
each jar contained one explant ; culture jars
were incubated in a temperature
controlled room at 27 °C ± 1 under complete darkness condition.
D . Callus induction
Uncontaminated and survived explants of each explant type (ST and Lp) were
transferred and
subcultured into corresponding
interval for at least 6 subcultures.
330
fresh medium every 6-8 weeks
Data of each explants type (ST and Lp) were calculated in every treatment at the
end of each subculture for at least 6 subcultures (6-8 weeks for each one) . Data were
taken as the average per explant for callus induction degree value. The average of
browning degree, the average of swelling degree and the average of callus induction
degree were scored visually as follows: (according to Pottino, 1981).
Negative results (-) = 1
below average results (+) = 2
Average results (++) = 3
Good results (+++) = 4
E . Embryogenic callus and somatic embryo development
White friable embryonic nodular callus developed from each explant type, shoot
tip(ST) and leaf primordia (Lp) and each subculture number (2,3,4,5 and 6) were
transferred and cultured on differentiation medium which consists of MS basal nutrient
medium supplemented with 200 mg/l glutamine, 0.4 mg/l thiamine-HCl mg/l and
0.1mg/l NAA (according to Mater, 1986).
Each treatment consists of 3 replicates and each replicate consists of 3 jars each
jar (150ml) each contained 20 ml of prepared medium. One pieces of white friable
embryogenic nodular callus (of about 0.1 g in weight and 1-2 mm in diameter) was
cultured on each culture jar. All culture jars were incubated in growth room at 27
±1°C under 16 hrs. daily exposure to low light intensity of 1000 lux illumination
(Mater, 1986).
White friable embryogenic nodular callus developed from each explant type(ST and
Lp) and each subculture number (2,3,4,5 and 6) were transferred and subcultured
onto fresh differentiation medium ( mentioned above) every 6-8 weeks intervals for
at least two subcultures.
Data of each explant type(ST and Lp) and each subculture number (2,3,4,5 and 6)
were collected at the end of each subcultures every 6-8 weeks intervals for at least two
subcultures. The parameters were tabulated as average of the two subcultures as follows:
- embryogenic callus percentage%.
- normal somatic embryos percentage (individual and multi somatic embryo).
- number of normal individual somatic embryo.
These shapes are described as follows:
331
Normal individual somatic embryo: (Individual embryo): Long of individual
embryoids were 5-10mm as described by Mater (1986). Small seedling with primary
root and shoot as described by George (1993). Mature embryos longer than 5mm as
described by Veramendi and Navarro (1997).
F . Germination of somatic embryos and development of plantlets
The normal somatic embryos(Individual and multi-somatic embryo) formed from
embryogenic callus developed from white friable embryonic nodular callus which
obtained from each explants type (ST and Lp) and each subculture number (2,3,4,5
and 6) cultured onto differentiation medium for at least two subcultures(6-8 weeks for
each one) were transferred and cultured onto germination and development medium
which consists of MS basal nutrient medium supplemented 200 mg/l glutamine, 0.4
mg/l thiamine-HCl and 0.1 mg/l NAA (according to Mater, 1986).
Each treatment included 3 replicates and each replicate included 3 jars (150ml) each
contained 20 ml of prepared medium. One normal somatic embryo (Individual or
multi-somatic embryo) was cultured on each culture jars. All culture jars were
incubated in growth room at 27±1°C under 16 hrs /day exposure to moderate light
intensity of 2000 lux illumination.
The normal somatic embryos(Individual) of each explants type (ST and Lp)and
each subculture number
(2,3,4,5 and 6)were repeatedly subcultured onto fresh
germination and development medium every 4 weeks intervals for at least two
subcultures. For various shapes of embryos the following data were recorded in every
treatment at the end of the second subcultures. The parameters were presented as the
average per plantlet as follows: - shoot length (cm)of normal individual somatic embryo .
- root number of normal individual somatic embryo .
G - Effect of subculture number (3 and 6) on genetic stability of plantlets
regenerated from normal somatic embryos developed from Sewi, Zaghloul
and Samani cvs
DNA-based tests for date palm identification include techniques such as RAPD
(Random Amplified Polymorphic DNA) technique .RAPD is possibly the simplest test
of all recently applied DNA-based tests for date palm identification. The RAPD
332
technique consists of the production of duplicate of segments of plant DNA and these
DNA segments are several million-fold amplified in a reaction called Polymerase
Chain Reaction (PCR). To duplicate the plant DNA, sets of very short nucleotides,
called primers that consist of the basic building blocks of DNA are required. These
primers, which bind selectively to the master-copy of plant DNA are commercially
available from several biotechnology companies. A thermo stable enzyme, called Taq
DNA polymerase, which carries out rapid temperature fluctuation cycles, is necessary
for the duplication process and consequently several million fold amplification of
DNA segments are obtained. Finally, amplified DNA segments are separated on either
agarose or poly acrylamide gels and visualized by staining. The detected
polymorphism between individual plants may result from either a DNA sequence
difference in the binding site for the primer on the plant DNA or a deletion of parts of
the plant DNA., both processes are visible as the absence of a particular RAPD band
after separation on a gel system, which allows the differentiation of cultivars.
Young leaves of plantlets regenerated from in vitro in subculture number 3 and 6
grown in rooting stage and young leaves of mother plants (selected healthy offshoots)
were collected randomly and used for DNA isolation from date palm cvs. Sewi ,
Zaghloul and Samani. Genomic DNA extraction and the method of Gel electrophoresis
as described by Maniatis et al.(1982). RAPD-PCR reactions were conducted using
thirty-six arbitrary 10-mer primers with the sequences indicated in Table (1).
H - Experimental design
The experiments were performed utilizing complete randomized block design with
factorial arrangement. The results were subject to analyzed for variance and the means
were compared using L.S.D at 5% level according to Snedecor and Cochran (1972).
Results and discussion
1. Callus induction degree value:
Data presented in Table (2) and Fig (1), (2) and (3) clearly showed the effect of
subculture number and explant type on callus induction degree value of date palm cvs.
Sewi , Zaghloul and Samani. Concerning the effect of subculture number data clearly
showed that, the highest significant value of callus induction degree was observed in
subculture number 5 (2.76). Followed significantly by value of callus induction degree
333
in subculture number 4, 6 ,3 and 2 (2.59,2.18,2.02 and 1.52 , respectively)with
significant differences in between. While, callus induction degree in subculture
number 1 showed the lowest significant value (1.06 ). Regarding to the effect of
explant type, data revealed that, shoot tip explants produce the higher significant value
of callus induction degree (2.28) compared with leaf primordia explants which showed
the lower significant value of callus induction degree (1.76).
Referring to the effect of cultivars on callus induction degree, data showed that, Sewi
cultivar gave the highest significant value of callus induction degree (2.21), while
Samani cultivar gave the lowest significant value of callus induction degree (1.87).
Callus induction degree value of Zaghloul cultivar came in between with significant
differences among them. Interaction between subculture number and explant type
showed that ,the highest significant value of callus induction degree was observed from
shoot tip in subculture number 4 and 5 (3.00 and 3.14 respectively), without significant
difference among them. Callus induction degree value obtained from leaf primordia
explants of Sewi and Zaghloul cultivars came next without significant difference in
between (1.90 and 1.78, respectively) while, leaf primordia explants of Samani cultivar
showed the lowest significant value of callus induction degree(1.60).
Similar results for callus induction degree value Dixon and Gonzales. (1994) , Zaid
(2003), Zaid et al., (2006) and Zein El-Din (2004) who indicated that the responses of
date palm explants to form callus formation degrees were found in small amounts at
the first subculture number and gradually increased towards the end of fourth
subculture. Also, Gadalla (2003) who indicated that shoot tip explant of dry date palm
cultivars appear to be the most effective explant to produce a highest significant value
of callus formation percentage compared with leaf primordia explant.
2. Embryogenic callus percentage:Data in Table (3) and Fig (1), (2) and (3) clearly showed, the effect of subculture
number and explant type of white friable embryonic nodular callus on percentage of
embryogenic callus cultured onto differentiation medium for 2 subcultures (6-8 weeks
for each) of date palm cvs. Sewi, Zaghloul and Samani. Regarding to the effect of
subculture number data showed that, the white friable embryonic nodular callus
obtained from subculture number 5 and cultured on differentiation medium (for 2
334
subculture 6-8 weeks for each ) gave the highest significant percentage of
embryogenic callus (42.78 %),followed significantly by percentage of embryogenic
callus developed on differentiation medium (for two subculture 6-8 weeks for each)
from white friable embryonic nodular callus obtained from subculture number 4 and 6
(30.28% and 32.22%, respectively ) without significant difference between them.
Percentage of embryogenic callus developed from white friable embryonic nodular
callus obtained from subculture number 3 (20.61 %)came next with significant
differences between them. Embryogenic callus developed on differentiation medium
(for two subculture 6-8 weeks for each) from white friable embryonic nodular callus
obtained from subculture number 2 showed the lowest significant percentage (9.39 %).
Regarding to the effect of explant type, data revealed that, white friable embryonic
nodular callus obtained from shoot tip explants produced the higher significant
percentage of embryogenic callus (37.20 %) compared with white friable embryonic
nodular callus from leaf primordia explants
which gave the lower significant
percentage of embryogenic callus (16.91 %).
Referring to the effect of cultivars on percentage of embryogenic callus, data
showed that, white friable embryonic nodular callus obtained from Sewi cultivar gave
the highest significant percentage of embryogenic callus (31.20 %), while, white
friable embryonic nodular callus obtained from Zaghloul and Samani cultivars gave
the lowest significant percentage of embryogenic callus (24.40 % and 25.57 %,
respectively) when transferred and cultured on differentiation medium for 2
subcultures 6-8 weeks for each.
With respect to the interaction between subcultures number ,explant type and
cultivars data revealed that, white friable embryonic nodular callus obtained from
shoot tip explants of Sewi cultivar gave highest significant percentage of embryogenic
callus in subculture number 5 (65.00%) when transferred and cultured onto
differentiation medium for 2 subcultures 6-8 weeks for each.
These findings are in agreement with those of Veramendi and Navarro (1997),
Sharma et al., (1984), Shakib et al., (1994) ,Yadav et al., (2001) and Gabr and Tisserat
(1985)who found that six of the date palm species, cultivars and clones cultured
produced embryogenic callus after 32 weeks in culture.
335
3. Percentages of normal somatic embryo
The normal somatic embryos (Individual and multi-somatic embryo)
Normal
individual somatic embryo: (Individual embryo): Long of individual embryoids were
5-10mm as described by Mater (1986). Small seedling with primary root and shoot as
described by George (1993). Mature embryos longer than 5mm as described by
Veramendi and Navarro
(1997). Normal multi-somatic embryo: (Secondary or
accessory embryos) which are 3-4 embryos can occur on base of the original embryo
as described by George (1993) and Zaid (2003).
Results in Table (4) and Fig (1), (2) and (3) showed the effect of subculture number
and explant type of white friable embryonic nodular callus on percentage of normal
somatic embryo (individual and multi-somatic embryo) of date palm cvs. Sewi,
Zaghloul and Samani. The formation of normal somatic embryo from embryogenic
callus cultured onto differentiation medium for 2 subcultures 6-8 weeks for each .
Concerning to the effect of subculture number data clearly showed that, embryogenic
callus developed from white friable embryonic nodular callus obtained from
subculture number 2 formed the highest significant percentages of normal somatic
embryos (76.67%).The production of normal somatic embryos from embryonic callus
developed from white friable embryonic nodular callus obtained from different
subculture number reduced significantly by increasing subculture number gradually
from 3,4,5 to 6 subculture number (70.28%,61.11,45.83 to 34.17 %, respectively) with
significant differences in between.
Concerning the effect of explant type data clearly showed that, results also showed
that, embryonic callus developed from white friable embryonic nodular callus obtained
from shoot tip explants formed higher significant percentage of
normal somatic
embryo (65.67%) than those formed from leaf primordia explants (49.56%). Referring
to the effect of cultivars on percentage of normal somatic embryo developed from
white friable embryonic nodular callus, data showed that, Sewi cultivar gave the
highest significant percentage of normal somatic embryo (60.50 %),compared with
those obtained from
Zaghloul and Samani cultivars which showed the lowest
significant percentage of normal somatic embryo (55.83 %and 56.50% , respectively )
without significant difference in between.
336
Regarding to the interaction between
subcultures number ,explant type and cultivars data revealed that, embryonic callus
developed from white friable embryonic nodular callus obtained from shoot tip
explants of Sewi ,Samani and Zaghloul cultivars in subculture number 2 formed the
highest significant percentages of normal somatic embryo (90.00% for each).
Similar results for production of Zaid (2003) found that abnormal shape percentage
of somatic embryos increased by increasing the subculture number for all cultivars of
date palm under investigation(Sewi, Bent-Esha and Hiane). Also, Furuya and Hosoki
(2004) found that many somatic embryos of Oenanthe javanica were developed into
normal plants.
4. Number of individual somatic embryo
Data tabulated in Table (5) and Fig (1), (2) and (3) showed the effect of subculture
number and explant type of white friable embryonic nodular callus on the number of
individual somatic embryo formed from embryogenic callus cultured onto
differentiation medium after2 subcultures 6-8 weeks for each of date palm cvs. Sewi,
Zaghloul and Samani. With regard to the effect of subculture number data clearly
revealed that, significant differences in number of individual somatic embryo for
different subcultures number. Embryonic callus developed from white friable
embryonic nodular callus obtained from subculture number 4 formed the highest
significant number of individual somatic embryo (27.50 embryo/ explant). This
number was reduced significantly with reducing the subculture number to 3 or 2
(17.44 and 7.11 embryo/explant, respectively) or with increasing the subculture
number to 5 or 6 (17.06 and 8.17 embryo/ explant, respectively). Concerning the effect
of explant type data clearly showed that, the number of individual somatic embryo was
affected significantly by explant type , embryonic callus developed from white friable
embryonic nodular callus obtained from shoot tip explants formed higher significant
number of individual somatic embryo (22.22 embryo/ explant) than those formed from
leaf primordia explants (8.69 embryo/ explant). Referring to the effect of cultivars on
the number of individual somatic embryo, data showed that, embryonic callus
developed from white friable embryonic nodular callus obtained from Sewi cultivar
formed the highest significant number of individual somatic embryo (18.43 embryo/
explant).Followed significantly by the number of individual somatic embryo formed
337
from embryonic callus developed from white friable embryonic nodular callus
obtained from Zaghloul and Samani cultivars (13.17 and 14.77 embryo/ explant,
respectively) without significant difference in between.
Concerning, the interaction between subculture number, explant type and cultivars
on number of individual somatic embryo, data revealed that, embryonic callus
developed from white friable embryonic nodular callus obtained from shoot tip
explants of Sewi cultivar in subculture number 4 formed the highest significant
number of individual somatic embryo (45.00 embryo / explant ).
5. Shoot length (cm) of plantlet developed from normal individual somatic embryo
The results in Table (6) and Fig (1), (2) and (3) clearly showed the effect of
subculture number and explant type of white friable embryonic nodular callus on shoot
length (cm)of individual somatic embryo cultured onto germination and development
medium after2 subcultures (4 weeks for each)of date palm cvs. Sewi, Zaghloul and
Samani. Regarding the effect of subculture number, data showed that, individual
somatic embryo formed from embryonic callus developed from white friable
embryonic nodular callus obtained from subculture number 4 showed the highest
significant increments in shoot length/plantlet (3.73cm) . This length of shoot
decreased significantly by decreasing the subculture number to 3 (3.16 cm)or by
increasing the subculture number to5 (2.97 cm) with significant difference in between
.Also, this length of shoot was decreased significantly by decreasing the subculture
number to 2 (2.74 cm). While, the lowest significant shoot length/plantlet was noticed
from individual somatic embryo obtained from subculture number 6 (2.50 cm).
In regard to the effect of different explant type, data indicated that, individual
somatic embryo formed from embryonic callus developed from white friable
embryonic nodular callus obtained from shoot tip explants formed the higher
significant shoot length/plantlet (3.48 cm) and leaf primordia explants gave the lower
significant shoot length/plantlet (2.56 cm) . Regarding to the effect of cultivars, data
indicated that, shoot length/plantlet was affected significantly by cultivars, data
indicated that, individual somatic embryo formed from embryonic callus developed
from white friable embryonic nodular callus obtained from Sewi cultivar formed the
highest significant shoot length/plantlet (3.18 cm). This length followed significantly
338
by shoot length of individual somatic embryo formed from embryonic callus
developed from white friable embryonic nodular callus obtained from Samani cultivar
(3.02 cm), with significant difference between them. While, Zaghloul cultivar formed
the lowest significant shoot length/plantlet (2.86 cm) .
Regarding, the interaction between subculture number, explant type and cultivars,
data revealed that, individual somatic embryo formed from embryonic callus
developed from white friable embryonic nodular callus obtained from shoot tip
explants of Sewi cultivar in subculture number 4 recorded the highest significant
shoot length/plantlet (5.20 cm).
6. Root number of plantlet developed from normal individual somatic embryo
The results in Table (7) and Fig (1), (2) and (3) clearly showed the effect of subculture
number and explant type of white friable embryonic nodular callus on root
number/plantlet of individual somatic embryo cultured onto germination and
development medium after2 subcultures (4 weeks for each)of date palm cvs. Sewi,
Zaghloul and Samani. For the effect of subculture number, data revealed that, the highest
significant root number/plantlet were observed among the root numbers formed from
individual somatic embryo formed from embryonic callus developed from white friable
embryonic nodular callus obtained from subculture number 5,4 and 6 (2.78 ,2.58, and
2.58 root/plantlet, respectively) without significant differences in between .The lowest
significant root number/plantlet were observed among the root numbers developed from
individual somatic embryo formed from embryonic callus developed from white friable
embryonic nodular callus obtained from subcultures number 2(2.11 root/plantlet).
For the effect of explant type data revealed that, individual somatic embryo formed
from embryonic callus developed from white friable embryonic nodular callus
obtained from shoot tip explants showed the higher number of root/plantlet compared
with those developed from leaf primordia explants (2.59 and 2.39 root/plantlet,
respectively). With regard to the effect of cultivars ,data showed that, no significant
difference was noticed between root number /plantlet formed from individual somatic
embryo formed from embryonic callus developed from white friable embryonic
nodular callus obtained from different cultivars under investigation Zaghloul, Samani
and Sewi(2.53,2.48 and 2.45 root/plantlet, respectively ) .
339
This is agree with Anjarne and Zaid (1993) demonstrated that high auxin
concentration, especially NAA, promoted root. Gadalla (2003) obtained that root length
was highly significant on medium supplemented with 0.1 mg/l NAA. El-Bahr (2007)
used shoot tip explants of the three cultivars of date palm. Culturing the in vitro derived
shoots on ¾ MS-medium contained 0.1 mg/l /NAA gave the best root formation.
7. Effect of subculture number (3 and 6) on genetic stability of plantlets regenerated
from normal somatic embryos developed from of Sewi, Zaghloul and Samani cvs.
Young leaves of plantlets regenerated from in vitro of subculture number 3 and 6
grown in rooting stage and young leaves of mother plants (selected healthy offshoots)
were collected randomly and used for DNA isolation from date palm cultivars Sewi ,
Zaghloul and Samani using 10-mer arbitrary primers to detect differences which may
be occurred through subculture number. Five arbitrary decamer oligmucleatide primers
were used; OP-B05, OP-B06, Op-B08, OP-B09 and OP-B10. As a general molecular
study on RAPD-PCR for mother plant(subculture number 0), plantlets developed from
normal somatic embryos obtained from subculture number 3 and plantlets developed
from normal somatic embryos obtained from subculture number 6 showed differences
between mother plant(subculture number 0) and plantlets developed from normal
somatic embryos obtained from subculture number 3 and from subculture number 6 .
All these similarities are shown in (Table 8, 9 and 10). In Sewi cultivar, the higher
similarity between mother plant (subculture number 0), plantlet developed from
normal somatic embryos obtained from subculture number 3 was 84.00 % and
between mother plant (subculture number 0), plantlet developed from normal somatic
embryos obtained from subculture number 6 was 60.00 %. Similarity decreased by
increasing the subculture number from 3 to 6 was (56.00 %).
In Zaghloul cultivar, the higher similarity between mother plant(subculture number 0),
plantlet developed from normal somatic embryos obtained from subculture number 3 was
87.40 % ,between mother plant(subculture number 0) ,plantlet developed from normal
somatic embryos obtained from subculture number 6 was 74.80 % and by increasing the
subculture number from 3 to 6 was (74.70 %).
In Samani cultivar, the higher similarity between mother plant(subculture number 0)
, plantlet developed from normal somatic embryos obtained from subculture number 3
340
was 90.40 % and the lower similarity between mother plant(subculture number 0)
,plantlet developed from normal somatic embryos obtained from subculture number 6
was 53.80 %. Similarity decreased by increasing the subculture number from 3 to 6
was (52.50 %).
From all previous results, the entire similarity matrix of plantlets regenerated from
normal somatic embryo developed from embryogenic callus of Sewi, Zaghloul and
Samani cvs. obtained from subculture number 3 and 6 showed more
similarity
comparing with the similarity between these plantlets and the mother plants of Sewi,
Zaghloul and Samani cvs. .Increasing the subculture number from 3 to 6 of
regenerated plantlets decreased the similarity between these plantlets and mother
plants. These results mean that genetic variations may be occurred through tissue
culture and this phenomenon called somaclonal variation.
RAPD technology appears very effective for identifying accessions of date palm,
although the overall exhibited polymorphism cultivated species (He and Quiros, 1991;
Yang and Quiros, 1993and Wolff and Van Run 1993).
This is agree with Aitchitt et al., (1993) who reported that optimized conditions for
regenerating random amplified polymorphic DNA (RAPD) markers using the
polymerase chain reaction (PCR) for date palm. They discussed the importance of
technique optimization to obtain reproducible and applicable RAPD patterns. Motawei
et al., (2003) who tested the pair wise comparison of amplification products, the
genetic similarity was estimated. The five date palm cultivars showed variation at the
DNA level. Therefore, the polymorphism detected and its reproducibility suggests that
RAPD markers can be used successfully for varietal identification and for studying the
genetic diversity among cultivars.
These results also are agree with Adawy et al., (2002) who used RAPD technique to
assay the genetic and found that all the tested primers exhibited inter varietal
polymorphism as well as inter varietal variation. El-Bahr (2007) who showed shoot tip
explants of date palm. cvs. Zaghloul, Samany and Amhat . The best results of both in
vitro and ex vitro results.
RAPD–based DNA fingerprints gave no evidence of
somaclonal variation during the in vitro propagation following the protocol used. The
RAPD profiles of tissue culture derived plantlets revealed high similarity to mother tree.
341
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344
Table 1. Random primer codes and their sequences for RAPD-PCR analysis.
Primer code
Sequence
OP- B 05
OP- B 06
5' - TGCGCCCTTC - 3'
5' - TGCTCTGCCC - 3'
OP- B 08 5' - GTCCACACGG- 3'
OP- B 09 5' - TGGGGGACTC- 3'
OP- B 10
5' - CTGCTGGGAC -3'
Table 2. Effect of subculture number and explant type on callus induction
degree value of date palm cvs. Sewi , Zaghloul and Samani.
Explant (B)
Subculture
no
(A)
ST
Lp
Cultivar (C )
Cultivar (C )
Mean
(A)
S
Z
Sm
Mean
S
Z
Sm
Mean
1
1.30*
1.07
1.00
1.12
1.00
1.00
1.00
1.00
1.06*
2
2.03
1.60
1.50
1.71
1.50
1.33
1.17
1.33
1.52
3
2.83
1.97
2.13
2.31
1.92
1.60
1.67
1.73
2.02
4
3.33
3.00
2.67
3.00
2.27
2.27
2.00
2.18
2.59
5
3.17
3.13
3.13
3.14
2.53
2.53
2.07
2.38
2.76
6
2.50
2.30
2.43
2.41
2.17
1.97
1.70
1.94
2.18
Mean (B)
Mean ( C)
Mean (B xC)
ST
Lp
2.28
1.76
S
Z
Sm
2.21
1.97
1.87
ST x S
ST x Z
ST x Sm
Lp x S
Lp x Z
Lp x Sm
2.53
2.18
2.14
1.90
1.78
1.60
Mean separation by L.S.D. at 0.05
A=0.14
B=0.08
C =0.10
AxB=0.20
AxBxC=0.35
AxC=0.24
BxC=0.14
*Values determined as described by Pottino (1981)
345
Table 3. Effect of subculture number and explant type of white friable embryonic
nodular callus cultured onto differentiation medium(after 2 subculturs 6-8 weeks for
each)on embryogenic callus percentage of date palm cvs. Sewi , Zaghloul and Samani.
Explant (B)
Subculture
no
(A)
ST
Lp
Cultivar (C )
Cultivar (C )
Mean
(A)
S
Z
Sm
Mean
S
Z
Sm
Mean
2
15.00
11.00
12.00
12.67
7.00
5.33
6.00
6.11
9.39
3
35.00
25.00
25.00
28.33
15.00
11.00
12.67
12.89
20.61
4
55.00
36.67
40.00
43.89
20.00
15.00
15.00
16.67
30.28
5
65.00
55.00
55.00
58.33
30.00
25.00
26.67
27.22
42.78
6
45.00
40.00
43.33
42.78
25.00
20.00
20.00
21.67
32.22
Mean (B)
Mean ( C)
Mean (B xC)
ST
Lp
37.20
16.91
S
Z
Sm
31.20
24.40
25.57
ST x S
ST x Z
ST x Sm
Lp x S
Lp x Z
Lp x Sm
43.00
33.53
35.07
19.40
15.27
16.07
Mean separation by L.S.D. at 0.05
A=2.43
B=1.53
C =1.88
AxB=3.43
AxC=4.20
BxC=2.26
AxBxC=5.94
346
Table 4. Effect of subculture number and explant type of white friable embryonic nodular
callus cultured onto differentiation medium (after 2 subculturs 6- 8 weeks for each) on
normal somatic embryo percentag of date palm cvs. Sewi , Zaghloul and Samani.
Explant (B)
Subculture
no
ST
Lp
Cultivar (C )
Cultivar (C )
Mean
(A)
(A)
S
Z
Sm
Mean
S
Z
Sm
Mean
2
90.00
90.00
90.00
90.00
70.00
60.00
60.00
63.33
76.67
3
85.00
80.00
73.33
79.44
65.00
56.67
61.67
61.11
70.28
4
75.00
71.67
65.00
70.56
55.00
50.00
50.00
51.67
61.11
5
55.00
50.00
45.00
50.00
40.00
40.00
45.00
41.67
45.83
6
40.00
35.00
40.00
38.33
30.00
25.00
35.00
30.00
34.17
Mean (B)
Mean ( C)
Mean (B xC)
ST
Lp
65.67
49.56
S
Z
Sm
60.50
55.83
56.50
ST x S
ST x Z
ST x Sm
Lp x S
Lp x Z
Lp x Sm
69.00
65.33
62.67
52.00
46.33
50.33
Mean separation by L.S.D. at 0.05
A=2.40
B=1.52
C =1.86
AxB=3.39
AxC=4.15
BxC=2.63
AxBxC=5.87
347
Table 5. Effect of subculture number and explant type of white friable embryonic nodular
callus cultured onto differentiation medium (after 2 subculturs 6-8 weeks for each)on
number of normal individual somatic embryo of date palm cvs. Sewi , Zaghloul and Samani.
Explant (B)
Subculture
no
(A)
ST
Lp
Cultivar (C )
Cultivar (C )
Mean
(A)
S
Z
Sm
Mean
S
Z
Sm
Mean
2
15.00
7.00
7.33
9.78
6.33
3.00
4.00
4.44
7.11
3
30.00
18.00
25.00
24.33
12.33
9.33
10.33
10.56
17.44
4
45.00
35.00
40.00
40.00
18.00
12.00
15.00
15.00
27.50
5
25.00
25.00
23.33
24.44
12.67
8.00
8.33
9.67
17.06
6
15.00
11.67
11.00
12.56
5.00
3.00
3.33
3.78
8.17
Mean (B)
Mean ( C)
Mean (B xC)
ST
Lp
22.22
8.29
S
Z
Sm
18.43
13.17
14.77
ST x S
ST x Z
ST x Sm
Lp x S
Lp x Z
Lp x Sm
26.00
19.33
21.33
10.87
7.00
8.20
Mean separation by L.S.D. at 0.05
A=2.17
B=1.37
C =1.68
AxB=3.07
AxC=3.75
BxC=2.37
AxBxC=5.31
348
Table 6. Effect of subculture number and explant type of white friable embryonic nodular callus on
shoot length(cm)of normal individual somatic embryo cultured onto germination and development
medium (after 2 subculturs 4 weeks for each) of date palm cvs. Sewi , Zaghloul and Samani.
Explant (B)
Subculture
no
(A)
ST
Lp
Cultivar (C )
Cultivar (C )
S
Z
Sm
Mean
2
3.40
3.07
2.93
3
3.97
3.23
4
5.20
5
6
S
Z
Sm
3.13
2.30
2.30
2.43
2.34
2.74
3.80
3.67
2.77
2.47
2.70
2.64
3.16
4.13
4.33
4.56
3.10
2.70
2.93
2.91
3.73
3.27
3.23
3.40
3.30
2.73
2.60
2.57
2.63
2.97
2.80
2.67
2.80
2.76
2.23
2.17
2.33
2.24
2.50
Mean (B)
Mean ( C)
Mean (B xC)
Mean
(A)
Mean
ST
Lp
3.48
2.56
S
Z
Sm
3.18
2.86
3.02
ST x S
ST x Z
ST x Sm
Lp x S
Lp x Z
Lp x Sm
3.73
3.27
3.45
2.63
2.45
2.59
Mean separation by L.S.D. at 0.05
A=0.19
B=0.12
C =0.14
AxB=0.26
AxC=0.32
BxC=0.20
AxBxC=0.45
349
Table 7. Effect of subculture number and explant type of white friable embryonic nodular callus
on root number of normal individual somatic embryo cultured onto germination and development
medium (after 2 subculturs 4 weeks for each) of date palm cvs. Sewi , Zaghloul and Samani.
Explant (B)
Subculture
no
(A)
S
ST
Lp
Cultivar (C )
Cultivar (C )
Z
Sm
Mean
S
Z
Sm
Mean
(A)
Mean
2
2.00
2.17
2.17
2.11
2.00
2.17
2.17
2.11
2.11
3
2.00
2.50
3.00
2.50
2.00
2.33
2.50
2.28
2.39
4
2.50
2.67
3.00
2.72
2.17
2.83
2.33
2.44
2.58
5
3.00
3.00
2.83
2.94
3.00
2.50
2.33
2.61
2.78
6
2.83
2.67
2.50
2.67
3.00
2.50
2.00
2.50
2.58
Mean (B)
Mean ( C)
ST
Lp
2.59
2.39
S
Z
Sm
2.45
2.53
2.48
ST x S
ST x Z
ST x Sm
Lp x S
Lp x Z
Lp x Sm
2.47
2.60
2.70
2.43
2.47
2.27
Mean (B xC)
Mean separation by L.S.D. at 0.05
A=0.24
B=0.15
C =N.S
AxB=0.34
AxC=0.42
BxC=0.27
AxBxC=0.59
350
Table 8. Similarity matrix between shoot tip explants mother plant(subculture number 0),
plantlet from subculture number 3 and plantlet from subculture number 6 of date palm cv. Sewi.
Similarity matrix
Mother plant
subculture number 0
Plantlet from
subculture number 3
plantlet from
subculture number 6
mother plant
subculture number 0
100.00
84.00
60.00
plantlet from
subculture number 3
84.00
100.00
56.00
plantlet from
subculture number 6
60.00
56.00
100.00
Table 9. Similarity matrix between shoot tip explants mother plant(subculture number 0), plantlet
from subculture number 3 and plantlet from subculture number 6 of date palm cv. Zaghloul.
Similarity matrix
mother plant
subculture number 0
Plantlet from
subculture number 3
plantlet from
subculture number 6
mother plant
subculture number 0
100.00
87.40
74.80
plantlet from
subculture number 3
87.40
100.00
74.70
plantlet from
subculture number 6
74.80
74.70
100.00
Table 10. Similarity matrix between shoot tip explants mother plant (subculture number0), plantlet
from subculture number 3 and plantlet from subculture number 6 of date palm cv. Samani.
Similarity matrix
mother plant
subculture number 0
Plantlet from
subculture number 3
plantlet from
subculture number 6
mother plant
subculture number 0
100.00
90.40
53.80
plantlet from
subculture number 3
90.40
100.00
52.50
plantlet from
subculture number 6
53.80
52.50
100.00
351
Fig. 1. In vitro micropropagation protocol for shoot tip and leaf primordia explants of
date palm cv. Sewi.
1- Shoot tip and leaf primordia explants.2- Callus induction degree
value. 3- Embryogenic callus.4- Normal somatic embryo development. 5- Germination of
somatic embryos and development of plantlets.6- Shoot length (cm) and Leaves number of
plantlet regenerated from normal individual somatic embryo.
352
Fig. 2. In vitro micropropagation protocol for shoot tip and leaf primordia explants of
date palm cv. Zaghloul.1- Shoot tip and leaf primordia explants. 2- Callus induction
degree value. 3- embryogenic callus 4- Normal somatic embryo development. 5Germination of somatic embryos and development of plantlets.6- Shoot length (cm) and
Leaves number of plantlet regenerated from normal individual somatic embryo.
Fig.3. In vitro micropropagation protocol for shoot tip and leaf primordia explants of
date palm cv. Samani.1- Shoot tip and leaf primordia explants. 2- Callus induction
degree value.3- Embryogenic callus. 4. Normal somatic embryo development. 5Germination of somatic embryos and development of plantlets.6- Shoot length (cm) and
Leaves number of plantlet regenerated from normal individual somatic embryo.
353
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354
OP 15
Application of in vitro culture technique for propagation of two
cultivars of Egyptian date palm
M.K. El-Bahr and Taha. H. S
Plant Biotechnology Department, National Research Centre, Dokki, Cairo Egypt.
E. mail of corresponding author mkbahr03@yahoo.com
Abstract
This study was carried out on two cultivars of Egyptian date palm i.e., Bartamoda
and Sakkoty (dry cultivars). Calli were obtained by culturing the different explants on
MS-medium supplemented with 30 mg/l 2,4-D+3 mg/l 2iP. Embryonic callus was
obtained by culturing the calli on MS medium supplemented with 10 mg/l 2,4-D+3
mg/l 2iP. Embryo formation were obtained by culturing the embryonic calli on MS
medium supplemented with 3 mg/l 2iP. Shoots formation was obtained by sub
culturing the embryos on 3/4 MS-medium supplemented with 0.1 mg/l each of BA
and NAA +0.5 g/l activated charcoal.
To obtain primary roots, the shoots were subcultured on solid 1/2 MS-medium
supplemented with 0.2 mg/l NAA. To obtain well developed root system the plantlets
(shoots with primary roots) of the different cultivars were transferred to a liquid
culture medium contained ½ MS+0.2mg/l NAA +10g/l PEG+ 2mg/l Glycin +5mg/l
Thiamin +1mg/l Biotin. This step could be considered as in vitro adaptation (Preacclimatization). The plantlets obtained were well developed and became ready for
ex-vitro acclimatization in the greenhouse .
To further molecular investigate the genetic stability and somaclonal variation of
in vitro derived plantlets, RAPD analysis was performed on the DNA of tissue
culture-derived plantlets of the two cultivars. According to PCR reactions, the results
of the RAPD profile of tissue culture-derived plantlets obviously revealed high
similarity to mother trees.
Introduction
The importance of palm tree is due to its high tolerance to environmental stresses
ranging from salinity to drought and high temperature, in addition to low maintenance
355
and yield harvesting costs. Therefore, date palm tree is an excellent candidate for
cultivation in arid regions of the world. In most of the date producing Arab Countries,
which are among the first world date producers, high quality dates are still imported.
So, development of an in vitro rapid mass propagation system is a unique solution to
face the shortage of offshoots required for expansion of palm cultivated area [3]. Plant
tissue culture techniques have developed into very powerful tool not only for quick
clonal multiplication of superior cultivars but also for elimination of disease
transmission [1, 10, 4, 14, 9, 2 ]. The successful production of date palm offshoots
using in vitro propagation techniques is due to the developed protocol. On the other
hand, it was mentioned by many investigators that successful rooting stage led to
successful acclimatization [7]. Randomly Amplified Polymorphic DNA (RAPD) has
been extensively applied on date palm for cultivar identification and to determine
phylogenetic relationship between them [13]. This study aimed to achieve an
applicable protocol for in vitro propagation of the two Egyptian cultivars of date palm
i.e., Bartamoda and Sakkoty.
Materials and Methods
Plant materials
Offshoots of the two identified cultivars were separated during fruiting stage from
attached mother plants growing in Upper Egypt (Aswan). These offshoots were used
as mother plant material, for initiation of in vitro culture. The suitable explants of
offshoot containing shoot tip and primordial leaves were excised more carefully.
Explants were surface sterilized by 70% ethanol for 10 sec., followed by 50% Clorox
(5.25 g/Cl) then dipping in 1.5 gm/l of HgCl2 for 5 min. Explants were then rinsed
three times using sterilized distilled water and ready for in vitro cultivation.
Callus induction, embryo’s and shoots formation
The two explants i.e., shoot tip and primordial leaf of the two cultivars were
cultured on MS medium supplemented with 30mg/l 2,4-D +3mg/l 2iP for callus
induction. Cultures were incubated in complete dark in a growth chamber at 26 ±1 °C.
Calli derived from the two explants of the two cultivars were subcultured on
modified MS-medium contained the main addition of 170 mg/l NaH2Po4 + 200 mg/l
KH2Po4 + 30 gm/l sucrose + 100 mg/l glutamine + 1 mg/l thiamin + 1.7 gm/l phytagel
356
and supplemented with 10 mg/l 2,4-D + 3mg/l 2iP for embryonic callus induction.
Embryonic callus derived from calli of the two explants were recultured on modified
MS-medium contained the main addition of 170 mg/l NaH2Po4 + 200 mg/l KH2Po4 +
30 gm/l sucrose + 100 mg/l glutamine + 1.7 gm/l phytagel and supplemented with 3
mg/l 2iP alone for embryos formation. Cultures were incubated under light condition
at 16/8 light/dark day, and the light intensity was 1000 lux. The normal and healthy
embryos were subcultured on ¾ MS-medium contained the main addition of 170 mg/l
NaH2Po4 + 200 mg/l KH2Po4 + 30 gm/l sucrose + 100 mg/l glutamine + 1.7 gm/l
phytagel and supplemented with 0.1 mg/l BA + 0.1mg/l NAA + 0.5 mg/l activated
charcoal for shoots formation. Cultures were incubated under light condition at 16/8
light/dark day, and the light intensity was 1000 lux.
Primary roots formation
After the in vitro shoot formation stage, the derived shoots of the two cultivars
(Bartamoda and Sakkoty) were transferred to1/2 MS-medium supplemented with
0.2mg/l of an auxin i.e., IAA , IBA or NAA as well as basal MS medium. This step
aimed
to study effects of these culture media on percentage of root formation,
number of roots and root length (cm). All cultures were maintained for 30 days under
light condition of 16/8 light / dark day, and the light intensity was 3000 Lux.
Roots formation (In vitro adaptation or pre-acclimatization).
To obtain well developed root system in vitro; two experiments were carried
out as follow:Effect of solidification of MS-medium.
The obtained plantlets from the previous stage were re-cultured onto different
types of MS-culture media (Solid, semi-solid and liquid media) as well as an aseptic
distilled water (as a control treatment) to study the effect of solidification of culture
medium
on their survival
percentage. Date palm plantlets of the two cultivars
(Bartamoda and Sakkoty) were used and the different types of MS-media were
supplemented with 0.2 mg/l NAA and 50 gm/l sucrose.
Effect of liquid MS-salt strength.
The obtained plantlets from the previous treatment were recultured on ¼, ½, ¾ and
full salt strength of liquid MS-medium supplemented with 0.2 mg/l NAA + 50 g/l
357
sucrose to study the effect of different MS-salt strength on survival percentage of the
plantlets. All cultures of root formation stages were incubated under light condition at
16/8 light/dark day, and the light intensity was 3000 lux.
RAPD analysis
Genomic DNA was isolated as the method mentioned by Doyle and Doyle [6].
Two random oligonucleotide primers (Operon technologies Inc., Alameda, California)
were used in RAPD analysis. Each primer was 10 bp lengths. The primers are OPK1
(5' TGC CGA GCT G 3') and OPK4 (5' TCG TTC CGC A 3'). PCR reactions were
carried out in 25 µl volumes containing 2 µl of the target DNA to each tube, 2µl of
each primer, 2 µl of dNTPs, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2.
The Taq-DNA polymerase concentration was 1.5 units per assay. Reaction was
conducted in a Perkin Elmer 2400 thermo cycler (Germany). Machine was
programmed for 45 cycles as follows: 4ºC/5 min (1 cycle), 94ºC/30 sec, 36ºC/30 sec,
2ºC/2 min (44 cycles) and 72ºC/7 min (1 cycle), then held at 4ºC. The amplification
products were size-separated by gel electrophoresis in 1% agrose gels with 1 x TE
buffer using a Pharmacia (GN. 100) submarine gel electrophoresis apparatus and
stained with ethidium bromide and visualized with UV transilluminator and
photographed. A 100-bp DNA ladder (Promega) was used as a DNA standard with
molecular sizes of 1200 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 bp.
Results and discussion
Callus induction, embryo’s and shoots formation
Calli were induced by culturing the two explants i.e., shoot tip and primordial leaf
of the two cultivars on MS medium supplemented with 30mg/l 2,4-D +3mg/l 2iP. to
proliferate embryonic callus , Calli derived from the two explants of the two cultivars
were subcultured on modified MS-medium contained the main addition of 170 mg/l
NaH2Po4 + 200 mg/l KH2Po4 + 30 gm/l sucrose + 100 mg/l glutamine + 1 mg/l
thiamin + 1.7 gm/l phytagel and supplemented with 10 mg/l 2,4-D + 3mg/l 2iP for
embryonic callus induction. To proliferate embryo’s ,the embryonic callus derived
from calli of the two cultivars were recultured on modified MS-medium contained the
main addition of 170 mg/l NaH2Po4 + 200 mg/l KH2Po4 + 30 gm/l sucrose + 100
mg/l glutamine + 1.7 gm/l phytagel and supplemented with 3 mg/l 2iP alone (Fig.1).
358
To recovery of shoots, the normal and healthy embryos were subcultured on ¾ MSmedium contained the main addition of 170 mg/l NaH2Po4 + 200 mg/l KH2Po4 + 30
gm/l sucrose + 100 mg/l glutamine + 1.7 gm/l phytagel and supplemented with 0.1
mg/l BA + 0.1mg/l NAA + 0.5 mg/l activated charcoal (Fig. 2). These results are in
accordance with our previous results [4, 7, 14].
Primary roots formation
After the in vitro shoot formation stage, the derived shoots of the two Egyptian date
palm cultivars (Bartamoda and Sakkoty) were transferred to MS-medium
supplemented with 0.2 mg/l of an auxins i.e., IAA , IBA or NAA as well as basal
MS medium. This step aimed to study effects of these culture media on percentage
of root formation, number of roots and root length (cm).
Data tabulated in Table (1) show the effect of the different culture media on roots
growth parameters formed on shoots of the two cultivars. The descending order of
the percentage of roots formation (91.5% and 82.4%) were recorded with
Bartamoda and Sakkoty cultivars, respectively, when their shoots cultured on MSmedium supplemented with 0.2 mg/l NAA. Among the four types of culture media,
the best results of both roots number and root length were also recorded with MSmedium supplemented with 0.2 mg/l NAA as compared with the other additions of
auxin. As observed before in case of root formation percentage, the optimum
estimation for both of root number and root length were recorded with shoots of
Bartamoda and Sakkoty cultivars, respectively (Fig. 3). Regarding the in vitro root
formation, 63% of rooting was obtained when shoots were cultured on full strength
MS-medium supplemented with 0.1 mg/l NAA [15, 16]. Furthermore, it was
reported that, transferring individual young plants of date palm to MS medium
supplemented with 0.1 mg/1 NAA enhanced root system production [12]. In this
connection, roots were induced on in vitro proliferated shoots of date palm cvs.
Zaghlool and Samany using MS-medium supplemented with 3 mg/l NAA + 0.5
mg/l Kin [5]. On the other hand, a reliable method for in vitro rooting on
proliferated shoots of date palm “Siwy” cv was reported [8]. It was found that, half
strength of solidified MS-medium was more efficient in promoting in vitro rooting
of proliferated shoots.
359
Root formation (In vitro adaptation or pre-acclimatization).
Obtaining a high percentage of survival plantlets during acclimatization stage of
date palm derived in vitro is very needed. The following two experiments were carried
out mainly to overcome the problem of transferring the plantlets (shoots with primary
roots) from in vitro to ex-vitro (greenhouse), e.g. obtain well developed root system on
the in vitro shoots [7].
Effect of solidification of MS-medium.
The obtained plantlets from the previous stage were re-cultured onto different types
of MS-culture media (Solid, semi-solid and liquid media) as well as an aseptic distilled
water (as a control treatment). The different types of MS-media were supplemented
with 0.2 mg/l NAA. The best culture medium for improvement the survival percentage
was MS-liquid medium as compared with solid or semi-solid of MS-media. The highest
values of the survival percentage were observed with plantlets of Bartamoda and
Sakkoty cultivars, respectively. This result was observed with plantlets of the two
cultivars when recultured on the four types of culture media used. The best results of
survival percentage were recorded after 30 days from re-cultivation and this percentage
was gradually declined by increasing the time of incubation (Table 2) .
Effect of liquid MS-salt strength.
The obtained plantlets from the previous treatment were recultured on ¼, ½, ¾ and
full salt strength of liquid MS-medium supplemented with 0.2 mg/l NAA to study the
effect of different MS-salt strength on survival percentage of the plantlets. Data
presented
in Table (3) show that, the best results of survival percentage
were
recorded with those plantlets growing on 1/2 salt strength of liquid MS- medium as
compared with the other strengths. Furthermore, the best results of plantlets survival
percentage were
recorded after 30 days from re-cultivation with Bartamoda and
Sakkoty cultivars , respectively (Fig.3). By increasing the time of culturing from (30
to 90 days) the survival percentage of plantlets was gradually decreased
Accordingly and for enhancement the efficacy of the protocol of the in vitro
propagation through achievement an elevated percentage of survival and development
during rooting and acclimatization stages, the plantlets (shoots with primary roots) of the
two cultivars were transferred to a liquid culture medium contained ½ MS+0.2mg/l NAA
360
+10g/l PEG+ 2mg/l Glycin +5mg/l Thiamin +1mg/l Biotin. This step could be considered
as in vitro adaptation (Pre- acclimatization). The plantlets obtained were well developed
and ready for transplanting to green house for ex-vitro acclimatization (Fig.4) .
RAPD analysis
Two random oligonucleotide primers (Operon technologies Inc., Alameda,
California) were used in RAPD analysis. Each primer was 10 bp lengths. To investigate
the genetic stability and somaclonal variation of the tissue culture-derived plantlets,
RAPD analysis was performed on the DNA of tissue culture-derived plantlets of the
two cultivars. The two primers gave sufficient and reproducible amplification products
Fig (5). Seven polymorphic bands were detected with primer k1 and tree with K4
.According to PCR reactions, the results of the RAPD profile of tissue culture-derived
plantlets obviously revealed high similarity to mother trees (Fig.5).
361
4. References
[1] Aaouine, M. (1998).The application of biotechnology to date palm, In: Agricultural
Biotechnology in International Development,Ives, C. and Bedford, B. (eds). CABI
publishing, UK. pp: 133-146.
[2] Abu El-Fadl, R.E. (2008). Studies on in vitro Propagation of Aswan Date Palm
Cultivars and Their Germplasm Preservation . Ph.D. Thesis, Genetic Engineering
and Biotechnology Research Institute (GEBRI), Sadat City, Minufiya University.
[3] Al-Khayri, J. M. (2007). Date palm Phoenix dactylifera L. micropropagation. In :
Protocol For Micropropagation of Woody Trees and Fruits. (Eds. S. M. Jain and
H. Haggmam), springer, 509-526.
[4] Bekheet, S.A.,Taha, H.S . and Saker, M.M. (2001). Factors affecting in vitro
multiplication of date palm. Biologia Plantarum 44(3) 431-433.
[5] Belal, A.H.
and El-Deeb, M.D. (1997). Direct organogenesis of date palm
(Phoenix dactylifera L.) in vitro. Assiut J. Agric. Sci. 28: 67-77.
[6] Doyle, J.J. and Doyle, J.L. (1987). A rapid DNA isolation procedure for small
quantities of fresh leaf tissue . Phytoch.Bull. 19,11-15.
[7] El-Bahr; M. K., Z. A. Ali and H. S. Taha. (2003). In vitro propagation of Egyptian
date palm c.v. Zaghlool:
II. Comparative anatomical studies between direct
acclimatized and in vitro adapted (pre-acclimatized plantlets). Arab. Univ. J.
Agric. Sci. 11 (2) 701-714.
[8] EI-Hammady, A. M., W.H.Wanas., M.Abo-Rawash, and A. A. Awad. (1999). In
vitro propagation of date palm. 2. Factors affecting rooting and acclimatization of
in vitro proliferated shoots of date palm "Sewy" cv. The international Conference
of Date palm, Assiut Univ., Egypt. pp 25-46
[9] Madboly, E.A.R. (2007). Biotechnology Studies on Date Palm via Tissue Culture
Techniques. Ph. D. Thesis, Pomology Dep., Fac. Agric., Cairo University, Egypt.
[10] Moursy, H.A. and Saker, M. M. (1998). Date palm problems and the need for
biotechnology. Bull. Fac. Agric., Univ. Cairo 49:315-330.
[11] Murashige, T. and Skoog, F.A. (1962). A revised medium for rapid growth and
bioassay with tobacco tissue cultures. Physiol. Plant, 15: 433-479.
362
[12] Omar, M.S. (1988). In vitro response of various date palm explants Date Palm
Journal, 2 (2): 371-389 (1988).
[13] Saker, M. M., Bekheet S. A. , Taha, H. S., Fahmy, A. S. and Moursy, H. A.
(2000). Detection of somaclonal variations in tissue culture-derived Date palm
plants using isoenzyme analysis and RAPD fingerprints. Biologia Plantarum, 43
(3):347-351.
[14] Taha, H.S. Bekheet, S.A. and El-Bahr, M.K. (2003). Alternative approach for
micropropagation of the date palm c.v. Zaghlool. Arab J. Biotech. 6 (1)103-112 .
[15] Tisserat, B. (1982). Factors involved in the production of plantlets from date palm
callus cultures. Euphytica, 31:201-214
.
[16] Tisserat, B. (1983). Development of new tissue culture technology to aid in
the cultivation and crop improvement in date palm. Proceeding of the First
Symposium on Date Palm, King Faisal Univ., Saudi Arabia, pp 126-140.
363
Table (1): Effect of MS-medium supplemented with 0.2 mg/l of IAA, IBA or
NAA on root growth parameters formed on shoots of the two date palm cultivars.
Egyptian date palm cultivars
Bartamoda
Culture media
Root
formation
(%)
No.
of roots
Sakkoty
Root
length
(cm)
Root
formation
(%)
No. of
roots
Root
length
(cm)
MS-basal
62±4.96
1.98±0.05 1.09±0.33
54±6.95
1.85±0.14
0.98±0.15
MS+ 0.2 mg/l
IAA
85±6.75
2.33±0.15 1.25±0.25
80±5.33
2.15±0.36
1.17±0.25
MS+ 0.2 mg/l
IBA
77±5.25
2.15±0.25 1.19±0.15
69±3.11
2.09±0.09
1.05±0.33
MS+ 0.2 mg/l
NAA
91±5.25
2.45±0.33 1.35±0.25
82±4.33
2.25±0.05
1.29±0.35
Each treatment is the average of 10 replicates ± SE. SE = Standard error
Table (2): Effect of solidification of MS–medium supplemented with 0.2
mg/l NAA on survival percentage of date palm plantlets. cv. Bartamoda and
Sakkoty, after 30, 60 and 90 days of re- cultivation.
Solidification of MS-media
Distilled water medium
Solid MS-medium
Semi-solid MS-medium
Liquid MS-medium
Time
of incubation
(days)
Survival percentage
Bartamoda
Sakkoty
30
64± 2.25
61± 2.89
60
55± 3.15
52± 2.59
90
46±3.15
43±2.68
30
77±4.21
71±4.15
60
70±2.58
65±1.56
90
56±2.56
52±2.33
30
80±4.26
75±3.56
60
74±2.18
69±2.16
90
62±2.54
55±1.66
30
85±4.65
79±3.21
60
77±3.55
72±3.12
90
65±2.27
59±2.59
Each treatment is the average of 10 replicates ± SE.
SE = Standard error
364
Table (3): Effect of ¼, ½, ¾ and full strength of liquid MS-medium
supplemented with 0.2 mg/l NAA on survival percentage of date palm
plantlets cv. Bartamoda and Sakkoty, after 30, 60 and 90 days of re-cultivation.
MS-liquid
Media
¼ Strength
½ Strength
¾ Strength
Full Strength
Survival percentage
Time of
Incubation
(days)
Bartamoda
Sakkoty
30
82±4.62
76±3.21
60
79±3.25
74±4.25
90
75±5.56
70±2.42
30
96±5.25
92±4.95
60
85±4.45
83±5.56
90
79±3.25
75±4.33
30
85±4.65
79±3.21
60
77±3.55
72±3.12
90
65±2.27
64±2.25
30
82±4.62
77±3.55
60
75±5.56
70±2.42
90
65±3.65
60±3.47
Each treatment is the average of 10 replicates ± SE.SE = Standard error
365
Fig. (1). Embryos formation on MS- medium supplemented
with 3 mg/l 2iP alone.
Fig.(2). Shoots formation on ¾ MS-medium supplemented
with 0.1 mg/l BA + 0.1 mg/I NAA + 0.5 g/l activated charcoal.
366
Fig. (3). In vitro primary roots formation on
MS-medium supplemented with 0.2 mg/I NAA.
Fig. (4). In vitro adapted plantlets (pre-acclimatization)
before ex vitro acclimatization in the nursery incubator
367
M
1
2
3
4
Fig. (5). RAPD profile of in vivo (lane 1) and in vitro (lane 2) of Bartamoda cultivar and
in vivo (lane 3) and in vitro grown plants (lane 4) of Sakkoty cultivar using primers K1.
M
1
2
3
4
Fig. (6). RAPD profile of in vivo (lane 1) and in vitro (lane 2) of Bartamoda cultivar and
in vivo (lane 3) and in vitro grown plants (lane 4) of Sakkoty cultivar using primers K4.
368
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370
OP 16
Importance of Inflorescence Tissues in Micropropagation of
Select Date Palm (Phoenix dactylifera L.) Genotypes
Larbi ABAHMANE
Plant Biotechnology Lab
INRA, Regional Centre of Agricultural Research
Marrakesh - Morocco
E-mail: abahmanel@yahoo.fr
Abstract
The current needs of Morocco in terms of date palm plants for the next decade are
about three million vitro plants. Since almost all oases are contaminated by Bayoud
disease, it’s essential that a great part of produced palms belongs to date palm genotypes
resistant to this disease. Commonly, date palm is micropropagated from explants excised
from offshoot shoot tips; but in the case of select genotypes resistant to Bayoud disease
and having good fruit quality, the needed numbers of offshoots for their
micropropagation is usually insufficient. In similar situation, the only way to start in vitro
multiplication of such genotypes is the use of floral tissues. This kind of plant material is
almost produced every year, abundant and free from internal bacterial contaminants.
Explants are taken from inflorescences at their emergence on the tree, disinfected
and inoculated into culture media. Obtained vegetative buds are mass propagated on
suitable media and then complete plantlets are regenerated. The whole process was
investigated from plant material collection to plant acclimatization and used to
micropropagate 14 select genotypes and 2 famous varieties with good fruit quality. No
vegetative disorders or abnormalities have been observed neither on vegetative buds
nor produced vitro plants using this micropropagation process. In fact, samples of
produced plants were transferred to soil at INRA’s experimental stations in Marrakech,
Zagora and Errachidia. Those vitro plants have started bearing fruit since 2005 and we
are now able to state on the true-to-typeness of produced vitro plants using this
technique. In the present paper, the different steps of micropropagation process by
using inflorescence tissues as well as the importance of its use in date palm select
genotypes micropropagation will be discussed.
371
Key words: Date palm, select genotypes, inflorescence tissues, masse propagation, in vitro.
Introduction
Date palm, Phoenix dactylifera L. (Family: Palmaceae) is a perennial long-lived,
dioecious, monocotyledon which is highly heterozygous. It is considered the key
species in the preservation of oasis ecosystems. Date palm cultivation is one of the
most economically important activities in the arid zones of the Middle East and North
Africa where it is cultivated not only for its valuable fruits but also to produce fuel,
fibres and to provide shelter for ground crops. In this region, 62 million of the 105
million date palm trees worldwide grow on an area of over 1 million hectares
(1,158,487 ha). World production of dates is approximately 7.43 million metric tons
and generates important commercial activities [14].
The Moroccan needs on date palm vitroplants has tremendously increased. In fact,
during the next decade three million of palm plants are needed to rehabilitate palm
groves and also to create new plantations. As almost all Moroccan palm groves are
infected by Bayoud disease, a soil borne fungus (Fusarium oxysporum fsp. albedinis),
the use of vitroplants from tolerant genotypes is of great importance. In this field, the
National Institute for Agricultural Research (INRA) has select many palm genotypes
that are tolerant to Bayoud and having good fruit quality. However, most of those
select palms are represented in the nature by single trees, usually without offshoots at
their bases, making their propagation by traditional means difficult. To overcome this
situation, a new technique based on use of floral tissues has been developed at the
INRA’s lab in Marrakech city. This process was well investigated and has been used
to micropropagate14 select date palm genotypes.
Some valuable works have been published on the use of such plant material. Most
of the work on floral tissues has been done on plant material excised from immature
inflorescences [11]; [12]; [10]; [8]. However, interesting results have also been
obtained using tissues excised from emerged inflorescences [17]; [18]; [19]; [1]; [2];
[3]; [4]; [5]; [6]. In the latest situation, inflorescences can be removed without severe
damages to the mother tree. In Morocco, the use of this technique has for main
objective the micropropagation of select genotypes in order to produce enough
372
plantlets to be used in resistance tests against Bayoud disease as well as in plant
behaviour studies in the field.
Research methodology
Plant Material Preparation
To start this method, plant material is often excised from emerged inflorescences
during the spring season. Mother trees are usually single trees of select genotypes with
no more offshoots. Those select genotypes are presumed resistant to Bayoud disease
and have good fruit quality. The inflorescences were harvested during the flowering
period from February to April. The disinfection protocol used was as follow:
1- The spathes are firstly washed with tip water and then dipped, during 10 minutes, in
a fungicide solution containing 3g/l of Moncozan (mancozeb).
2- Then, they’re opened under aseptic conditions and the pedicels were carefully
collected and dipped during 15 minutes in a solution of sodium hypochlorite
50% (figure 1).
3- Finally the pedicels were washed three times with sterile distilled water before
transferring on culture media.
4- Plant material can be soaked in an antioxidant solution (ascorbic acid: 100 mg/l and
citric acid: 150 mg/l) until its inoculation on culture media.
Segments of pedicels (1.5 - 2 cm in length) with at least 2 flowers were used as explants.
Culture Medium
Culture medium used in the starting stage consisted of Gamborg and Eveleigh [15]
basal medium as modified by Gresshoff and Doy [16] for macro-elements, Gamborg
and Eveleigh [15] basal medium for microelements and iron sources of Murashige and
Skoog [20] basal medium. Beyond these mineral salts, the following chemicals were
incorporated in the culture medium (mg.l-1): Myo-inositol (100), Adenine (25),
Tyrosine (250), Glycine (2), Biotin (0.01), Thiamin-HCl (1), Nicotinic acid (0.3),
Sucrose (40000), Agar (8000) and PVP-40 (2000).
The medium was adjusted to pH 5.7 after addition of all compounds. The cultures
were incubated in darkness at 27 ± 1°C during the illuminated period and 22 ± 1°C
during the dark period. The photoperiod was 16 hours per day. Transfers to fresh media
373
were done at one month intervals. Culture medium was dispensed in test tubes (25 x 150
mm) at 15 ml per tube before autoclaving during 20 minutes under 1 bar pressure.
Results and discussion
Shoot Formation
In general, it was noticed that use of culture media with high auxins/cytokinins
ratios leads to root formation and carpel development; in contrast, low ratios
enhance floral pieces multiplication usually followed by shoot formation [4]; [18].
In addition, high concentrations seem to be necessary for callus induction when
emerged inflorescences are used as a source of plant material [6]. However, callus
formation can be induced on floral explants excised from immature inflorescences
and cultured on media containing (mg/l): 2,4-D (0.5), IBA (0.5) and BA (0.2l) [12].
Reversion of date palm floral buds to a vegetative state, occurring under tissue
culture conditions, may have many pathways based on the given literature:
• Induction of inflorescence buds reversion: this way requires the use of floral tissues
excised from immature inflorescences before pedicels formation. This method has the
main disadvantage of a limited number of inflorescence buds available per tree [11].
• Induction of floral buds reversion: this method has the advantage of working with
floral buds at different growth stages [11].
• Induction of vegetative budding from floral parts: it presents the major advantage of
working with plant material which is abundant and easy to collect from the mother
tree [3]; [17]; [18].
• Regeneration of vegetative buds (or somatic embryos) from callus induced on floral
parts [17], [6].
Shoot Multiplication
Regenerated shoots were transferred on culture medium containing half strength of
MS basal medium salts supplemented with low concentrations of growth regulators as
previously reported by Abahmane [4]. Hence, a satisfactory rate of multiplication can
be obtained by using a culture medium containing MS salts at half strength
supplemented with NAA (0.2 mg/l), 2-iP (0.5 mg/l) and BA (0.5 mg/l). At the
beginning of shoot multiplication stage, buds growth was slow and the multiplication rate
was no more than 1.2. After the second subculture, this situation has completely changed.
374
In fact, shoot multiplication was satisfactory and reaches a rate of 2.3 every 4 to 5 weeks
(figure 2). Shoots behaviour was similar to that of buds regenerated directly by
organogenesis technique from explants offshoot shoot tips.
The obtained results were in accordance with those reported by Loutfi [19] on several
female varieties and some males, using a culture medium containing MS salts
supplemented with 0.5 mg/l of NAA, 1 or 2 mg/l of BA and 1 mg/l of 2-iP. However,
a diminution in the rate of multiplication was observed after some subcultures.
Reduction of BA concentration to 1 mg/l permitted sustained bud multiplication even
after many subcultures. Similar results have been reported by Al Khateeb [9] on shoot
multiplication of date palm cv. Sukary. He stated that low hormone concentrations
promoted formation of new buds while high concentrations resulted in abnormal
growth and no sign of new budding or shoot formation was observed. According to his
study, the best combination that gives good multiplication rate contained, mg/l:
Kin(0.2), 2-iP(0.1), BA(0.1), IAA(0.1), NOA(0.1), NAA(0.1).In addition, Drira [11]
used a culture medium containing 0.2 - 0.5 mg/l of NAA and 2 to 3 mg/l of BA to
multiply vegetative buds of Allig cultivar.
Shoot Elongation and Rooting
At the end of multiplication stage, shoots were transferred on culture medium
containing MS salts at half strength supplemented with 0.1 to 0.2 mg/l of NAA. In order
to produce complete plantlets, the leaves were left intact (without cutting) to promote
their elongation. In the following subcultures, clusters of small plantlets were formed and
were separated to individual plantlets. On the previous medium, elongated shoots
produced also new leaves as well as many roots (Figure 3).
Elongation of regenerated buds can be easily induced with some genotypes when
others require many transfers on the same medium in order to obtain well elongated buds.
The same results were reported in a previous study on some select date palm genotypes
micropropagated from inflorescence tissues [4]. Some authors have reported that rooting
of elongated shoots can be better when IBA, at 1 mg/l, was used instead of NAA [12].
Furthermore, Loutfi [18] reported good shoot elongation on culture medium containing
MS salts supplemented with 2 mg/l of NAA, 0.5 to 1 mg/l of 2-iP. The author stated
that use of gibberellins (GA3) at 0.01 mg/l permits good elongation of shoots. To
375
obtain vigorous plantlets with formed roots, the use of culture media containing 60 g/l
of sucrose, 0.5 mg/l of NAA, 0.2 mg/l of BA and 0.1 mg/l of 2-iP is required [18].
Plant acclimatization
The use of specialized conditions in growth rooms leads to formation of plantlets
of abnormal morphology, anatomy and physiology [13]; [22].
After ex vitro
transfer, they may be easily impaired by sudden changes in the environmental
conditions. To correct those abnormalities, a period of acclimatization is necessary
[21].
The produced plantlets were transferred in plastic bags under greenhouse. The soil
consisted in a mixture of peat moss and gravels at equal volume (v/v). The temperature
under greenhouse was maintained at 28 ± 2°C and the relative humidity at 70%. To
increase relative humidity around the newly transferred plantlets, a micro-tunnel
covered by transparent plastic was used. Every 2 to 3 days, plantlets were sprayed with
a fungicide (Pelt 44) to prevent crown and leaves rots. Under these conditions,
plantlets having 2 to 3 leaves, a well formed and closed crown and 3 to 4 roots showed
a high percentage of surviving of about 90%. Using such protocol, hundreds of well
acclimatized plants belonging to many genotypes were obtained (figure 4).
It was noticed that plant quality is genotype dependant; hence genotypes like
Mabrouk and INRA-3002 produced vigorous and well-formed plantlets of desired
characteristics while others such as Al Amal and INRA-1445 produced plants of
inferior quality (open crown, curved leaves, weak plantlets).
In such situation,
plantlets should be kept in the lab until they have acquired the desired characteristics.
The same results have been reported by Abahmane [7] on date palm micopropgation
from offshoot shoot tip tissues via organogenesis technique.
Water supply must be monitored very carefully during the first month of
acclimatization. Too much water can lead to plantlet rots and too little moisture in the
soil can decrease the relative humidity around the plants and cause their rapid wilt.
Field Transfer
Samples of produced vitroplants belonging to three select genotypes were transferred
to soil at INRA’s experimental stations in Marrakech, Zagora and Errachidia respectively
in 2001, 2003 and 2005. The growth of those plants was normal and similar to those
376
micropropagated from offshoot tissues. The first fruit set was observed on vitroplants at
Marrakech experimental station in 2005 four years after plantation. For those transferred
to Errachidia, the first fruit set was noticed in 2007 after only three years of planting.
The produced fruits were similar to those of mother trees for both select genotypes
Mabrouk and INRA-1007. Fortunately, this is the first time, in Morocco, that vitroplants
are produced from floral tissues of many select genotypes, transferred to soil and start
bearing fruits at different localities (figure 5). Hence this technique can be used for mass
propagation of select or rare date palm genotypes that have no more offshoots.
Beyond genetic conformity, this experiment has the following objectives:
- Study of agronomic behavior of those select genotypes in the three localities
(Marrakech, Zagora and Errachidia).
- Study of susceptibility of those genotypes against Bayoud disease at Zagora station
which soil is highly infested by Fasarium oxysporum fsp. Albedinis (Bayoud).
Conclusions and prospects
An efficient regeneration protocol for rare and select date palm genotypes with
limited numbers of offshoots (or that have no more offshoots) has been established.
The protocol starts from floral tissues excised from emerged inflorescences. Suitable
media enhancing tissues reversion from floral to vegetative state and consequently
initiating vegetative buds have been used. Obtained shoots were successfully
multiplied and complete plantlets regenerated. Well acclimatized vitroplants have been
produced from several select genotypes. Vitroplants transferred to soil showed normal
growth and start to produce fruits since 2005. Produced fruits were true to type to
those of mother trees.
Using this process, 14 select genotypes (resistant to Bayoud disease and having
good fruit quality) that have no more offshoots have been micropropagated. By using
the described technique, the mother tree can be multiplied without severe damages.
Refining of this process for application in multiplication of further select genotypes
will continue in the future.
377
References
[1] Abahmane, L. 1998. Date palm (Phoenix dactylifera L.) micropropagation by
using young flowers. Proceedings of International Conference on Date Palm,
Marrakech, Morocco, February 16-18, pp: 256-260.
[2] Abahmane, L. 2003. Date palm (Phoenix dactylifera L.) micropropagation by using
inflorescence tissues. Proceedings of the International Conférence on date palm..
Proceedings of International Conference on Date Palm, College of Agric. And Vet.
Med., King Saud University, pp: 911-924.
[3] Abahmane, L. 2005a. Les tissus inflorescentiels: Une nouvelle source de matériel
végétal pour la micropropagation des clones sélectionnés de palmier dattier
(Phœnix dactylifera L.). Proceedings of International Symposium on Sustainable
Agricultural Development of Oasian Ecosystems. Erfoud, Morocco, March 0810, pp: 99-105.
[4] Abahmane, L. 2005b. Micropropagation par tissus inflorescentiels du palmier
dattier (Phœnix dactylifera L.): Un outil efficace pour la sauvegarde des génotypes
rares. Al Awamia 113 (2): 49-60.
[5] Abahmane, L. 2007. Micropropagation of selected clones from inflorescence
tissues and its role in the date palm (Phoenix dactylifera L.) improvement
programme. Proceedings of the 4th International Symposium on the Date Palm,
King Faisal University - Al Hassa, Saudi Arabia, May, 5-8 , 2007 (in press).
[6] Abahmane, L. 2010. Micropropagation of Date Palm (Phoenix Dactylifera L.)
Selected Genotypes from Inflorescence Tissues by Using Somatic Embryogenesis
Technique. Acta Hort. (ISHS) 882:827-832.
[7] Abahmane, L. 2010. Date Palm Micropropagation via Organogenesis’’ In Jain
SM, Al-Khayri J.M. and Johnson D.V. (eds) Date palm Biotechnology,
Springer, pp:69-91.
[8] Abul-Soad, A.A. and Mahdi, M.S. 2010. Commercial production of tissue culture
date palm (Phoenix dactylifera L.) by inflorescence technique. Journal of Genetic
Engineering and Biotechnology 8(2): 39-44
[9] Al Khateeb, A.A. 2006. Role of cytokinin and auxin on the multiplication stage of
date palm (Phoenix dactylifera L.) cv. Sukry. Biotechnology 5(3): 349-352.
378
[10] Bhaskaran, S. and Smith, R.H. 1992. Somatic embryogenesis from shoot tip
and immature inflorescences of Phoenix dactylifera L. cv. Barhee. Plant Cell
Rep 12:22-25.
[11] Drira, N. 1985. Multiplication végétative du palmier dattier (Phoenix dactylifera L.)
par les néoformations induites en culture in vitro sur des organes végétatifs et
floraux prélevés sur la phase adulte. Thèse de doctorat d'Etat Es Sciences Naturelles
Fac. Sc. Tunis, 121p.
[12] Drira, N. and Benbadis, A. 1985. Multiplication végétative du palmier dattier
(Phoenix dactylifera L.) par réversion, en culture in vitro, d'ébauches florales de
pieds femelles. J. Plant Physiol. 119: 223-235.
[13] El Bahr M.K., Ali Z.A., Taha H.S. 2003. In vitro propagation of Egyptian date
palm cv. Zaghlool: II. Comparative and anatomical studies between direct
acclimatized and in vitro adapted pre-acclimatized plantlets. Arab Univ. J. Agric.
Sci. 11:701-714.
[14] FAO. 2009. http://faostat.fao.org
[15] Gamborg, O.L. and Eveleigh, D. 1968. Culture methods and detection of glucanases
in suspension cultures of wheat and barley. Can. J. Biochem. 46: 417-421.
[16] Gresshoff, P.M. and Doy, C.H. 1972. Development and differentiation of haploid
Lycopersicon esculentum. Planta 107: 161-170.
[17] Loutfi, K. 1989. Multiplication végétative du palmier dattier (Phoenix dactylifera
L.) à partir de la culture in vitro d'explants inflorescentiels. Thèse Doctorat 3ème
cycle Université Cadi Ayyad Marrakech – Morocco, 105p.
[18] Loutfi, K. 1999. Organogenèse et embryogenèse somatique à partir des tissus
floraux du palmier dattier (Phoenix dactylifera L.) cultivés in vitro. Aspects
histologiques et caryologie des vitroplants. Thèse doctorat Es Sciences,
Université Cadi Ayyad Marrakech, Morocco, 150p.
[19] Loutfi, K. and Chlyah, H. 1998. Multiplication végétative du palmier dattier à
partir de segments d'inflorescences cultivés in vitro: Effet de différentes
combinaisons hormonales et capacités organogénétiques de divers cultivars,
Agronomie 18 (8-9): 573-580.
[20] Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and
379
bioassays with tissue cultures. Physiol. Plant. 15: 473-497.
[21] Pospisilova, J., Ticha, I. and Kadlecek, P. 1999. Acclimatization of micropagated
plants to ex vitro conditions. Biotech Plantarum 42:481-497.
[22] Saker, M., Bekheet, M. and Taha, H.S. 2000. Detection of seasonal variations in
tissue culture derived date palm plants using isosyme analysis and RAPD
fingerprints. Biol. Plant. 43:347-351.
380
Figure 1: Plant material preparation and disinfection for in vitro inoculation
Figure 3: Shoot elongation and rooting of select date palm genotype INRA-3002
381
Figure 4: vitroplants from Al Amal select date palm genotype, produced
from inflorescence tissues, under greenhouse
Figure 5: vitroplants of Mabrouk select genotype at
Zagora (Top) and Marrakech (Bottom) experimental stations
382
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384
OP 17
Growth promotion of date palm plantlets ex vitro by inoculation of
rhizosphere bacteria
Hala M. A. Farrag, Abeer.H.E. Abd-El Kareim and Rasmia S.S.Darwesh
Central Laboratory of Date Palm for Research and Development, (ARC) Giza, Egypt
Abstract
This study was aimed to use phyto hormones to produce by some strains like
(Azospirillum brasilense, Bacillus megaterium and Klebsiella pneumoniae) as a
protocol in date palm (Phoenix dactylifera L.) cv. Bartomouda to enhance ex vitro
plants during adaptation stage. Plantlets which acclimatized at the greenhouse (six
month) were subjected with three bacteria to evaluate the useful effect of plant growth
promoting were produced by these different bacteria on the growth parameters of the
plantlets and chemical contents. Three treatments were used 10, 20 and 30 cm/l with
irrigation water with 2.5 g/l NPK two times at the week, and two periods were used
(six month for each), control treatment (no bacteria) was used. Results showed that
plant height (cm), number of leaves/plant, root length (cm), number of roots/plant,
indole mg/g (f.w.) and chlorophyll mg/g (f.w.) were most enhanced with the three
types of bacteria at the three levels 10, 20 and 30 cm/l. The highest significant results
were occurred by Klebsiella pneumoniae consecutive by Bacillus megaterium and
Azospirillum brasilense compared to control treatment. Little effect of the level 10
cm/l, meanwhile the level of 30 cm/l has left the greatest results of growth parameters
in the two seasons. Greatest contents of leaves were found in indole IAA mg/g f.w.
and chlorophyll a and b with three types of bacteria and varies concentrations, highly
content have been produced by the level of 30 cm/l. The present study has clearly
shown that the application of Azospirillum brasilense, Bacillus megaterium and
Klebsiella pneumoniae might play a significant role in improving the growth response
of date palm thereby producing good quality planting stock. These plants may perform
better growth, survival and more fruits production.
Key words: Date palm, Azospirillum brasilense, Bacillus megaterium and Klebsiella
pneumoniae, indole, chlorophyll
385
Introduction
Bio fertilizers are the formulations of living microorganisms, which are able to fix
atmospheric nitrogen in the available form to plants, either by living freely in the soil
or being associated symbiotically with plants [1]. They are capable of mobilizing
nutritive elements from non-usable form to usable form through biological processes
[2] Biological nitrogen fixation is carried out by both symbiotic and free living
bacteria and blue green algae.
Azospirillum promoted epidermal cell differentiation in root hairs that increased the
number of potential sites for Bradyrhizobial infection [3]; as a result more nodules
were developed [4].
Gutierrez-Manero el al [5] reported that Bacillus licheniformis
and B. pumilus were active in auxin–like (IAA-1) compounds production. The isolates
produced 1.736 and 1.790 mg IAA-1/L culture growth medium, respectively. The
results were particularly in agreement with, [6] who identified isolates as members of
genera Klebsilla sp., Azospirillum, Azotobacte , Enterobacter and Pseudomonas.
Dong et al [7] revealed that culture medium for Bacillus megaterium contained IAA
and
GA3. Addition of tryptophan (IAA-precursor), increased growth regulator
concentration. Neelam et al [8] identified different isolated bacteria as Bacillus and
Pseudomonas which can produce IAA in addition, it has the ability to solubilize of
phosphate. Sivaprasad et al [9] noticed that Azospirillum spp. associated with rice were
isolated and tested for its efficiency and found these isolates produced IAA; In vitro, at
concentrations from 30 to 55 mg/100mL. In the same purpose, Tensingh Baliah et al
[10] also found that incorporation of Azospirillum strains in the soil resulted in
increasing the soil content of GA3 to 850% over
control. Bacterial strains
(diazotrophs) like Azospirillum brasilense, Bacillus megaterium and Klebsiella
pneumoniae instead artificial fertilization as a protocol in date palm adaptation. Many
phytohormones like auxins, cytokinins, gibberellins (GA3) and abscisic acid (ABA) or
their derivatives can be produced by microorganisms. Numerous studies have shown
the synthesis of plant growth regulators (PGRs) by bacteria in soil and play big role in
biological activities which markedly enhanced by microbial interactions in the
rhizosphere of plants [11]. The present investigation aimed to use bacterial strains as a
386
production of many growth regulators to stimulate and development of date palm
plantlets after acclimatization stage
Materials and methods
This experiment was occurred in the greenhouse of the Central Laboratory of Date
Palm Researches and Development Giza, Egypt throughout the period from 2010-2011
at to investigate the effects of bacterial strains (diazotrophs) like Azospirillum
brasilense, Bacillus megaterium and Klebsiella pneumoniae instead artificial
fertilization as a protocol in date palm adaptation.
Source of bacterial strains
Representative bacterial strains used throughout this study were: Klebsiella
pneumoniae and Azospirillium brasilense which were obtained from a previous
study carried out in the Environmental Studies and Research Unit (ESRU)
Department of Agricultural Microbiology, Faculty of Agriculture, Cairo University,
Giza by Farrag [12]. The other strain Bacillus megaterium was obtained from the
Culture Collection of Microbiological Resource Center, Faculty of Agriculture, Ain
Shams University, Cairo, Egypt.
Purification and maintenance of the isolates
The selected strains were purified by streaking on agar plates containing the
selective medium:
I . Nitrogen fixation deficient medium (NFDM) for Klebsiella [13]
II. Semi-solid malate medium for Azospirillium [14]
III. Bacillus medium [15]
These selective media were surface inoculated with single strain and incubated at
28±2°C for 48hr to 7 days (5-7 times or more) until pure colonies were obtained.
Characteristics of colonies for each tested strains of every culture medium
developed on agar plates were carefully examined, which has different colors.
Maintenance of the selected strains was carried out by sub-culturing on several
selective agar media as slants, which were then kept in refrigerator at (4°C). Strains
were separately grown in liquid medium for preparation of batch cultures by
inoculating 10 mL of 105-106 cells mL-1 in selective culture media and incubated in a
rotary shaker at 100 rpm and temperature of 30ºC to reach to the population density of
387
>107 cfu mL-1(colony forming/ unit) pH was adjusted to 7.0. Growth and population
of tested strains were followed by
measuring optical density (OD) in
spectrophotometer at 580 nm.
Plant material
Plants of date palm Phoenix dactylifera cv. Bartomouda which derived from tissue
culture, these plants derived by somatic embryogenesis for 12-16 months, plantlets
cultured on rooting media 3/4 MS [16] media supplemented with 0.05mg/l BA+
0.1mg/l NAA+ 6g/l agar+ 30g/l sugar) for two months. Then, the plantlets were
cultured on ¼ MS media supplemented with 0.1mg/l NAA for pre-acclimation stage
for 3-6weeks. Plantlets which at length 10-15cm, contained 2-3leaves and 5-6
roots/plant sterilized with fungicide for 10minutes, then washing with water then
cultured on pots containing a mixture of peat moss+ perlite at 2:1and putting under
tunnels at 90% humidity for three months. After 6 months these plants which
described (2-4 leaves, 10-15cm for shoot length and 3-5 roots/plantlet) were subjected
with Azospirillum brasilense, Bacillus megaterium and Klebsiella pneumoniae
supernatants at three concentration (10, 20 and 30cm/l) for each one with irrigation
water two times in the week, three replicates were used at three plantlets for each one.
This experiment was carried out for two seasons 6 months for one season. Also, all
treatments were used as solution with irrigation water and 2.5g/l NPK. After the end of
this experiment the following estimation were take:
1- Plant height (cm)
2- Number of leaves/plant
3- Root length (cm)
4- Root numbers/plant
5- Total indoles
6- Chlorophyll a, b and carotene
Total indole mg/l: as described by Salim et al. [17] and the concentration was
calculated as mg indole acetic acid/100 g fresh weight.
Chlorophyll:
A (mg/g) = 9.784 E 660 – 0.99 E 640
Cl. B (mg/g) = 21.426 E 640 – 4.65 E 660
Carotene (mg/g) = 4.695 E 440 – 0.268 (a + b)
388
Results and discussion
Data in Table (1) showed the effects of bacterial supernatants (Azospirillium
brasilense, Bacillus megaterium and Klebsiella pneumonia) on plant height (cm) and
number of leaves/plantlet of date palm at two seasons.
Plant height
Data analysis at two seasons showed that plant height significantly increased by
increased the concentrations of bacterial supernatant from zero to 30mg/l. It was
noticed that the highest concentration of three bacteria supernatants produced the
highest plant length in both seasons. Klebsiella pneumonia supernatant at 30mg/l gave
the highest plant length in both seasons (18.4 and 23.6 cm in the 1st and 2nd
respectively), compared with other used concentrations. Respecting to the effect of
substances, Bartomouda plant height was significantly affected by bacteria
supernatants as compared with control in both seasons.
However, significant differences could be detected between Azospirillium brasilense
(18.3cm at the 1st and 19.2cm at 2nd seasons) and Bacillus megaterium (18.4cm at the
1st and 21.7 cm at 2nd seasons) supernatants, also significant differences detected with
Klebsiella pneumonia supernatant (20.2cm at the 1st and 30.2cm at 2nd seasons).
The interaction between the types of bacteria supernatants and their concentrations
indicated that 30mg/l of Klebsiella pneumonia supernatant in two seasons gave the
highest Bartomouda plant height (22.0cm at the 1st and 33.5 cm at 2nd seasons)
comparing with other interactions.
Number of leaves
Data in Table (1) cleared that the increasing of concentration of bacteria
supernatants significant production in number of leaves/plant as the highest production
was obtained by using 30mg/l which gave 3.4 and 3.7leaves/plant in the 1 st and 2nd
seasons respectively.
Concerning the types of bacteria, data showed that using Klebsiella pneumonia
supernatant was the most effective in the number of leaves/plant which gave (3.6 at the
1st and 3.9leaves/plant at 2nd seasons) followed by Bacillus megaterium supernatant
gave (3.2 at the 1st and 3.5leaves/plant at 2nd seasons) with significant differences in
389
between. The lowest values were produced by the control treatment (no bacteria
supernatant used) as 2.2 and 2.3 leaves/plant in the 1 st and 2nd respectively.
The interaction between types of bacteria supernatants and their concentrations,
observed that the highest number of leaves/plant (4.4 at the 1st and 4.8leaves/plant at
2nd seasons) obtained as the result of using Klebsiella pneumonia supernatant.
However, control treatment produced the lowest number of leaves/plant which gave
(2.2 at the 1st and 2.3leaves/plant at 2nd seasons). Several studies have been performed
with most focusing on the effect of different types of bacteria on the growth
parameters, Sheng [18] on cotton stated that Bacillus edaphicus NBT strain was
increased plant height, Medina et al. [19] on Alpinia purpurata, El-Barougy et al. [20]
on soybean and Mafia et al. [21] on Eucalyptus globules they found that plant height
and number of leaves were increased with Azospirillum brasilense, Azotobacter
chroococcum and Bacillus megaterium and subtilis, recently Sandeep et al. [22] on
Amaranthus gangetcus, proved that Azotobacter chroococcum was raised the plant
height, root length and leaves number.
Root length
Data presented in Table (2) indicated that the increasing in bacteria supernatants
levels significantly increased root length (cm), as the highest values of root length
resulted from 30mg/l which gave (12.3 and 12.9 cm) in the 1st and 2nd season
respectively, followed by 20mg/l which gave (11.6 and 12.1cm) with significant
differences in between.
The highest stimulation effect occurred by using Klebsiella pneumonia supernatant
(14.3cm at the 1st and 14.8 at 2nd seasons) followed by Bacillus megaterium
supernatant (12.8 cm at the 1st and 13.3cm at 2nd seasons) with significant differences
in between followed by Azospirillium brasilense supernatant (12.6 cm at the 1st and
13.2cm at 2nd seasons). The interaction between type of bacteria supernatants and their
concentrations indicated that 30mg/l of Klebsiella pneumonia supernatant in two
seasons gave the highest significant Bartomouda root length (15.6 cm at the 1 st and
16.2cm at 2nd seasons) comparing with other interactions.
Respecting to the effect of type of bacteria, data cleared that Klebsiella pneumonia
supernatant was the most effective in number of roots/plant (6.7 at the 1st and 7.1
390
roots/plant at 2nd seasons) followed by followed by Bacillus megaterium supernatant
(6.1 at the 1st and 6.4 roots/plant at 2nd seasons) with significant differences in between
followed by Azospirillium brasilense supernatant (5.4 at the 1st and 5.9 roots/plant at 2nd
seasons) and finally control treatment (3.8 at the 1st and 4.3 roots/plant at 2nd seasons)
with significant differences among them. Regarding to the concentration, it was clearly
noticed that the highest concentration 30mg/l of three bacteria supernatants produced
the highest Bartomouda roots number in both seasons (6.4 at the 1st and 6.8 roots/plant
at 2nd seasons). Whereas, the highest significant results of number of roots was (7.9 at
the 1st and 8.3 roots/plant at 2nd seasons) obtained as the result of using Klebsiella
pneumonia supernatant at 30 cm/l. The observed promotion in roots of plants in this
study could be attributed to the cumulative effects of those types of bacteria. Similar
results were obtained by Shishido et al. [23] on Pinus contorta showed that roots length
and numbers were increment with Bacillus polymxa, Wahyudi et al. [24] on soybean
stated that Bacillus sp were increased root length and numbers.
Total indoles mg/g f.w.
Data in Table (3) illustrated that the positive effect of three types of bacteria and
levels on the total indoles mg/g for two seasons. Clearly shown that total indole was
varies with the different types of bacteria, largest significant content of total indoles
were occurred by treatment of Klebsiella pneumonia supernatant, consecutive by
Bacillus and Azospireillum at the 1st season, meanwhile, at the second season the
gradual increasing of indoles from Klebsiella followed by Azospirellum and Bacillus,
significant differences were found. To regard the levels of three types of bacteria, the
level 30 mg/l have left the highest significant values of indoles, while the other levels
10 and 20 mg/l were hadn't significant differences among them at the 1 st season. At the
2nd season the significant differences were found between levels 30 and 10 mg/l.
Highly interaction occurred at 30 mg/l of Klebsiella at the 1st, on the other hand great
significant interaction was found at the level 30 mg/l and three types of bacteria at the
2nd season. In this respect Bent et al. [25] on pinus contorta, Mirza et al. [26] on
sugarcane in vitro, they found that Klebsiella oxytoca increased IAA.
391
Total chlorophyll a and b
Total chlorophyll a and b content (Table 4) was found to be great in the plant leaves
with different three types of bacteria and three levels 10, 20 and 30 mg/l. the
increasing in bacteria supernatants levels significant gradually rising contents in
chlorophyll a and b at the 1st and 2nd respectively. The highest values were found by
the level 30 mg/l at two seasons respectively.
For the types of bacteria, Klebsiella supernatant had the greatest significant results
of chlorophyll a and b respectively at the 1st and 2nd sequenced by other types of
bacteria. Meenakshisundaram et al. [27] on Delonix regia , Ravikumar et al. [28] on
Jatrova curcas they stated that Azospirellum strain and azotobacter were increasing
chlorophyll content.
392
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395
Table (1): Effect of different bacterial supernatant on plant height (cm) and
number of leaves/plant of date palm plants at the 1st and 2nd seasons
Plant height (cm)
Bacterial strains
First season
Second season
10
20
30
mean
10
20
30
mean
Control
12.5
11.4
11.9
11.9
13.1
11.9
12.5
12.5
Azospirillium brasilense
16.3
18.9
19.7
18.3
16.8
19.5
21.2
19.2
Bacillus megaterium
16.4
18.7
20.0
18.4
17.1
20.9
27.1
21.7
Klebsiella pneumoniae
18.7
19.9
22.0
20.2
27.7
29.3
33.5
30.2
Mean
16.0
17.2
18.4
18.7
20.4
23.6
L.S.D
A= 0.5
B= 0.6
AB=1.1
A= 0.9
B= 0.8 AB=1.5
Leaves number/plant
First season
Second season
Control
2.2
2.2
2.3
2.2
2.3
2.4
2.3
2.3
Azospirillium brasilense
2.6
2.8
3.2
2.9
2.9
3.2
3.7
3.3
Bacillus megaterium
2.8
3.3
3.6
3.2
3.0
3.4
4.0
3.5
Klebsiella pneumoniae
3.0
3.5
4.4
3.6
3.1
3.8
4.8
3.9
Mean
2.7
3.0
3.4
2.8
3.2
3.7
L.S.D
A= 0.1
B=0.1
AB=0.2
A=0.2
B=0.3
AB=0.5
Table (2): Effect of different bacterial supernatant on root length (cm) and roots
number/plant of date palm plants at the 1st and 2nd seasons
Root length (cm)
Bacterial strains
First season
Second season
10
20
30
mean
10
20
30
mean
Control
6.1
6.4
6.8
6.4
6.6
6.8
7.1
6.8
Azospirillium brasilense
11.5
13.0
13.3
12.6
12.1
13.6
14.1
13.3
Bacillus megaterium
11.7
13.0
13.5
12.8
12.3
13.5
14.1
13.3
Klebsiella pneumoniae
13.1
14.1
15.6
14.3
13.5
14.6
16.2
14.8
Mean
10.6
11.6
12.3
11.1
12.1
12.9
L.S.D
A= 0.3
B= 0.3
AB=0.5
A= 0.3
B=0.3
AB=0.6
Roots number/plant
First season
Second season
Control
3.7
3.8
4.0
3.8
4.1
4.2
4.4
4.3
Azospirillium brasilense
3.8
6.0
6.5
5.4
4.2
6.5
6.9
5.9
Bacillus megaterium
4.8
6.4
7.1
6.1
5.1
6.6
7.5
6.4
Klebsiella pneumoniae
5.3
7.0
7.9
6.7
5.6
7.3
8.3
7.1
Mean
4.4
5.8
6.4
4.8
6.1
6.8
B=0.5
AB=0.8
A=0.4
L.S.D
A=0.4
396
B=0.5
AB=0.8
Table (3): Effect of different bacterial supernatant on indoles
mg/g f.w. of date palm plants at the 1st and 2nd seasons
Indoles mg/g f.w.
First season
Second season
10
20
30
Mean
10
20
30
Mean
Con
1.1
1.0
1.0
1.0
1.1
1.0
1.1
1.0
Azospirillum
2.6
2.7
3.3
2.9
2.6
4.3
4.4
3.8
Bacillus
2.9
2.9
2.8
2.9
3.1
3.5
3.0
3.2
Klebsiella
2.9
3.3
4.5
3.6
3.2
3.7
5.3
4.1
Mean
2.4
2.4
2.9
2.5
3.1
3.5
L.s.d.
A= 0.5
B= 0.5
AB=0.9
A= 0.5
B=0.6
AB=1.1
Table (4): Effect of different bacterial supernatant on chlorophyll
a and b of date palm plants at the 1st and 2nd seasons
Chlorophyll a mg/g f.w.
First season
Second season
10
20
30
Mean
10
20
30
Mean
Con
0.7
0.7
0.8
0.7
0.7
0.7
0.6
0.7
Azospirillum
1.3
1.4
2.2
1.6
1.3
1.5
2.1
1.6
Bacillus
1.4
1.7
2.4
1.8
1.4
1.8
2.3
1.8
Klebsiella
1.6
2.1
2.3
2.0
1.8
2.2
3.2
2.4
Mean
1.3
1.5
1.9
1.3
1.6
2.1
L.s.d.
A= 0.1
B= 0.2
AB=0.3
A= 0.1
B=0.1
AB=0.2
Chlorophyll b
First season
Second season
Con
0.2
0.2
0.3
0.2
0.3
0.2
0.2
0.2
Azospirillum
0.1
0.8
0.9
0.6
0.1
1.1
1.9
1.0
Bacillus
0.7
1.1
0.2
0.7
0.9
1.3
0.1
0.8
Klebsiella
0.5
0.4
2.0
1.0
0.7
0.7
2.1
1.2
Mean
0.4
0.6
0.9
0.5
0.8
1.1
L.s.d.
A=0.1
B=0.1
AB=0.2
397
A=0.3
B=0.3
AB=0.5
Control
Bacillus megaterium
Azospirillum brasilens
Klebsiella pneumoniae
Fig (1): Effect of different bacterial supernatant on growth
parameters of date palm plants at the 1st and 2nd seasons
398
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400
OP 18
Studies on storage under minimal growth condition of date palm callus explants
*
Zeinab E. Zaid., *Maiada M. El-Dawayati., ** EL-Sayed I. Baker, and ** Amina H. Gomaa
*
The Central Laboratory for Date Palm Researches and Development, Agriculture
Research Center, Cairo, Egypt. Zemmz2005@yahoo.com
**
Department of Pomology, Faculty of Agriculture, Cairo University, Egypt.
Abstract
In vitro conservation of date palm callus explant under minimal growth condition was
investigated. In this study pieces approximately about (1cm x1cm)of develop callus of
Gundila cv. developed from uncontaminated shoot tip explants cultured on callus
formation medium were transferred and recultured on conservation media which consists
of MS basal nutrient medium + 0.5 mg/l (BA) + 0.1 mg/l NAA +3.0 g/l activated
charcoal. Different sugar (sucrose, sorbitol or Mannitol) with their concentrations (0.1,
0.3, 0.5 and 0.7M) were added to nutrient media. Culture jars of each conservation
treatment were divided into two groups, the culture jars of the first group were conserved
under complete darkness at 5oC and the culture jars of the second group were conserved
under complete darkness at 15oC .Data were taken at the end of each conservation period
(6 and 12 months) . Conserved callus explants at 5°C for 12 months showed more friable
callus degree value and embryonic callus degree value compared with those conserved
within 6 months. Callus explants conserved on conservation media supplemented with
0.1 and 0.3M of different sugars showed the highest survival percentage. Conservation
media supplemented with 0.3M of different sugars (sucrose, sorbitol and mannitol) at
15oC showed more friable callus degree values, embryonic callus degree values and
number of mature somatic embryos. All callus explants conserved for 6 months were able
to survived when they were recultured on recovery medium for four weeks and incubated
under normal condition while, 90.73%of callus explants conserved for 12 months were
able to survived when they were recultured on normal growth medium and incubated
under normal conditions for four weeks.
Key words: Date palm, Conservation, Callus, Minimal growth condition, Sugar, ABA
401
Introduction
Tissue culture provides new methods for storing the plant material needed for many
purpose such as delayed planting until climatic conditions are again favorable,
conserve stocks of horticultural and agriculturally interesting species or varieties,
retain genotypes for as long as they are needed in immediate plant breeding programs
and preserve the widest possible use in future (George, 1993).
When culture regularly sub cultured over long period of time, they accumulate
somaclonal variation and also there is additional risk of introducing contaminants into
the culture with regular program of subculture. Reducing the frequency of the
subculture, or stopping it altogether would reduce both the possibility of somaclonal
variation and the risk of contamination (Benson, 1994).A great deal of interest has
been expressed in recent years concerning the application of tissue-culture or in vitro
techniques to plant genetic conservation (Dodds, 1991).
In vitro storage under slow growth conditions delays the necessity for sub culturing
and consequently allows efficient utilization of labor year round (Gollagunta et al.,
2005). As for most in vitro techniques, the successful implementation of minimal
growth technology requires the establishment of specific protocols for each type of
explant and species under consideration (Watt et al., 2000).
This study was carried out to investigate storage of date palm callus explants under
minimal growth condition.
Materials and methods
This work was aimed to study the in vitro conservation of date palm callus explant
under different minimal growth conditions and to examine the recovery of the
conserved explants when they were returned and recultured on normal growth medium
and incubated under normal growth conditions in order to achieve the best minimal
growth conditions for in vitro conservation of different date palm explants.
Effect of different sugar concentrations on callus explants of date palm Gundila cv.
conserved at 5oC or 15oC under complete darkness for (6 and 12 months
In this experiment callus of Gundila cv. developed from uncontaminated shoot tip
explants cultured on callus formation medium as mentioned before, (friable callus
which composed embryonic callus) were divided into pieces each piece was
402
approximately about 1cm x1cm.These callus pieces were used as callus explants in
this experiment. The callus explants were conserved on conservation media which
consists of MS basal nutrient medium (1962) + 0.5 mg/l (BA) + 0.1 mg/l NAA +3.0
g/l activated charcoal with addition of different sugars (sucrose, sorbitol or Mannitol)
concentrations (0.1, 0.3, 0.5 and 0.7M).
The pH of conservation medium of each treatment was adjusted to 5.7 ± 0.1 prior to
addition of 8.0 mg/L agar. The medium of each treatment was distributed into culture
jars (150 mL) where each one contained 40 mg/L. The culture jars were immediately
capped with polypropylene closure and then the medium was sterilized by autoclaving
at 121oC and 15 Ibs/in2 for 20 min. The culture jars of each conservation treatment
were divided into two groups The culture jars of the first group were conserved under
complete darkness at 5oC. The culture jars of the second group were conserved under
complete darkness at 15oC for 6 and 12 months.
Each treatment = 3 replicates and each replicate = 3 culture jars and each jar contain
one pieces of develop callus explant
Data were calculated after 6 and 12 months about the following changes on the
conserved callus explants:1. The average degree value of embryonic callus / explant
This data were scored visually as mentioned above according to Pottino (1981).
2. The average number of mature somatic embryos / explants.
3. Survival percentage.
The survival percentage and viability of callus explants were evaluated at the end of
each conservation period (6 and 12 months).The explants of each conservation
treatment were transferred at the end of different conservation period and recultured on
normal growth medium for somatic embryos differentiation which consists of MS
basal nutrient medium supplemented with 0.5 mg/l BA + 0.5 mg/l kinetin and 30.0 g/l
sucrose, medium solidified by adding 5.0 g/L agar. The pH of medium was adjusted to
5.7±0.1 prior to agar addition .The medium was distributed into culture jars (150 mL)
where each one contained 40 mL. The culture jars were immediately capped with
polypropylene closure and then medium was sterilized by autoclaving at 121 oC and 15
Ibs/in2 for 20 min. Culture jars of each conservation treatment were divided to three
403
replicates .Each replicate consists of three cultures jars .Each jar contained one
conserved callus explants . Culture jars of each conservation treatment were incubated
under normal growth conditions for somatic embryos differentiation at 27 ± 2 oC under
light with 1500 lux for 16 hrs. and 8hrs.dark for 4 weeks to determine the survival
percentage of the conserved callus explants.
Layout of the experiments
The randomized factorial design was used and data were subjected to analysis of
variance. Separation of means among treatments was determined using L.S.D test at
5% according to Snedecor and Cochran (1972).
Results and discussion
The following experiment was aimed to study in vitro conservation of embryonic
callus date palm explant under minimal growth conditions by using osmotic agent and
to examine the recovery of the conserved explants when they were returned and
reculture under normal growth condition in order to achieve the best minimal growth
condition for in vitro conservation of different date palm explants.
a - Effect of different sugar concentrations on callus explants of date palm Gundila
cv. conserved at 5oC under complete darkness for (6 and 12 months)
1. Embryonic callus degree value
Data in Table (1) determined that different sugar type added to conservation media
had a significant effect on the embryonic callus formed from the callus explants of
Gundila cv. The highest significant degree value of embryonic callus formed from
callus explants conserved on medium supplemented with sucrose (1.61) followed
significantly by the degree value of embryonic callus formed from the callus explants
conserved on conservation medium supplemented with mannitol (1.15), The callus
explants conserved on medium supplemented with sorbitol cannot able to form any
embryonic callus (1.00).
There was no significant effect for different sugar concentrations added to
conservation media on the formation of embryonic callus from callus explants.
According to the effect of conservation period (6 and 12 months) on the embryonic
callus formation data showed a significant difference between the two conservation
periods. Conserved callus explants for 12 months resulted the higher significant degree
404
value of embryonic callus (1.50), while callus explants conserved for 6 months cannot
able to form embryonic callus (1.00).
The interaction between the effect of different sugar type and different sugar
concentrations showed that all callus explants conserved on medium supplemented
with sorbitol at different concentrations (0.1 ,0.3, 0.5 or 0.7M) or on
medium
supplemented with mannitol at 0.3M did not able to develop to embryonic callus. The
highest significant degree value of embryonic callus formed from callus explants
conserved on conservation medium supplemented with 0.3M sucrose (1.83).
Referring to the interaction between the effect of different sugar type added to
conservation medium and the conservation period (6 and 12 months), data showed that
all callus explants conserved on medium supplemented with sucrose, sorbitol or
mannitol for 6 months cannot able to develop embryonic callus. While with increasing
the conservation period to 12 months, the degree values of embryonic callus formed
from the callus explants conserved on media supplemented with sucrose or mannitol
were increased significantly, The degree value of embryonic callus formed from callus
explants conserved for
12 months on medium supplemented with sucrose was
superior than the degree value of embryonic callus formed from the callus explants
conserved on medium supplemented with mannitol (2.22 and 1.30) respectively.
2. Number of mature Somatic embryo
All embryonic callus formed from callus explants of Gundila cv. conserved on
conservation media supplemented with different sugar concentration under
investigation and incubated at 5°C under complete darkness for different conservation
period (6 and 12 months) failed completely to developed to mature somatic embryos.
3. Survival percentage
Explants of each conservation treatment were transferred at the end of different
conservation period (6 and 12 months) and recultured on normal growth medium for
somatic embryos differentiation which consists of MS basal medium supplemented for
with 0.5 mg/l BA + 0.5 mg/l kinetin and 30 g/l sucrose +5.0 g/L agar and incubated
under normal growth condition at 27 ± 2oC under light with 1500 lux for 16 hrs. and
8hrs.dark for 4 weeks to determine the survival percentage.
405
Data in Table: (2) revealed clearly that the effect of different sugar type added to
conservation media on survival percentage of callus explants gave significant
differences among different treatments.
All callus explants conserved on media supplemented with sucrose were able to
survive completely with 100% of survival percentage followed significantly by the
survival percentage (88.88%) of the callus explants conserved on media supplemented
with sorbitol. While, the lowest significant (58.32%) survival percentage was obtained
from callus explants conserved on media supplemented with mannitol.
Concerning to the effect of different sugar concentrations added to conservation
media on the survival percentage data indicated that increasing sugar concentration on
conservation media during conservation period parallel with the significant reduction
in the survival percentage .Callus explants conserved on media supplemented with
0.1M sugar gave the highest significant survival percentage followed by the survival
percentage of callus explants conserved on medium supplemented with 0.3M sugar
(90.73% and 88.88% respectively) without significant difference in between. While
the lowest significant survival percentage (70.36%) was obtained when callus explants
conserved on medium supplemented with 0.7M sugar
Data indicate that there was a significant effect for the conservation period (6 and
12 months) on the survival percentage. The survival percentage of callus explants for 6
months was significantly higher than the survival percentage of callus explants
conserved for 12 months (89.81 % and 74.99% respectively).
The interaction between the effect of different sugar type and different sugar
concentrations added to conservation media on the survival percentage gave
significant differences among different treatments. Data determined that all the callus
explants conserved on conservation media supplemented with different sucrose
concentrations (0.1, 0.3, 0.5 and 0.7M) and media supplemented with sorbitol at 0.1 M
and 0.3 M. were able to survive completely with survival percentage 100%, on other
hand, the callus explants conserved on media supplemented with mannitol showed
variable effect in the survival percentage, the survival percentage decreased with
increasing the mannitol concentration on medium
406
The lowest significant survival percentage (38.88%) was achieved when the callus
explants conserved on conservation medium supplemented with 0.7M. mannitol.
The interaction between the effect of different sugar type added to conservation
media and the conservation period (6 and 12 months) had a significant effect on the
survival percentage. Callus explants conserved on medium supplemented with sucrose
for 6 months or for 12 months also callus explants conserved on medium
supplemented with sorbitol for 6 months were able to survive completely as the
survival percentage was 100% While the survival percentage of callus explants
conserved on medium supplemented with sorbitol for 12 months the survival
percentage was decreased to 77.77%.
All callus explants conserved on medium supplemented with mannitol for 6
months exhibited a lowest significant reduction in the survival percentage
(69.43%), this survival percentage decreased to 47.21% when the callus explants
conserved for 12 months.
Regarding to the interaction between the effect of different sugar concentrations
added to conservation media and the conservation period (6 and 12 months) on the
survival percentage, data showed that callus
explants conserved for 6 months on
medium supplemented with 0.1M sugar gave the
highest
significant
survival
percentage (96.29%). While, callus explants conserved for 12 months on medium
supplemented with 0.7M sugar gave the lowest significant survival percentage (74.07%).
There was a significant effect for the interaction among different sugar type,
different sugar concentrations and conservation period (6and 12 months) on the
survival percentage
b. Effect of different sugar concentration on callus explants of date palm Gundila
cv conserved at 15 oC under complete darkness for 6 and 12 months
1. Embryonic callus degree value
Data in Table (3) clearly revealed that different sugar type added to
conservation media affected significantly the embryonic callus formed from callus
explants, Conserved callus explants on medium supplemented with sorbitol
exhibited the highest significant degree value of embryonic callus followed by the
degree value of embryonic callus formed from callus explants conserved on
407
medium supplemented with sucrose (2.25 and 2.04 respectively) without
significant difference in between. While the lowest significant degree value of
embryonic
callus
formed
from
callus
explants
conserved
on
medium
supplemented with mannitol (1.66).
Data clearly showed that different sugar concentrations gave significant effect
on the embryonic callus formed from the callus explants. The highest
concentrations of sugar 0.5M or 0.7M recorded the lowest significant degree
values of embryonic callus formed (1.51, 1.29 respectively) without significant
difference in between, on the other hand the highest significant degree values of
embryonic callus formed from
callus explants conserved on medium
supplemented with 0.3M sugar (2.36) followed significantly by the degree value
of embryonic callus formed from callus explants conserved on medium
supplemented with 0.1M sugar (2.03).
Concerning to the effect of the conservation period (6 and 12 months) on the
embryonic callus formation data revealed that increasing the conservation period
to 12 months increased significantly the degree value of the embryonic callus
formed from callus explants (2.09) more than the degree value of the embryonic
callus formed from callus explants conserved for 6 months (1.05).
According to the interaction between the effect of different sugar type and
different sugar concentrations on the embryonic callus formed from callus
explants, data recorded that callus explants conserved on medium supplemented
with sorbitol, sucrose or mannitol at 0.7M gave the lowest significant degree value
of embryonic callus (1.60, 1.44 and 1.44 respectively) without significant
differences in between. Conservation medium supplemented with 0.3M sorbitol
was the most effective in giving the highest significant degree value (2.88) of
embryonic callus formed from callus explants.
2. Number of mature somatic embryos
Data in Table (4). showed the effect of different sugar type which added to
conservation media on the number of mature somatic embryos produced from
callus explants of Gundila cv. conservation medium supplemented with sorbitol
gave the highest significant number of mature somatic embryo (1.62) followed
408
significantly by the number of mature somatic embryo produced from callus
explants conserved on medium supplemented with sucrose (0.77). While callus
explants conserved on medium supplemented with mannitol failed completely to
produce any mature somatic embryo.
Somatic embryo produced from callus explants affected significantly with
different sugar concentration added to conservation media. Increasing sugar
concentration on conservation medium from 0.1M to 0.3M increased significantly
the number of mature somatic embryo from 0.98 to 1.68 respectively. While,
increasing sugar concentration on conservation medium to 0.5M or to 0.7M
reduced significant the number of mature somatic embryo to 0.31 and 0.20
respectively without significant difference in between.
Concerning to the effect of the conservation period (6 months and 12 months)
on the number of mature somatic embryos produced from the callus explants, data
observed clearly that the number of mature somatic embryos produced from callus
explants conserved for 12 months was significantly higher than the number of
mature somatic embryos produced from callus explants conserved for 6 months
(1.10 and 0.48 respectively).
Regarding to interaction between the effect of different type of sugar and
different sugar concentrations, data showed a significant effect on the number of
mature somatic embryo produced from the callus explants. Conserved callus
explants on medium supplemented with 0.3M sorbitol gave the highest significant
number of mature somatic embryo (3.16) followed significantly by the number of
mature somatic embryo produced from callus conserved on medium supplemented
with 0.1M sorbitol and the number of mature somatic embryo produced from
callus explants conserved on medium supplemented with 0.3M sucrose (2.02 and
1.88 respectively) without significant difference in between, Conserved callus
explants on medium supplemented with 0.7M
sucrose or on medium
supplemented with 0.1, 0.3, 0.5 and 0.7M mannitol cannot able to develop any
mature somatic embryos.
Referring to interaction between the effect of the type of sugar and the
conservation period (6 and 12 months) on the mature somatic embryos produced
409
from callus explants, data revealed that the number of mature somatic embryos
produced from
callus explants conserved for 6 or 12 months
on medium
supplemented with sorbitol (1.12, and 2.10 respectively) were significantly higher
than the number
of mature somatic embryos produced from callus explants
conserved on medium supplemented with sucrose (0.33 and 1.21 receptivity).
3. Survival percentage
Explants of each conservation treatment were transferred at the end of different
conservation period (6 and 12 months) and recultured on normal growth medium for
somatic embryos differentiation which. consists of MS basal medium supplemented
for with 0.5 mg/l BA + 0.5 mg/l kinetin and 30.0 g/l sucrose +5.0 g/L agar and
incubated under normal growth condition at 27 ± 2oC under light with 1500 lux for 16
hrs. and 8hrs.dark for 4 weeks to determine the survival percentage.
Data in Table (5) showed that different sugar type added to conservation media
gave significant effect on the survival percentage of the callus explants. All callus
explants conserved on medium supplemented with sucrose or sorbitol were able to
survive as the survival percentage was 100% while, 86.10% of callus explants
conserved on medium supplemented with mannitol able to survive.
Different sugar concentrations added to conservation media did not affect
significantly the survival percentage of callus explants.
It could be observed from data that the conservation period (6 and 12 months)
affected significantly the survival percentage of callus explants. All callus explants
conserved for 6 months able to survive (100%).While, 90.73% of callus explants
conserved for 12 months able to survive.
Concerning to interaction between the effect of different sugar type and the
conservation period (6 and 12 months) on the survival percentage of callus explants
data indicated that, all callus explants conserved on medium supplemented with
different type of sugar (sucrose, sorbitol and mannitol) for 6 months succeeded in
surviving (100%). All callus explants conserved on medium supplemented with
sucrose or with sorbitol for 12 months were able to survive completely (100%). While,
72.21% of the callus explants conserved on medium supplemented with mannitol for
12 months able to survive
410
From all the previous results about the conservation of callus explants of Gundila
cv. on conservation media supplemented with different sugar concentrations and
conserved at 5°C or 15°C under complete dark condition for at least 12 months, data
clearly showed that callus explants conserved on media supplemented with sucrose at
5°C and callus explants conserved on media supplemented with sorbitol at 15°C gave
the highest embryonic callus degree values.
In this concern, Broncema et al., (1997) reported that Zygotic embryos of maize
produced less callus on sorbitol than on sucrose containing medium, so that overall
lower growth of cultures grown on sorbitol compare to cultures grown on sucrose
might explain the longer subculture intervals possible on sorbitol containing medium.
The previous results also revealed that all embryonic callus conserved on
conservation media supplemented with different sucrose concentration except 0.7M
and concentration media supplemented with different concentration of sorbitol at 15°C
for at least 12 months able to develop to mature somatic embryos.
Also from the previous result it could be concluded that all callus explants
conserved on conservation media supplemented with sucrose and conservation media
supplemented with 0.1 or 0.3 M sorbitol at 5C for at least 12 months, also all callus
explants conserved on conservation media supplemented with sucrose or sorbitol at
15°C for at least 12 months able to survive.
In these respect, Schenk and Hildebrand (1972) reported that high sucrose
concentrations (above 4-5%) begin to have an inhibitory but nontoxic effect on plant
cell growth. High sucrose levels can therefore be used to maintain cultures in a
dormant condition for long period. Tarmizi et al., (1993) indicated that high sucrose
concentrations is one of the approaches in minimal growth stage which after the
possibly of reducing requirements for subcultures of oil palm polyembroys cultures.
The treated cultures could be maintained for at least 6 months. Also, Danso and Ford LIoyd (2004) mentioned that high sucrose concentration is known to have adverse
effect on morphogenesis.
Bekheet et al., (2001) showed that health shoot bud cultures of date palm were
obtained after 6 months of storage on medium containing 40mg/L sorbitol. However,
this period extended for 9 months in the case of callus cultures.
411
From the previous results it could be clearly showed that all embryonic callus
formed from callus explants conserved for 6 or 12 months on conservation media
supplemented with different concentrations of mannitol at 15°C failed completely to
developed to mature somatic embryos.
Also, callus explants conserved on mannitol conservation media at 5 or 15°C for at
least 12 months showed the lowest survival percentage compared with those obtained
from callus explants conserved on sucrose and sorbitol media.
In these concerns, Westcott (1981) reported that mannitol concentration of 6-10%
exhibited toxic effect on solanum species. Mannitol was also toxic to cassava cultures
if the storage temperature lower than 20°C (Roca et al., 1989). Low temperature and
high mannitol concentration is probably toxic for Calocasia esculanta (Bessembinder
et al.,1993). Harding (1994) found that growth in vitro potato micro plant on high
mannitol medium cause hyper methylation of DNA. Also, from the previous results it
could be clearly revealed that callus explants conserved on mannitol media at 5°C
and15°C for at least 12 months showed the lowest survival percentage compared with
those conserved on sucrose and sorbitol media Also callus explants conserved on
mannitol media at 5°C showed the lower survival percentage compared with those
conserved at15°C. At the same time increasing the concentration of mannitol in
conservation media decreased the survival percentage.
In these concerns Negash et al., (2001), found that low incubation temperature of
15°C combined with low concentration of mannitol in the growth medium (1% or 2%)
reduced growth of inset cultures while maintaining high survival frequency. Low
amount of mannitol in the culture medium increase the survival of plant material from
conserved nodal cuttings of Dioscorea alata during recovery process (Borges et al.,
2004). Mannitol had a positive effect on survival of Colocasia esculanta at 28/24°C
over 12-h photoperiod (Bessembinder et al., 1993).
412
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414
Table 1. Effect of different sugar concentrations on embryonic callus degree values formed
from callus explants of Gundila cv. conserved at 5 oC under dark for 6 and 12 months.
(C) Conservation Period (month)
(B)
Sugar
M/L
6
12
(AB)
Mean
0.1
1.00
2.22
1.61
0.3
1.00
2.66
1.83
0.5
1.00
2.00
1.50
0.7
1.00
2.00
1.50
1.00
2.22
1.61a
0.1
1.00
1.00
1.00
0.3
1.00
1.00
1.00
0.5
1.00
1.00
1.00
0.7
1.00
1.00
1.00
1.00
1.00
1.00 c
0.1
1.00
1.11
1.05
0.3
1.00
1.00
1.00
0.5
1.00
1.44
1.22
0.7
1.00
1.66
1.33
Mean (A)
1.00
1.30
1.15 b
Mean (C)
1.00 b
1.50a
(A)
Sugar
Sucrose
Mean (A)
Sorbitol
Mean (A)
Mannitol
(B) Sugar M/L
0.1
0.3
0.5
0.7
1.22
1.27
1.24
1.50
(B)
Sugar M/L
(C) Conservation Period (month)
6
12
0.1
1.00
1.44
0.3
1.00
1.55
0.5
1.00
1.48
0.7
1.00
1.55
L.S.D 0.05 for
A
0.40
AB
0.28
B
N.S
AC
0.20
C
0.11
BC
N.S
ABC
N.S
* Values determined as described by Pottino (1981).
415
Table 2. Effect of different sugar concentrations on survival percentage of callus explants
of Gundila cv. conserved at 5 oC under dark for 6 and 12 months(when recultured on
normal growth medium and incubated under normal growth conditions for 4 weeks.)
(C) Conservation Period (month)
(B)
Sugar
M/L
6
12
(AB)
Mean
0.1
100
100
100
0.3
100
100
100
0.5
100
100
100
0.7
100
100
100
100
100
100 a
0.1
100
100
100
0.3
100
100
100
0.5
100
66.66
83.33
0.7
100
44.44
72.22
100
77.77
88.88 b
0.1
88.88
55.55
72.21
0.3
66.66
66.66
66.66
0.5
66.66
44.44
55.55
0.7
55.55
22.22
38.88
Mean (A)
69.437
47.217
58.32 c
Mean (C)
89.81 a
74.99 b
(A)
Sugar
Sucrose
Mean (A)
Sorbitol
Mean (A)
Mannitol
(B) Sugar M/L
0.1
0.3
0.5
0.7
90.73a
88.88a
79.62b
70.36c
(B)
Sugar
M/L
(C) Conservation Period (month)
6
12
0.1
96.29
85.18
0.3
88.88
88.88
0.5
88.88
81.48
0.7
85.18
74.07
L.S.D 0.05 for
A
5.56
AB
11.12
B
6.42
AC
7.86
C
4.54
BC
9.08
ABC
15.73
416
Table 3. Effect of different sugar concentrations on embryonic callus degree values formed
from callus explants of Gundila cv. conserved at 15 oC under dark for 6 and 12 months.
(C) Conservation Period (month)
(B)
Sugar
M/L
(A)
Sugar
6
12
(AB)
Mean
0.1
2.33
2.66
2.48
0.3
2.33
2.99
2.66
0.5
1.33
1.88
1.60
0.7
1.33
1.55
1.44
1.83
2.27
2.04 a
0.1
2.55
2.77
2.66
0.3
2.66
3.10
2.88
0.5
1.77
1.99
1.88
0.7
1.33
1.88
1.60
2.07
2.43
2.25 a
0.1
1.22
1.55
1.38
0.3
2.11
2.33
2.22
0.5
1.44
1.77
1.60
0.7
1.22
1.66
1.44
Mean (A)
1.49
1.82
1.66 b
Mean (C)
1.05b
2.09 a
Sucrose
Mean (A)
Sorbitol
Mean (A)
Mannitol
(B) Sugar M/L
0.1
0.3
0.5
0.7
2.03b
2.36a
1.51c
1.29c
(B)
Sugar M/L
(C) Conservation Period (month)
6
12
0.1
2.03
2.32
0.3
2.36
2.80
0.5
1.51
1.88
0.7
1.29
1.69
L.S.D 0.05 for
A
0.29
AB
0.58
B
0.33
AC
N.S
C
0.23
BC
N.S
ABC
N.S
* Values determined as described by Pottino (1981).
417
Table 4. Effect of different sugar concentrations on the number of mature somatic embryos
produced from Gundila cv. callus explants conserved at 15oC under dark for 6 and 12 months
(C) Conservation Period (month)
(B)
Sugar
M/L
(A)
Sugar
6
12
(AB)
Mean
0.1
0.00
1.88
0.94
0.3
1.33
2.44
1.88
0.5
0.00
0.55
0.27
0.7
0.00
0.00
0.00
0.33
1.21
0.77 b
0.1
1.38
2.66
2.02
0.3
2.44
3.88
3.16
0.5
0.33
1.00
0.60
0.7
0.33
0.88
0.60
1.12
2.10
1.62 a
0.1
0.00
0.00
0.00
0.3
0.00
0.00
0.00
0.5
0.00
0.00
0.00
0.7
0.00
0.00
0.00
Mean (A)
0.00
0.00
0.00 c
Mean (C)
0.48 b
1.10 a
Sucrose
Mean (A)
Sorbitol
Mean (A)
Mannitol
(B) Sugar M/L
0.1
0.3
0.5
0.7
0.98 b
1.68 a
0.31 c
0.20 c
(B)
Sugar M/L
(C) Conservation Period (month)
6
12
0.1
0.46
1.51
0.3
1.25
2.10
0.5
0.11
0.51
0.7
0.11
0.29
L.S.D 0.05 for
A
0.30
AB
0.61
B
0.35
AC
0.43
C
0.25
BC
N.S
ABC
N.S
418
Table 5. Effect of different sugar concentrations on survival percentage of callus explants
of Gundila cv. conserved at 15 oC under dark for 6 and 12 months(when recultured on
normal growth medium and incubated under normal growth conditions for 4 weeks.)
(A)
Sugar
(C) Conservation Period (month)
(B)
Sugar M/L
6
12
(AB)
Mean
0.1
100.00
100.00
100.00
0.3
100.00
100.00
100.00
0.5
100.00
100.00
100.00
0.7
100.00
100.00
100.00
100.00
100.00
100.00 a
0.1
100.00
100.00
100.00
0.3
100.00
100.00
100.00
0.5
100.00
100.00
100.00
0.7
100.00
100.00
100.00
100.00
100.00
100.00 a
0.1
100.00
77.77
88.88
0.3
100.00
88.88
94.44
0.5
100.00
77.77
88.88
0.7
100.00
44.44
72.22
Mean (A)
100.00
72.21
86.10 b
Mean (C)
100.00 a
90.73 b
Sucrose
Mean (A)
Sorbitol
Mean (A)
Mannitol
(B) Sugar M/L
0.1
0.3
0.5
0.7
96.29
98.14
96.29
90.74
(C) Conservation Period (month)
(B)
Sugar M/L
6
12
0.1
100.00
92.59
0.3
100.00
96.29
0.5
100.00
92.59
0.7
100.00
81.48
L.S.D 0.05 for
N.S
AB
4.49
A
6.35
AC
N.S
B
N.S
CB
3.66
C
N.S
ABC
419
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OP 19
A new concept for production and scaling up of bioactive compounds
from Egyptian date palm (Zaghlool) cultivar using bioreactor.
Taha H. S*., S.A. Bekheet and M. K. El-Bahr
Plant Biotechnology Department, National Research Centre, Dokki, Cairo ,Egypt.
e.mail of corresponding author: Hussein.taha2@yahoo.com.
Abstract
A promising and successful protocol for enhancement and production of total
phenolic and peroxides compounds from Egyptian date palm cultivar Zaghlool cells in
stirred tank reactor was established. The influence of cultivation of cell cultures in
combining with
Aspergillus niger extract, and Methyl-Jasmonate
elicitors
incorporation feeding medium on cells growth patterns and production of active
compounds was investigated. The maximum value of cell growth parameters and
highest content of bioactive compounds were resulted from elicitation of modified
MS-medium with Aspergillus niger extract at 0.1% in combination with Methyl
ـJasmonate (100 µM) as compared with other concentrations
after 10 days of
cultivation. The chemical analyses of the different cell lines were spectrophotometerically performed. This study clearly indicates that combining of different
elicitors achieved of secondary metabolites process in date palm cell cultures and that
Aspergillus niger and Methyl-Jasmonate plays a critical role in elicitation of date
palm cell cultures.
Key words: Date palm , elicitors, phenolic and peroxidase compounds, bioreactor
Introduction
Date palm (Phoenix dactylifera L.) is a dioecious fruit tree native to the hot arid
regions of the world, mainly grown in the Middle East, and North Africa. Since
ancient time this majestic plant has been recognized as the “tree of life” because of its
integration in human settlement, wellbeing, and food security in hot and barren parts
of the world, where only a few plant species can flourish (Al-Khayri,2007). Date palm
trees provide the most sustainable agro-ecosystems in harsh dry environments
providing raw materials for housing, furnishings, and many handcrafts in addition to
421
supplying nutritious delicious fruits that can be consumed fresh, dried, or processed,
providing a nutritious source of sugars, minerals, and vitamins. Economically, date
palm provides a major source of income for local farmers and associated industries in
communities where it is grown. Biotechnology is a set of rapidly emerging and far
reaching new technologies with great promise in areas of sustainable food production,
nutrition security, health care and environmental sustainability. Our vision is to use
powerful tools of biotechnology to help convert the countries diverse biological
resources to useful products and processes that are accessible to its masses for
economic development and employment generation (Shri et al., 2011). Bioreactors
have several advantages for mass cultivation of plant cells. i) it gives better control for
scale up of cell suspension cultures under defined parameters for the production of
bioactive compounds. ii) constant regulation of conditions at various stages of
bioreactor operation is possible iii) handling of culture such as inoculation or harvest is
easy and saves time Iv) nutrient uptake is enhanced by submerged culture conditions
which stimulate multiplication rate and higher yield of bioactive compounds. v) large
number of plantlets are easily produced and can be scaled-up (Fulzele & Heble, 1994 ).
Phenolics are intermediates of phenylpropanoid metabolism (Cvikrová, 1996) and
precursors of lignin (Lewis and Yamamoto,1990) and phenylpropanoid phytoalexins
(Kessmann, 1996). Their deposition in cell walls is an important defense mechanism
after pathogen infection (Bolwell, 1986). The plant cells cultivated in vitro synthesize
phenolic compounds, however, in some cases changes in the quality and quantity of
the substances were recommended (Zagoskina, 1983). This is probably due to
specificity of the tissue cultures as artificial biological system in which the basic
function of phenols is to interfere in cell proliferation (Ozyigit, 2007). Longenbeck
(1983) reported that substitution pattern of phenols was affected according to the rate
of IAA degradation. Also he reported that phenols were found to react with hydrogen
peroxide produced during IAA degradation, thereby protecting cellular constituents
from its toxic effect.
Peroxidase is an enzyme known to play a very crucial role in scavenging free
radicals within the plant system (Regalado, 2004) in addition to their involvement in
various metabolic activities. Outside the plant system this enzyme has several
422
commercial applications, the major one being its use as an important component in
chemical diagnostics and laboratory experiments (Ayala, 2000). A wide range of
chemicals can be modified using peroxidase and hence has varied applications in
waste water treatments to remove phenolics, synthesis of various aromatic compounds.
On other hand Booij et al. (1993) reported that the changes in soluble peroxidases
correlated well with budding and the modification of peroxides activities and
expression of iso-peroxidase always preceded the morphological appearance of buds.
So, evaluation and determination of peroxides during subculture can lead us to a better
understanding of the physiological processes as well as establish optimum conditions
for the culture of date palm.
Elicitors are molecules that stimulate defense or stress–induced responses in plants
(Radman et al., 2003). However the broader definition of elicitors is include both
substances of pathogen origin and compounds released from plants by the action of
pathogen (endogenous elicitors). Further, the nature elicitors can be divided into two
types; biotic and abiotic. The biotic elicitors have biological origin, derived from the
pathogen or from the plant itself while abiotic elicitors haven’t a biological origin and
are grouped in physical factors and chemical compounds (Kumar and Shekhawat,
2009). One of the biotic elicitors is Aspergillus niger, Zhao et al. (2000) reported that,
using combined elicitor treatment of an Aspergillus niger mycelium and tetramethyl
ammonium bromide with C.roseus (L.) Don cell cultures, was enhanced the
accumulation of ajmalicine content as compared with control medium.
On other hand, Fritz et al. (2010) reported that jasmonic acid (JA) and methyl
jasmonate (MeJA), are plant hormones involved in chemical and physiological defense
responses. Moreover, Balbi and Devoto (2008) stated that JA and MeJA are oxylipins
(oxygenated fatty acids) that originate from linolenic acid released from chloroplast
membranes by lipase enzymes and subsequently oxygenated by lipoxygenases (LOXs)
to hydroperoxide derivatives. Elicitation or stress stimulus leads to a rapid release of
α-linolenic acid from the lipid pool of the plant cell which through an intracellular
signal cascade elicits secondary metabolite production important for plant defense
(Memelink et al., 2001). α-Linolenic acid is converted by a lipoxygenase, an Allene
Oxide Synthase (AOS) and an Allene Oxide Cyclase (AOC) into the intermediate 12423
oxo-phytodienoic acid. This compound is converted into JA through the action of a
reeducates and three rounds of β-oxidation (Menke et al., 1999; Mueller, 1997).
The main objective of this investigation is focusing on
the effect of different
concentrations among of Aspergillus niger and Methyl-Jasmonate as biotic elicitors on
primordial leaf cell growth parameters and, achieves the accumulation rate of phenolic
and peroxidase compounds in suspension cultures of Egyptian date palm (Zaghlool)
cultivar using bioreactor.
Material and methods
Plant materials: Female date palm (Phoenix dactylifera L.) offshoots of Egyptian
date palm cultivar (Zaghlool) were secured from Rashed in North Egypt which
separated during fruiting stage from attached mother plants, the measurements and
parameters of the offshoots were (120-150 cm) in height, (35 cm) in diameter and (4550 Kg.) in weight. These offshoots were used as mother plant materials, for initiation
of in vitro cultures.
Sterilization: Sterilization statement of the obtained shoot tip was carried out
according the described method by Taha et al. (2010).
Nutrient media and callus production: Explant of sterilized primary basal leaves
excised the base of shoot tip were cultured on solidified Murashige and Skoog (1962)
nutrient medium (MS) supplemented with 1.7 g/1 phytagel and 30 g/l sucrose. The
MS-nutrient medium was fortified by 170 mg/l Na H2Po 4 .2H 2O + 200 mg/l H2PO4 +
I mg/l thiamine HCI and 3 g/l activated charcoal and augmented with 3 mg/l 2,4-D +
3 mg/l 2iP and 5 mg/l BA (The best medium for callus production according to Taha et
al. (2010) . The pH of the culture medium was adjusted to 5.7 with 0.1 M NaOH or 0.1
M HCI before adding phytagel. The culture medium was dispensed into small jars
(150 ml), each one contained 40 ml . Cultures medium was autoclaved at 121°C ± 1
for 20 min. Cultures were incubated in darkness in a growth chamber at a constant
temperature of 28 °C ± 1 then incubated under light condition (2000 Lux) from cool
white fluorescent lamps, and sub cultured every six weeks on new fresh medium.
After three subcultures, white calli were initiated and observed.
424
Cell production: The obtained calli from the previous experiment was saved and resuspended in an agitated liquid MS medium containing 1 mg/l 2,4-D + 1 mg/l 2iP
+3mg/l NAA according to the best results obtained by Taha et al.(2010).
Establishment of bioreactor experiments: The available 2-L turbine stirred tank
bioreactor (STB) in National Research Centre (NRC) was used with a working volume
of 1.5- 1.7 L (B. Braun, Biotech, International, Germany). The culture was aerated
through a stainless steel sparger. The flow rate was set up according to the type of
experiment and maintained at the normal level with a mass flow control system until
the end of the culture period. Two six bladed turbine impellers (D=45 mm) were used
for mixing; rotation speed was 120 rpm. The temperature was maintained at 26 °C
with thermostatic outlet spongy sheet rounded the vessel. Aeration was performed by
filtered sterile air at the rate of 0.5l/min Dissolved oxygen concentrations were
measured
with
sterilizeable
oxygen
electrode
(Ingold).
Dissolved
oxygen
concentration was monitored with a sterilizeable pO2 electrode. For maintaining
different levels of dissolved oxygen concentrations in the bioreactor broth, volume of
the inlet air was dosed by a mass flow controller connected with software and pO2
electrode. The bioreactor was inoculated with one part of suspension culture and five
parts of medium, and the cell cultures were kept at 25 °C. The MS-nutrient medium
containing cell lines were provided into glass tank bioreactor under sterilized air
condition. The following parameters affecting on either mass cell culture and/or
enhancement of phenolic and peroxides compounds in lyophilized suspension culture
of Zaghlool date palm cultivar were investigated as the follow:•
Effect of controlled pH medium at the degree of 5.7 using either 0.2 N NaOH
or 0.2 N HCL using ADI 1030 Bio-controllers (Applikon) equipped with sterilizeable
pH–electrode (Ingold) and peristaltic pumps for alkali and acid addition .
•
Effect of uncontrolled of pH medium.
Each experiment had been done for two weeks after inoculation. At the end of
fifteen days of inoculation, the obtained cells were harvested and chemically analyzed
for accumulation of phenolic and peroxides compounds and those concentrations. The
percentage of these target compounds were recorded as control treatment.
425
Effect of two types of biotic elicitors at different concentrations on enhancement
of cell growth parameters and phenolic & peroxides compounds production.
Effect of Aspergillus niger: The fungus of Aspergillus niger was obtained from The
Department of Plant Pathology of the National Research Centre.
Preparation of
fungus elicitor was carried out according to the method described by Taha (2002). In
this experiment, 0 %, 0.1 %, 0.2 % and 0.3% of suspended Aspergillus niger (P.C.V)
were added to the culture media.
Effect of Methyl-Jasmonate. In this experiment, 0, 50, 100 and 200 µM of methyJasmonate were used.
Measurement of cell growth parameters.
The following parameters were carried out using sampling unit (2 ml) every 2
days for two weeks.
1-Fresh weight (w/v).
2-Dry weight (w/v).
Extraction and determination of total phenolic compounds: Extraction and
determination of total phenolics in different date palm cell lines were determined using
Folin–Ciocalteau reagents (Singleton and Rossi, 1965). Date palm cell line extracts of
(40 µl) or gallic acid standard were mixed with 1.8 ml of Folin–Ciocalteu reagent
(prediluted 10-fold with distilled water) and sanded at room temperature for 5 min,
then 1.2 ml of sodium bicarbonate (7.5%) was added to the mixture. After one hour at
room temperature, absorbance was measured at 765 nm. Results were expressed as ng
gallic acid equivalents (GAE)/ g DW sample (Shui and Leong, 2006).
Determination of peroxides compounds:
The peroxides activity in lyophilized cell cultures (cell and liquid medium) was
monitored for a total peroxides as described method by Wititsuwannakul (1997).
Data analysis:
The all experiments were designed in a completely randomized design. The
obtained results were statistically analyzed using standard error (SE) according to the
method described by Snedecor and Cochran (1980).
426
Results
Bioreactor experiments: Effect of different conditions affecting on date palm cell
growth parameters.
1-Effect of controlled and uncontrolled pH medium
The effects of controlling and uncontrolling of pH MS- liquid medium containing 1
mg/l 2-4-D and 1 mg/l 2iP + 3 mg/l NAA on Zaghlool cell growth parameters (fresh
and dry weights) are presents in Fig. (1).
The schedule time of measurement of
different cell growth parameters i.e., fresh and dry weights was intervals of two days
for 16 days. Culturing of date palm cell culture was carried out in 2-L turbine stirred
tank bioreactor (STB) in with a working volume of 1.5- 1.7 L. The highest values of
fresh weight for controlled pH MS-medium 175.6 and 112.3 (w/v) were recorded in
the 10
th
and 8th days, respectively. However those recorded 215.8 and 175.3 (w/v) in
the 10 th and 12 days, respectively for uncontrolled pH MS-medium.
Regarding dry weights, the highest records 18.37 and 15.86 (w/v) were recorded
with uncontrolled and controlled pH of MS- media, respectively. It can be concluded
that setup of Zaghlool date palm cell culture in STB for 10 days in MS-liquid modified
with uncontrolled pH medium recognized the maximization of different cell growth
parameters compared with controlled pH medium.
2-Effect of Aspergillus niger (AN) .
The effect of elicitation of uncontrolled MS-medium with different concentrations
of Aspergillus niger (AN) as biotic elicitor on
optimize of different conditions
affecting on maximization of date palm cell growth parameters was investigated.
Filtered and sterilized mycelium of Aspergillus niger as biotic elector at different
concentrations 0,0.1,0.2,0.3 (%) was used. Sterilized filtrate of AN was used for
enhancement of both cell growth parameters as well as accumulation rate of total
phenolic and peroxidase compounds in suspension culture of Zaghlool date palm. Date
tabulated in Table (1) clearly show that the best result of fresh (245.4, 235.12,
223.17and 197.8 w/v) and dry (24.12, 22.17, 20.15 and 18.2 w/v) weights were
recorded with 0.1, 0.2, 0.0 and 0.3 (%) of AN, respectively. The best results of fresh
and dry weights were recorded at the 10
th
427
day of cultivation compared with other
schedule times. Further the optimum concentration of AN for achievement of different
cell growth parameters was 0.1 % compared with other concentrations.
3-Effect of Methyl-Jasmonate (MJ) .
Furthermore, MJ at different concentrations i.e., 0,50, 10 and 200 µM were used
for scaling up and production of mass cell cultures, as well as achievement of active
compounds in suspension culture of Zaghlool date palm cultivar. Further MJ at
different concentrations were incorporated with uncontrolled MS-medium containing
0.1 % AN as shown in Table (2). The highest value of fresh and dry weights 364.12,
331.45, 275.5, 249.75 & 39.28, 35.37, 31.05 and 28.15 were recorded within fortified
of modified MS medium with 100, 200, 50 and 0 µM of MJ for fresh and dry weights
of cell cultures, respectively. In general these experiments clearly indicated that
supplementation of uncontrolled MS liquid medium with 0.1 % of AS and 100 µM of
MJ enhanced of mass cell production from Zaghlool date palm cultivar using STB
(Fig. 2) for 10 days compared with other concentrations and cultivation times.
4- Bioactive compounds determination
The total phenolic (ng (GAE) /g) and peroxides (u/mg) compounds of lyophilized
Zaghlool date palm cell cultures were determined within the previous experiments.
Illustrated data in Fig.(3) clearly presents the effect of modified liquid MS-medium
with either controlled or uncontrolled of pH media or elicitation of culture medium
with 0.1, 0.2 or 0.3 (%) of AS or with 50, 100 or 200 µ M of MJ as biotic electors on
achievement of total phenolic and peroxides compounds production in different cell
lines of Zaghlool date palm cultivar. The highest values of total phenolic (25.35
ng(GAE/g) and peroxides (5432 u/g) compounds were recorded with uncontrolled pH
of MS-medium augmented with 0.1 % of AS and 100 µM of MJ compared with other
supplementation and concentrations.
In conclusion this study clearly indicates that combining of different elicitors
achieved of secondary metabolites process especially total phenolic and peroxides
compounds in date palm cell cultures; and that Aspergillus niger and MethylJasmonate plays a critical role in elicitation of date palm cell cultures. Moreover,
scaling up and production of mass cell cultures and production of active compounds
from date palm cell cultures using serried tank bioreactor 2 L was established.
428
Discussion
The scaling up of mass cell cultures and maximization of bioactive products
through bioreactors must be follow up. Further bioreactors have two advantages over
flasks for culturing plant cells. The 1st is that better control can be exerted on the
system (i.e., pH and dissolved gas concentrations can be controlled). The 2nd is that
since most bioreactors are scalable, they are better able to reproduce on a larger scale
those conditions which were observed on a smaller scale to be the most desirable for
culture performance. With the aim of implementing an industrial scale process, the
behavior of cell culture in bioreactors has been receiving much investigative attention
(Zhong, 2001 and Huang et al. 2002). However, understanding how to improve cell
cultures through national modification of the reactor environment remains a challenge.
One of the methods frequently used to increase the productivity of plant cell culture
is use of so-called elicitors (Singh, 1996). Elicitors can be all types of compounds, that
provoke (the increase of) the production of phytoalexins (Muller, 1956 and Kue,
1972). Phytoalexins are antibiotically active compounds, and by that important factors
in the resistance of plants to microbial attack (Darvill and Albersheim, 1984). Many
secondary metabolites belong to the group of phytoalexins. So, if the right elicitor can
be found, it is
possible to enhance the production of the desired secondary metabolite (Eilert,
1987). In addition, Wijnsma et al.(1985) reported that, the anthraquinons in Cinchona
ledgeriana cell cultures were increased when the cells were treated with 0.5 mg/ml of
Aspergillus niger as elicitor. JA actives stress response in cell by two ways (1) JA
produced at the wound site serves as a mobile signal to activate responses in systemic
tissues. (2) wound-induced production of a mobile signal other than JA activates
synthesis of the hormone in systemic tissues (Abraham and Howe, 2009).
Furthermore and in close of our obtained results Zabetakisa et al., (1999) mentioned
that elicitation through MJ increased tropane alkaloid from Datura sramonium more
in comparison with fungal elicitor and oligoalacturonide. As well as Taha (2003)
established an efficient protocol for enhancement of total alkaloids production from
suspension cultures of A. belladonna L. using various concentrations of Aspergillus
niger. He reported that the optimum augmentation of liquid MS-medium was 1 mg/l of
429
NAA and BA and extract of A. niger at the concentration of 10 % (~ 0.5 mg/ ml), gave
the highest value for cell growth and total alkaloid accumulation in the different type
of cell cultures following 10 days of cultivation.
In general, the obtained results may be due to enhancement and achievement and
production of total phenolic and peroxidase from cell cultures of Zaghlool date palm
cultivar using advanced techniques of scaling up through bioreactors such as STB.
The highest values of total phenolic compound accumulation 25.37 ng(GAE)/ g of
lypholized date palm cell cultures was recorded with elicitation of uncontrolled MS
medium with 0.1 % of AS and 100 µM . The obtained results are in agreement with
Taha et al. (2011) who reported that the maximum value of cell growth parameters and
highest content of inulinase activity (0.395 u/ml) were resulted from elicitation of
augmented MS-medium with Aspergillus niger extract at the level of 0.2 % in
combination with Methyl Jasmonate (150 µM) as compared with other concentrations
after 2 weeks of cultivation. In addition the obtained results are in agreement with
those obtained by Del-Rio (2003) who reported that the highest phenol levels were
detected after 120 days in leaves, stems and roots (in that order) of Olea europaea L.
fruits. This is because leaves are the principal producers of phenolic compounds, the
pathway of shikimic acid, which acts as the precursor of phenolic compounds,
beginning in their photosynthetic cells. Several studies in different plant species have
shown that various phenolic compounds are synthesized and accumulate in different
tissues of the leaf (Botýa, 2001; Del Rio, 2000). However in contrast of our obtained
results the result of the present study further revealed that phenolic content increased
quantitatively with the increase in age of suspension from main to the 3
rd
week of
cultivation may be this due to the hyperactivity of oxidative enzymes
(Cochrane,1994). An increase in production of phenolic compounds has been
associated with a decrease in growth, a decline in protein synthesis.
Regarding, plant peroxidases, Obinger et al. (1996) reported that phenol oxidizing
enzymes widely used as markers in the plant kingdom, due to their high
polymorphism. In agreement of our obtained results, Azeqour et al. (2002) mentioned
that leaves of date palm contains highly active peroxidases. However since plant
peroxidases are involved in many functions such as growth, vegetative development,
430
resistance against biotic and abiotic stresses (Gonzalez-Verdejo, 2006; Mc Innis,
2006), the exact role of these enzymes is not yet elucidated in date palm. Plant cell and
organ cultures grown in vitro usually exhibit changes in physiological and biochemical responses upon exposure to biotic and abiotic elicitors (Sircar and Mitra
2008). Elicitation is a process of induced or enhanced synthesis of secondary
metabolites by the plant cells to ensure their survival, persistence and competitiveness.
431
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Table (1). Effect of elicitation of uncontrolled pH liquid MS-medium with
different concentrations of Aspergillus niger on enhancement of fresh and dry
weights of Zaghlool date palm suspension cultures. Culture was carried out using
serried tank bioreactor 2 L for 2 weeks at 26 ºC under 16/8 day light condition.
MS medium* supplemented with different concentrations of Aspergillus niger (%).
Days
0
0.1
0.2
0.3
durati
F.W
D.W
F.W
D.W
F.W
D.W
F.W
D.W
0
61.84±3.15
8.66±1.52
95.38±3.25
9.16±1.69
64.25±2.95
8.74±1.53
59.4±2.63
8.42±1.42
5
100.5±5.22
10.82±1.79
125.63±5.93
12.63±2.48
119.6±4.87
17.3±2.23
107.6±3.52
11.2±2.15
10
223.17±7.5
20.15±2.24
245.43±8.25
24.12±3.54
235.12±7.96
22.17±2.56
197.8±5.18
18.2±2.36
15
185.3±6.13
17.42±2.15
215.37±7.61
21.7±3.15
205.14±6.85
19.63±3.15
191.6±6.45
15.87±2.05
on
*The pH of MS- medium was set up as uncontrolled. Each treatment was the average of 3 replicates ±
Standard Error F.W: Fresh weight (w/v) ; D.w: Dry weight (w/v).
Table (2).Effect of incorporated of uncontrolled pH liquid MS-medium containing
0.1 (%) of Aspergillus niger with different concentrations of Methyl-Jasmonate on
enhancement of fresh and dry weight of Zaghlool date palm suspension cultures.
Culture was carried out using serried tank bioreactor 2 L for 2 weeks at 26 ºC.
MS medium* supplemented with 0.1 % of Aspergillus niger (%) and different concentrations of
Methyl-Jasmonate ( µM).
Days
duration
0
50
100
200
F.W
D.W
F.W
D.W
F.W
D.W
F.W
D.W
0
66.25±3.42
8.73±1.55
65.18±3.53
8.07±1.15
64.35±2.95
8.12±1.76
65.25±2.25
7.95±1.25
5
100.63±5.43
11.32±1.93
112.13±4.65
13.25±2.15
135.12±5.25
15.17±2.09
125.15±3.98
15.33±2.95
10
249.75±7.95
28.15±3.05
275.25±6.33
31.05±3.17
364.12±8.14
39.28±4.56
331.1±5.22
35.37±2.85
15
235.13±6.25
25.45±2.89
264.85±5.89
28.19±3.85
325.13±8.09
35.62±3.97
305.52±4.87
32.49±2.97
Each treatment was the average of 3 replicates ± Standard Error ,*The pH of MS- medium was setup as
uncontrolled.
F.W: Fresh weight (w/v) ; D.w: Dry weight (w/v).
437
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hŶĐŽŶƚƌŽůůĞĚƉ,&͘t;ŵŐͿ
hŶĐŽŶƚƌŽůůĞĚƉ,͘t;ŵŐͿ
Fig. (1). Effect of controlled and uncontrolled the degree of pH MS agitated liquid
medium containing 1 mg/l 2,4-D + 1 mg/l 2iP+3mg/l NAA on cell growth
parameters
(fresh and dry weights)of Zaghlool date palm cultures, cultured in STB and
incubated under 16/8 of day light condition at 26 ± 1 ºC.
Fig.(2). B-Braun Biotechnology International stirred tank bioreactor (2 L).
438
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Fig.(3). Effect of controlled and uncontrolled pH of MS-medium , or elicitation of
uncontrolled pH liquid MS-medium with either Aspergillus niger (As %) or MethylJasmonate (µ M) incorporated with 0.1 % of AS on accumulation of total phenolic (ng
(GAE)) and peroxides (u/ mg) of date palm lyophilized cell cultures. Cultures were carried
out using STB 2 L for 2 weeks at 26 ºC, and incubated under 16/8 of day light condition.
439
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440
OP 20
Potential Applications of Gene Silencing or RNA Interference (RNAi)
to the Control of Disease and Insect Pests of Date Palm
Charles L. Niblett and Ana M. Bailey
Venganza, Inc., 840 Main Campus Drive, Raleigh, NC 27606 USA
Abstract
Gene silencing or RNA interference (RNAi) is a recently-discovered, natural,
regulatory and defense mechanism in plants, animals and other organisms. It has great
potential to control plant pests such as fungi, insects, nematodes and parasitic plants. A
gene is targeted that is essential for the survival or development of the plant pest. A
DNA construct containing an inverted repeat of the essential gene is transformed into a
susceptible host plant. Plant transcription produces a double-stranded RNA (dsRNA)
of that essential gene, which the plant recognizes as a foreign molecule. The plant’s
protective ribonuclease enzyme, or dicer, hydrolyzes the dsRNA into small interfering
RNAs (siRNAs). The pest feeding on the transgenic plant ingests the siRNAs, causing
the pest’s RNAi mechanism to hydrolyze the messenger RNA of its own essential
gene. This prevents expression of or “silences” that essential gene in the pest, which
either dies or is debilitated, and the transgenic plant is resistant to that pest. RNAi has
been shown to provide resistance against insects (Diabrotica, Helicoverpa), bacteria
(Agrobacterium, Staphylococcus), nematodes (Heterodera, Meloidogyne) and parasitic
plants (Orobanche, Striga, Triphysaria). We propose that RNAi would provide
effective and durable resistance to serious pests and diseases of date palm, including
the red palm weevil (Rhynchophorus ferrugineus), bayoud disease (Fusarium
oxysporum f. sp. albedinis), Al-Wijam, and other serious pests of date palm.
Introduction
Date palm (Phoenix dactylifera) has been a cultivated tree crop for at least 5,000
years [1]. It is a very important plant throughout the world, and is perhaps the most
important plant in Saudi Arabia and throughout the Middle East. It has high
socioeconomic importance, due not only to its food value, but also its capacity to
provide many other products such as shelter, fiber, clothing, aesthetic beauty and
441
furniture [2]. It has high natural tolerance to very adverse growing conditions,
including drought, salinity and high temperatures [3]. In 2007 nearly 1.1 million ha of
date palm were harvested, yielding 6.91 million tons. The major producers were Egypt
(19%), Iran (15%) and Saudi Arabia (14%) [4].
Each year plant pests cause serious economic losses throughout the world in palm
species, especially in date and coconut palms. In date palm up to 30% of production can
be lost to pests and diseases [5], including the red palm weevil, (RPW Rhynchophorus
ferrugineus), the Bayoud disease (Fusarium oxysporum f. sp. albedinis) [6,7] and
phytoplasma diseases [8,9,10]. Depending on the level of infestation, the RPW can
cause losses up to $130 million annually in the Middle East countries alone, and
additional millions of dollars of losses on coconut and other palm species [11,12].
Because date palm is a long-lived plant and because it is genetically heterogeneous and
difficult to propagate, it is essential to develop durable resistance against these
important pests and to incorporate it into horticulturally desirable cultivars.
Recent advances in biotechnology, both in date palm and in new disease resistance
strategies, provide encouraging opportunities for developing pest resistant date palms.
Tissue culture from somatic embryos of date palm has been advanced significantly,
and large scale micro-propagation [13] is now possible. In addition, transformation of
embryonic date palm callus cells has been achieved [2,14] using biolistic (particle
bombardment). Successful transformation was demonstrated both by β-glucuronidase
(GUS) expression (histochemical staining) and direct detection by polymerase chain
reaction (PCR). However, transgenic plants have not yet been regenerated. Even
greater success has been achieved in the oil palm (Elaeis guineensis), where stable
transformation has been achieved both by biolistics and by Agrobacterium tumefaciens
[15,16,17], and transgenic plants have been regenerated.
RNAi is a recently discovered mechanism for regulating gene expression. It
functions in diverse organisms including plants [18,19,20] bacteria [21,22,23,24,25],
nematodes, [26,27,28], hydra [29], humans [22], parasitic plants [30] and plant viruses
[31,32,33,34]. It holds great promise to control human, plant and animal diseases
[22,29]. RNAi is considered an ancient defense mechanism whereby the host organism
recognizes as foreign a double-stranded RNA (dsRNA) molecule and hydrolyzes it
442
with a ribonuclease named dicer. This hydrolysis produces small and specific RNA
fragments of 21–28 nucleotides called small interfering RNAs (siRNAs). The siRNAs
then combine with constitutive proteins to form the RNA-induced silencing complex
(RISC). The RISC diffuses in the cell, and its resident siRNA hybridizes to the specific
messenger RNAs (mRNAs) with sequences complementary to that of the siRNA. The
new double-stranded region stimulates the hydrolysis of that mRNA by dicer to
produce more siRNAs. This process is repeated each time the siRNA hybridizes to its
complementary mRNA, effectively destroying and preventing that mRNA from being
translated, thus “silencing” the expression of that specific gene [18].
Stimulated by these discoveries, Venganza, Inc. developed a new technology for
plant disease control called host-mediated silencing of pest genes (HMSPG).
Venganza has filed a patent on HMSPG (www.venganzainc.com), and has used
HMSPG to develop plants resistant to several fungal pathogens in addition to the
oomycete Phytophthora species shown in the figures below. The molecular approach
is to target an essential gene of the pest by producing a DNA construct containing an
inverted repeat of that essential gene. The susceptible host plant is transformed with
the inverted repeat construct, and transcription in the plant produces a dsRNA with the
sequence of the targeted and essential pest gene. The plant recognizes the dsRNA as a
foreign molecule, and the plant’s protective dicer enzyme hydrolyzes the dsRNA into
siRNAs. When the pest attacks the transgenic plant, it ingests those siRNAs, which
then cause the RNAi mechanism within the pest to hydrolyze the mRNA of the pests’
own essential gene. Silencing the pest gene stops the infection because the pest dies or
is no longer pathogenic (depending on the choice of the gene), and the transgenic plant
is now resistant to that disease. Venganza first demonstrated HMSPG in tobacco using
the cutinase gene from P. nicotianae, because cutinase is essential for pathogenicity in
Phytophthora [35]. Interestingly, this essential gene sequence from P. nicotianae also
was effective in conferring resistance against several related pathogenic fungi.
Research Methodology
The materials and methods used in the following research are described in the
Niblett patent application [36], in the individual references cited, and in “Molecular
Cloning: A Laboratory Manual” [37].
443
Results and Discussion
As shown in Figure 1, plant A, typical of those transformed with the cutinase gene
construct (pVZA100), is resistant to P. nicotianae, whereas the untransformed or wild
type plant B is susceptible. This resistance was effective against both Races 0 and 1 of
P. nicotianae. Similar results have been obtained with other fungal pathogens on dicot
and monocot hosts.
Figure 2 demonstrates that the molecular mechanism of the resistance is RNAi.
Panel A shows the effect of pVZA100 transformation on P. nicotianae. Lane 1 is a 35
nt marker and Lane 2 shows the intact 620 nt cutinase mRNA from a wild type culture.
RNAs from four transformed cultures (A-D; lanes 3-6) contain 21-25 nt siRNAs that
hybridized to the cutinase probe, demonstrating that the cutinase mRNA has been
hydrolyzed in the transformed cultures. Furthermore, the P. nicotianae isolates
containing the intact mRNA were pathogenic, whereas those transformed with
pVZA100 and containing the siRNAs were nonpathogenic. Cultures of P. nicotianae
re-isolated from resistant transgenic tobacco plants showed the same siRNA profiles as
those in Figure 2 A-D. The nonpathogenic cultures of P. nicotianae that had been
transformed with pVZA100 or re-isolated from resistant transgenic tobacco were
transferred monthly on growth media for three years and never regained pathogenicity,
indicating that the RNAi is long-lasting, if not permanent.
Panel B of Figure 2 shows direct evidence for RNAi activity in tobacco plants
transformed with pVZA100. Lane 1 is the 35 nt size marker. RNAs from wild type and
pCAMBIA1201 control transformed plants (lanes 2 and 3) showed no hybridization
because cutinase is a fungal gene, not present in plants. However, the four tobacco
lines transformed with pVZA100 (lines 4, 23, 26 and 27, and their T1 seed progeny; 41
etc.) all contained the cutinase siRNAs, and were resistant to P. nicotianae, as in Figure
1. These lines were randomly selected from about 50 individual transformation events.
Figure 3 shows that the tobacco plants transformed with pVZA100 also were
resistant to the blue mold disease caused by Peronospora tabacina. Peronospora and
Phytophthora are related taxonomically as members of the same Order
(Peronosporales), but they are in different families (Peronospora = Peronosporaceae
and Phytophthora = Pythiaceae). Therefore the pVZA100 construct also confers broad
444
resistance to a distantly related fungal pathogen. Figures 4 and 5 provide additional
evidence that HMSPG confers a broad type of resistance. In Figure 4 the cutinase gene
from P. nicotianae (pVZA100) provides resistance against P. sojae, in soybean. This
resistance was effective against all
seven races of P. sojae tested.
In Figure 5, pVZA100 (Row 2) confers resistance against P. infestans in potato as
compared with Row 1, which contains wild type plants and those transformed with
pCAMBIA1201 alone). Rows 3 and 4 show resistance in potato plants transformed
with pVZA300 and pVZA400, which contain the elicitin and ribosomal RNA (rRNA)
genes, respectively, from P. infestans. Note that the level of resistance conferred
against P. infestans by pVZA100 is not as high as with pVZA300 and pVZA400. This
may reflect the lower sequence identity (82%) between the cutinase gene of P.
nicotianae and that of P. infestans. The resistance conferred by pVZA100, 300 and
400 was effective against both mating types A1 and A2 of P. infestans. The pVZA300
construct used here contains a gene from both P. infestans and a plant insect pest,
indicating that a construct containing two genes remains functional and is effective
against its target gene in P. infestans. We have recently used "gene stacking" on a
single construct to confer resistance to three different fungal pathogens.
HMSPG and similar strategies are widely applicable to a broad spectrum of plant
pests. For example, inverted repeats of genes from multiple plant viruses were used to
obtain resistance to two and four different viruses [33,34], respectively. HMSPG also
is effective against insects, nematodes, parasitic plants, and bacteria, which is the
capability we propose to implement here against the pests of palm. Corn plants
transformed with a construct containing an inverted repeat from a vacuolar ATPase
gene from Western corn rootworm showed significant reduction in damage from this
insect [38], and cotton plants were protected from the cotton bollworm when
transformed with an inverted repeat from a bollworm cytochrome P450 gene [39].
Inverted repeats of nematode parasitism genes also have been effective in
controlling both root knot [40] and cyst nematodes [28]. Working with the parasitic
plant Triphysaria versicolor, a species of broomrape, Tomilov, et al [41] demonstrated
that lettuce plants containing an inverted repeat of the GUS gene could silence an
445
active GUS gene in the T. versicolor when it fed on the lettuce. Furthermore, feeding
that same “silenced” T. versicolor on lettuce expressing the GUS gene, silenced the
GUS gene in lettuce. This demonstrated that the “silencing principle” (siRNA) moves
back and forth between lettuce and T. versicolor. Using Orobanche aegyptiaca,
another species of broomrape, Aly, et al [30] demonstrated that tomato plants
transformed with an inverted repeat of the mannose 6-phosphate reductase gene of O.
aegyptiaca showed a 58% greater mortality of the broomrape tubercles that developed on the
transgenic tomatoes.
With bacteria, siRNAs have been effective in vivo and in vitro against the coagulase
enzyme of the human pathogen Staphylococcus aureus [25], and the crown gall disease
of plants caused by Agrobacterium tumefaciens was controlled in tobacco, Arabidopsis
and tomato plants transformed with inverted repeats of the A. tumefaciens genes iaaM
and ipt, which encode precursors for auxin and cytokinin biosynthesis [21,24].
Because HMSPG is effective against bacteria there is reason to be optimistic that it
will be effective against phytoplasmas. Because phytoplasmas cannot be cultured we
performed a preliminary experiment for this proposal to test the efficacy of HMSPG
against Xanthomonas campestris pv. campestris (Xcc), a serious bacterial pathogen of
cabbage and other vegetables. We used the in vitro incubation assay that we developed
for fungi [42] to identify candidate genes for HMSPG. With fungi, spores or mycelium
are incubated in the dsRNAs and siRNAs, and viability is measured by colony formation
or infectivity. Here we prepared dsRNAs from the Xcc 23S rRNA and enolase as
candidate genes. The bacteria were incubated directly in the dsRNAs or in siRNAs
prepared from the dsRNAs by digestion with ribonuclease III and then plated on YDC
medium to measure viability by colony formation. The control dsRNA and siRNAs were
prepared from the β-glucuronidase (GUS) gene of Escherichia coli. Our data (not
shown) indicates that Xcc dsRNAs and siRNAs reduced colony formation by 24 to 55%,
as compared to about 10% reduction in the controls treated with GUS ds- or siRNAs.
Similar reductions in viability were obtained with candidate fungal genes that
subsequently provided strong resistance in transgenic plants. Therefore, as demonstrated
with fungi, both the 23S rRNA and enolase genes have high potential for conferring
resistance to Xcc in planta, and likely to other bacterial and phytoplasma pathogens.
446
Advantages of RNAi and HMSPG
A major concern voiced against transgenic plants is the possible expression of a
protein that might cause an allergic response in consumers. Therefore, a major asset of
our RNAi strategy is that no protein is expressed. We further ensure this in our construct
design by avoiding both 5’ terminal and internal ATG initiation codons and by inserting
one or more stop codons in all six possible reading frames. Potential off-target effects on
host plant genes or other species are minimized by designing constructs to produce
siRNAs with a maximum of 15 contiguous base pairs of identity to known coding
sequences. This is a conservative strategy, given that an upper limit of 18 contiguous
base pairs is generally considered adequate to avoid off-target effects [43].
Using conventional breeding techniques it may be difficult or impossible to achieve
disease resistance in an important crop species when genes for resistance to a
particular pest do not exist or that crop is difficult or very time-consuming to breed for
resistance because of sterility, ploidy differences or incompatibilities (e.g. date palm,
bananas, potatoes, etc.). Also, when plant resistance genes are identified and
transferred into desirable varieties, that resistance may not be durable because of the
presence of diverse genotypes of the pest, or because the pest may mutate and rapidly
“defeat” that resistance gene.
HMSPG has now been demonstrated to be effective against all fungi for which it
has been tested. Evidence for Oomycetes is presented above. From ongoing projects
we have evidence in transgenic monocots and dicots for resistance to a Basidiomycete
and three species of Ascomycetes, while others have shown RNAi activity in the
ascomycetes Fusarium graminearum (Gibberella zeae) and Aspergillus flavus [44],
and Nowara et al. [45] and Yin et al. [46] have recently shown it to be effective against
the obligate parasites Blumeria and Puccinia, respectively. As noted above, HMSPG
also is effective against insect, bacterial and nematode pests.
Durability of RNAi-Derived Resistance or HMSPG
Venganza and others have demonstrated that sequence identity to the target gene
must be about 70-80% to provide high level resistance (Fig. 5). Therefore, it would
require mutations altering 20-30% of an essential gene sequence for a pest to overcome
or develop resistance to HMSPG. Such mutation in an essential gene would likely be
447
lethal to the pest. Furthermore, even if a pest does mutate sufficiently to overcome the
action of a single siRNA, the many other siRNAs also produced from that same
essential gene construct [47] will be present to hydrolyze the mRNA transcript of that
essential gene at many additional sites, and only a single hydrolysis is necessary to
cause the desired silencing of that essential gene. Hence, this form of resistance is
recalcitrant to mutation and should provide durable resistance to all mating types, races,
strains, biovars and pathovars of the important pests of date palm that we have
described. Our recent discovery of near identity among the sequences of essential genes
in fungal pathogens from the US and Africa strongly supports this concept.
Because of their interest in HMSPG, the National Agricultural Research
Organization of Uganda negotiated a contract with Venganza, Inc. in 2009 to identify
candidate genes potentially effective for the control of Fusarium oxysporum cubense
(FOC) and Mycosphaerella fijiensis (MF), which cause Fusarium wilt and black
sigatoka, respectively, in bananas. Using our in vitro assay we identified candidate
genes for conferring resistance to these pathogens. Genes known to be essential for
fungal survival were selected. Because the FOC genome has not been sequenced, the
sequenced genome of the closely related Fusarium graminearum (Gibberella zeae =
GZ) was used to identify sequences for PCR oligonucleotide primers to amplify gene
segments from genomic DNA of FOC and MF, which like GZ are both ascomysetes.
The well-annotated genomic sequence of Neurospora crassa, another ascomycete, and
accessible through the National Center for Biotechnology Information (NCBI), also
was used for comparison, as were the expressed sequence tags (ESTs) for FOC and
MF available through NCBI. PCR primers were designed for 14 FOC and 12 MF
candidate genes, and the amplicons obtained in all 26 cases from genomic DNA of
both Ugandan and Florida isolates of FOC and MF. The dsRNAs transcribed from the
gene segments were effective against both Ugandan and Florida isolates of FOC and
MF. Sequencing of the cloned amplicons from both Ugandan and Florida isolates of
Foc and Mf revealed that the nucleotide sequences of the same essential genes were
essentially identical, demonstrating the highest possible level of conservation among
these essential genes, and therefore the broad applicability of HMSPG to disease
control on both continents, and probably worldwide.
448
Future Considerations
HMSPG now provides to agriculture and plant breeders an entirely new and unique
source of genes for pest resistance - the essential genes of the pests themselves. Using
HMSPG to develop resistant plant varieties is potentially much more rapid than
conventional breeding because HMSPG inserts specific genes for resistance into
proven and accepted plant varieties with no other changes. Several genes may be
stacked on a single or on multiple constructs, thereby conferring durable resistance to
several pests with a single transformation event. Where sequences of essential genes
are not available for a particular pest, we have demonstrated that sequences of closely
related pests can be used to prepare PCR primers and those amplicons sequenced to
confirm the gene’s identity. Therefore, we conclude that HMSPG can be applied
immediately to the control of serious pests affecting date palm.
449
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454
Figure 2. Hybridization of a of cutinase
probe
to
PAGE-separated
RNA
extracted from transgenic and wild-type
cultures of P. nicotianae and
tobacco plants.
Figure 1. Resistance to P.
nicotianae
conferred
to
ful
transformation was demonstrated both
by β-glucuronidase (GUS) expression
(histochemical
staining)
and
direct
Figure 3. Resistance to tobacco
Blue Mold (Peronospora tabacina)
conferred by transformation with
pVZA100 (plant B). Plant A is an
untransformed control plant.
Figure 4. Resistance to P. sojae in
soybean plants by transformation with
pZA 100
455
Figure 5. Resistance to late blight (P.
infestans mating type A2) conferred to potato
by transformation with pVZA100 (Row 2),
pVZA300 (Row 3) or pVZA400 (Row 4),
compared to wild type plants, and those
transformed with pCAMBIA 1201 (Row 1).
456
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458
OP 21
ƮBiophysical
and Engineering Contributions to Plant Research`
Artmann GM. , I. Digel, P. Linder, and Mrs. Aysegül Temiz Artmann
University of Applied Sciences Aachen, Institute for Bioengineering, Heinrich
Mussmann Strasse 1, D- 52428 Juelich, Tel.+49 241 6009 53866, E-mail:
artmann@fh-aachen.de, Digel@fh-Aachen.de, Linder@fh-Aachen.de, and
a.artmann@fh-aachen.de, Admin. Office: Kayser@fh-aachen.de.
Abstract
In the past decades, much scientific attention has been spent to the development of
biomedical engineering devices applicable to humans in health and disease. At the
same time, biology of eukaryotic cells including stem cells developed to an extend that
nowadays we are able to think about curing diseases which we could not think of any
time before. Many of us, scientists as well as engineers, are proud of these
developments. Our Institute of Bioengineering at Juelich, a city with strong
background in bioengineering research located close to the city of Aachen, Germany
(www.biomedtech.de) contributed its share. We did it with dedication and profound
background in engineering and natural sciences.
Introduction
Mankind needs food, drinking water, and healthy environments to live in. In the
past few years our Institute for Bioengineering shed parts of its attention to
technological solutions in crop sciences, plant growths, and plant disease identification
and -fighting. We intend to apply our background to palm tree research, palm tree
gender differentiation, (the RPW pest combating) and palm tree disease recognition.
The topics of our talk will include 1) automated scanners and data achieved so far of
date palm tree leaves. This will include judging the potential of such scanners to
differentiate date palm tree gender and phenotype. Plants including date palm trees
display a huge variety of geometries, boundary conditions, necessities. Any plant
scanner, if portable or not, not only lives from its hardware design but very much and
to a huge extend from the science and engineering one puts into the ´brain´ of such
machines. Thus, automated scanner generations designed for certain applications as
date palm tree research must be custom designed and can only be marketed as such.
459
One of the key goals will be finding technological solutions in particular for palm tree
researches which are applicable in the field (plantations). Considering such goal, one
of our plant scanners was developed as portable machine, capable of scanning leaves
(date palm tree leaves) in any plantation and location. With a little wider focus in plant
research we will talk on implications of automated C3/C4 plant differentiation
(rice/corn), on water uptake of plants, time constants of water transport in leaves, plant
leave volume and geometry determination for water uptake and precise biomass
quantification as well as for plant stress research. Finally, we would like to emphasize
that combining engineering, biophysics and plant research is a most valuable tool for a
better living, a healthy environment and sufficient food for all people on our planet.
Automated Plant Leaf Scanners - Methods and Techniques
Any classification for gender or phenotype based on leaf analysis, respectively,
requires the identification of parameters sensitive to differences between two different
types of samples. Thus, experiments based on very different technology have to be
carried out before designing a scanner to observe whether a particular parameter and
method is of interest for such classification. Surface topology detection is a very first
method which can be carried out. Principally it can be achieved by scanning a leave
passing through a Laser line light path from above the leave and at the same time from
below. Mounting both profiles together a cross-section of a strip of a leaf (70µm wide)
can be detected. Using appropriate software a 3D Image of this leaf can be reproduced
on the computer screen. Such image can be analyzed further for characteristic surface
features potentially usable for plant differentiation purposes. Such analysis would
imply the assumption that different internal structures are represented at the surface of
a particular leaf (figure 1).
Regular bright field microscopy may be chosen as well for plant differentiation
(figure 2, above left). This requires a leaf to be permeable for regular light which is not
always the case. However, if applicable, such technique is very useful to identify
internal leaf structures. However, not only methods determine the complexity of a
scanner software. A leaf may be incorrectly introduced into the scanner. A handy
software needs to be able to recognize and correct such matter in order to calculate
correct distances of for example vascular bundle distance (figure 2,3).
460
Methods like auto fluorescence microscopy may be also helpful (figure 3).
Chlorophyll exhibits a wonderful auto-fluorescence. Such images would enable
detecting pattern of chlorophyll distribution. Overlaying such images with topographic
images in the computer hows for example chlorophyll distributions relative to vascular
bundles. Additionally, very new technology may be applied as Optical Coherence
Tomography. Such imaging shows cross sections through a leaf at a resolution of 10
µm (figure 3, right).
Thus, the set of methods implemented into the scanner defines its purpose and
applicability. There is no scanner for everything at once. Very detailed preinvestigations will have to be performed in order to reach two goals 1) to detect what
you want to detect, and 2) to use the most appropriate method in order to make the
scanner as inexpensive as possible.
The convenience of handling a scanner also depends on the use of bar-code readers
for plant identification (figure 4, middle), touch screen (figure 4, above), battery driven
scanners, internet and USB connection for data storage and remote data analysis which
theoretically could be done anywhere in the world. A scanner for greenhouse usage
(figure 4, left) maybe larger but must be waterproof and withstand warm and humid
environments. It should be easily transportable even on an uneven ground. Portable
scanners for field studies must be robust, battery driven, easy to handle and easy to
store and transport (figure 4, right).
Conclusions
Tests with palm tree leaves have just started yet and scan data are in the process to
be analyzed. The final goal of future project for palm tree gender and species
recognition will be to develop optical scanning technology to be applied to date palm
tree leaves for in–situ screening purposes. Depending on the software used and the
particular requirements of the users the technology potentially shall be able to identify
palm tree diseases, palm tree gender, and species of young date palm trees by scanning
leaves. Being aware of the complexity of the subject we suggest a limit for a
successful detection of ninety percent, thus, in nineteen percent of all detections the
technology should allow a correct classification. This is an ambitious goal which will
be approached by methods of contemporary high tech optical technology implanted in
461
modern scanner machines as indicated above. Semi-automated scanners should
identify micro pattern characteristics by extracting (neural network applications)
characteristics of Date Palm Tree Leaves (Phoenix dactylifera L.) and shall be finally
designed for field screenings. In parallel, molecular biological and genetic data should
be extracted and analyzed. The final technology should be fast and applicable in-situ.
There is still a road to go to reach such ambitious goals. However, if these inventions
were successful then they could provide a significant progress in date palm tree
culturing and date production. We are ready to take the challenge.
Figure 1: Left) Scheme of a plant leaf. The red arrow indicates the vascular
bundle distance. Typical pattern (black) as well as chlorophyll distributions
(green) appear between bundles. After pattern analysis based on image analysis
and neural network applications characteristics of such pattern may lead to a
differentiation between two types of plants the leaves were harvested from.
Middle) Scheme of the internal structure of a C3 plant leave (rice) as compared to
a C4 plant leave (right). Differences between the internal structures may appear
as typical pattern on surfaces of plant leaves enabling to differentiate plant leaves
using scanning procedures.
462
Figure 2: Screenshot of a plant scanner for the detection of distances between
vascular bundles. Images 1-5 from top, left to right, and to below: 1) Transmitted
light image through a leaf (white lines indicate a vascular bundle, 2) 2D Fourier
Transformed Image (angle to the horizontal line of the image indicates the
inclination angle of the leaf with respect to the Laser line which should be
oriented perpendicular to the leaf. 3) Image corrected for an inclination angle of
90° relative to the Laser line and vascular bundles (green lines), 4) maximum
detection for vascular bundles, and 5) location of the bundles. Below, right, the
average vascular bundle distance, 41.47 µm, is shown.
Figure 3: Same leaf as above inclined to the Laser line by 15°. Left: inclined
original. Middle: 2D Fourier transformation, right: for orientation corrected
image of the leaf with bundles (green).
463
Figure 4: Left Image: Auto-fluorescence image (top view) of a fig tree leave.
White: High chlorophyll content, Black: Vacuoles - no chlorophyll. Middle
image: Birch tree leaf (view from below) showing stomata openings. Right image:
Cross sectional image obtained with Optical Coherence Tomography (OCT).
OCT is a non-invasive optical detection method resulting in cross sectional
images of the leaf at a resolution of ~ 10 µm. The photograph shows a cross
section of a rice leaf. Black dot-areas above indicate the size and opening
characteristics of stomata. The optical penetration depth depends on optical and
leaf parameters and can reach up to 1 mm.
Figure 5: Prototypes of plant scanners. Right: Phytoscan alpha 70, for green
house studies (battery, internet, USB), Left: Mobile scanner prototype for field
studies portable in a suitcase.
464
Aه ت ا ء ا! وا> ' 12أ! ث ا ت
Artmann GM. (Prof.Dr. habil.)1,2, Ilya Digel (Dr.)1, Linder P. (MSc. Biomed.
Eng., cand. PhD)1, Mrs. Aysegül Temiz Artmann (Prof. Dr. Dr.)1,2
E-mail: artmann@fh-aachen.de
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466
OP 22
Identification of sex-specific DNA markers for Date Palm
(Phoenix dactylifera L.) using RAPD and ISSR techniques
Rania A. A. Younis1, Omayma M. Ismail2 and S. S. Soliman3
1Genetics Department, Faculty of Agriculture, Ain Shams University, Cairo, Egypt.
2,3Technology of Horticultural Crop, National Research Center (NRC), Cairo, Egypt.
Abstract
In this paper we have attempted to identify sex-specific DNA markers for some date
palm cultivars using molecular technique (RAPD and ISSR) to facilitate the selection
and identification of good male pollinators for further utilization in breeding programs
to increase the yield and to improve some quality traits of fruits. To our knowledge,
this is the first report of an analysis of gender genetic identification in date palm using
both RAPD and ISSR analyses which gave three positive specific markers for females
and two for males in RAPD analysis in addition to five positive specific markers for
males in ISSR analysis. On the other hand, the level of polymorphism across cultivars
was 70% and 87% as revealed by RAPD or ISSR, respectively.
Keywords: Date Palm, RAPD, ISSR, DNA markers, Gender identification
Introduction
The date fruit produced largely in the hot arid regions of South West Asia and
North Africa, is marketed all over the world as a high-value confectionery and fruit
crop and remains an extremely important subsistence crop in most of the desert
regions. The major date producers in the world are located in the Middle East and
North Africa[1].
Date palm (Phoenix dactylifera L.) is an important crop in Egypt where the total
number of fruitful female palm is about 10,735.646 palm trees producing about
1,121.890 tones according to the statistics of the Central Administrations of
Horticultural, Ministry of Agriculture (2004).
The establishment of a date palm plantation is a long term and costly investment.
There are risks in any production process that plants produced may not be true-to-type,
i.e. genetically identical to the mother plant. A range of different approaches is
467
available for detecting true-to-type plants. The methods differ in their sensitivity,
technical complexity, ease of use, and stage at which they can be applied. For date
palm, the conventional technique is the morphological screening, but this technique is
applicable when the fruits are ripe. In the last decade, a variety of molecular
applications have been applied to identify plants, including the use of molecular
markers, i.e.; RAPD and microsatellite techniques [2].
The pollen of the date palm has been found to exert a direct influence on the size,
shape and color of the seed, and also on the size of the fruit, on the speed of
development of the fruit and on the time of ripening of the fruit.
This
direct
influence of the male parent on the development of the date fruit is precise and definite
and varies with the particular male used to fertilize the female flowers. Each male
exerting approximately the same effect on fruit of all varieties and exerting the same
effect in different years. This direct effect of the pollen on the parts of the seed and
fruit lying outside the embryo and endosperm is called metaxenia
[3]
.Therefore, it is
important to select and identify superior male in term of fertilization.
In all date palm, a major problem for farmers is to identify the sex of saplings at an
early stage so that they can cultivate in their orchards a sufficiently large number of
productive female trees with only a minimal number of male trees. However, attempts
to identify the sex of the dioecious species at an early stage have remained
frustratingly unsuccessful. In recent years, there have been serious efforts to
understand the genetic basis of sex determination in plants and to develop methods to
identify sex at an early stage by using molecular marker tools [4]; [5]; [6].
Data based on molecular markers such as RFLPs and RAPDs have been used to
characterize date palm genotypes
[7] [8] [9] [10]
. In this paper we have attempted to
identify sex-specific DNA markers for date some palm cultivars using molecular
technique (RAPD and ISSR) to facilitate the selection and identification of good male
pollinators for further using in breeding programs to increase the yield and improve
the physical and chemical characters of fruits.
468
Materials and methods
Plant materials
Four dry dates cultivars (Sakoty, Bertmoda, Malkabi, Dagana) and three males
(Dagana, Malkabi,Sakoty) recognized as date superior pollinators. Collected from
Com Ambo farm at Aswan, Horticultural Services for Ministry of Agriculture, Egypt.
DNA preparation
Three leaves were collected from each plant and three plants per cultivar were
subjected to molecular analysis. Leaves (200 mg) were ground to a powder using
liquid nitrogen in microfuge tubes then DNA were isolated using AxyPrep multisource
Genomic DNA Mini-Prep Kit (Axygen Bioscience, USA, cat. No. Ap-MN-MSGDNA-50) according to manufacturer manual. DNA samples of each cultivar were
analyzed individually to detect intra-cultivar variations and bulked to detect intercultivar variations.
PCR conditions and electrophoresis
An initial screening of 30 RAPD decamer and 20 ISSR primers (successfully
utilized in other plant species,
[11] [12]
,
) was performed in order to test their readability
and amplification profiles for polymorphism. After this screening procedure, seven
RAPD and seven ISSR primers were selected (Table1).
PCR for both analysis was performed in 25 μl volume containing 2.5mM MgCl2
0.2mM dNTPs, 20 μM primer, 50 ng genomic DNA and 1 U Taq DNA polymerase
(Bioron, Germany). All reactions were performed in a Perkin Elmer 2400 thermal
cycler. RAPD program was performed as 1 cycle of 94oC for 4 min and 40 cycles of
94oC for 1 min, 35oC for 1 min and 72oC for 2 min, then, a final extension step 72oC
for 8 min. The ISSR program was performed as 1 cycle of 94oC for 4 min and 35
cycles of 94oC for 1 min, 44o for 45 sec, 72o C for 1.5 min, and, a final extension step
of 72oC for 8 min. To visualize the PCR products, 15 μl of each reaction was loaded
on 1.8% agarose gel. The gel was run at 90 V for about 1 hr and visualized with UV
trans illuminator and photographed using UVP gel documentation system. For each
amplification, a negative control reaction without DNA template was included. PCR
reaction, that generated high level of polymorphism across both types of analyses, was
repeated twice in order to verify the reproducibility of scored polymorphic bands. This
469
procedure allowed only those bands present in all replicated experiments to be scored
as markers. Amplicon size were estimated using both 100-bp and 1-kb DNA standards
(Bioron, Germany).
Data analysis
Reproducible bands visualized on the gels were scored using a binary code (1/0) for
their presence or absence for both RAPD and ISSR based on the UVP gel
documentation system (Gel Works ID advanced software, UVP).
Results and discussion
In this work, the utility of RAPD and ISSR markers in the sex determination
analysis of Palm germplasm was studied. According to the documented publications,
this is the first report of an analysis of gender genetic identification in date palm using
both RAPD and ISSR analyses. The optimal number of primers, required to
discriminate among genomic DNA of seven cultivars, depends on the level of
polymorphism generated by type from the palm samples. In other hand, from the same
7 primers A12 of molecular analysis (eg., RAPD, ISSR, etc.).
Identification of RAPD markers
At the polymorphism level, a high level of polymorphism was generated utilizing
the 7 RAPD primers (Figure 1 and Table 2). A total number of 109 RAPD bands
across all cultivars, were obtained. Of these, 76 bands were polymorphic (70%). The
highest number of amplicons was generated from Malkabi male palm (75 amplicons),
while Dajna cultivar
generated the lowest (66 amplicons). The highest number of
amplicons was generated from primer
D5 (24 amplicons), while the lowest was
generated from primer D20 (6 amplicons). A number of 25 amplicons were useful
gender-specific markers in which 13 of them were scored for the presence of a unique
band for a given cultivars (positive marker), while 12 were scored for the absence of a
common band (negative marker). The highest number of cultivar-specific markers
(seven) was scored for Dajna cultivar while the lowest number of cultivar-specific
markers (one) was scored for Sakoty male palm and Bertamoda cultivars. Primer D5
generated the highest number of cultivar-specific markers (nine), while primers D10
and D20 generated the lowest (one) (table 3). In conclusion, all RAPD primers used in
the present study allowed for enough distinction among the seven palm cultivars.
470
Overall comparison among cultivars across the seven primers revealed the power of
RAPD in distinguishing among palm cultivars grown in the same location. These
markers can be used in subsequent experiments to detect molecular markers for genes
with male and female identification in palm cultivars. A low number of RAPD
amplicons per primer was sufficient to produce useful fingerprints for palm cultivar
discrimination [13]. Based on the amplification products, seven individual primers were
selected and the amplification polymorphism against DNA samples from three male
and four female palm trees were examined. Of the seven primers, only three primers
A10, A12 and D10 yielded a clear and characteristic amplification products,
approximately 490, 750 and 800 bp in size, respectively only in females and not in
males (Fig.1 and table 6). These bands were consistent and present in all four females
analyzed while primersA12 and D10 yielded a clear and characteristic amplification
markers, approximately 370 and 675 bp long, respectively, only in males and not in
females (Fig 1and table 6). These bands were consistent and present in all three males
analyzed from the palm samples.
Identification of ISSR markers
ISSR is a class of molecular markers based on inter-tandem repeats of short DNA
sequences. These inter repeats are highly polymorphic, even among closely related
genotypes, due to the lack of functional constraints in these non-functioning regions.
Similarly, a high level of polymorphism was generated utilizing the seven ISSR primers
(Figure 2 and Table 4). A total number of 89 ISSR bands were obtained. Of these 77
bands were polymorphic (87%) and only 30 were monomorphic (34%). The highest
number of amplicons was generated from Dagana male palm (50 amplicons), while
Bertamoda cultivar generated the lowest (35 amplicons). The highest number of
amplicons was generated from primers HB11 and HB12 (16 amplicons), while the
lowest was generated from primer HB10 (8 amplicons) (Fig 2,Table 4). A number of 30
amplicons were a specific markers in which 22 of them were scored for the presence of
a unique band for a given cultivars (positive markers), while 8 were scored for the
absence of a common band (negative marker). The highest number of cultivar-specific
markers (seven) was scored Dajna cultivar, while the lowest number of cultivar-specific
markers (two) was scored for Bertamoda cultivar (Table 5). In conclusion, all ISSR
471
primers used in the present study allowed for enough distinction among the seven palm
cultivars as used before in previous work
[2] [14]
. Based on the amplification products,
seven primers were selected and the amplification polymorphism against DNA samples
from three male and four female palm trees were examined. Of the seven primers, only
five primers HB9, HB10, HB12, 814, 844A yielded a clear and characteristic five
markers (approximately 340, 1010, 375, 590 and 920 bp long), respectively, only in
males and not in females (Fig.2 and table 6). These bands were consistent and present
in all three males analyzed from the palm samples. On the other hand, from the same
seven primers there were no female- specific markers.
Our results provide the exciting possibility of being used to address several issues,
including developing DNA probes to determine sex in palm dates to increase
understanding of the evolution of date palm.
472
References
[1] Zaid Abdelouahhab 2002. Date Palm Cultivation, FAO Date production support
programm, copyright@fao.org.
[2] Hamama L., N. Cornee, V. Leclerc, F. Marionnet, M.Javouhey and R. Letouze,
2003. Date Palm (phoenix dactylifera) Offshoot Identification by PCR-ISSR
Markers. Acta Hort 616: 453-457.
[3] Walter T. Swingle,(1928). Metaxenia in the date palm Possibly a Hormone Action
by the Embryo or Endosperm. The Journal of Heredity 19(6):257-268.
[4] Mulcahy, D.L., N.F. Weeden, R. Kesseli and S. Carroll. 1992. DNA probe for Ychromosome of Silene latifolia, a dioecious angiosperm. Sex Plant Reprod. 5:86-88.
[5] Hormaza, J.I., L. Dollo and V.S. Polito. 1994. Identification of a RAPD marker
linked to sex determination in Pistacia vera using bulked segregant analysis. Theor.
Appl. Genet. 89:9-13.
[6] Biffi, R., F.M. Restivo, F. Tassi, A. Carboni, G.P. Marziani, A. Spada and A.
Falvigna. 1995. A restriction fragment length polymorphism probe for early
diagnosis of gender in Asparagus officinalis L. HortScience 30:1463-1464.
[7] Sedra My.H, Lashermes P, Trouslot P, Combes MC, Hamon S (1998).
Identification and genetic diversity analysis of date palm (Phoenix dactylifera L.)
varieties from Morocco using RAPD markers. Euphytica 103: 75-82.
[8] Ben Abdallah A, Stiti K, Lepoivre P, Du Jardin P (2000). Identification de
cultivars de palmier dattier (Phoenix dactylifera L.) par l’amplification aléatoire
d’ADN (RAPD). Cahiers Agricultures. 9: 103 -107.
[9] Trifi M, Rhouma A, Marrakchi M (2000). Phylogenetic relationships in Tunisian
date palm (Phoenix dactylifera L.) germplasm collection using DNA amplification
fingerprinting. Agronomie 20: 665-671.
[10] Trifi M (2001). Polymorphisme et typage moléculaire de variétés tunisiennes de
palmier dattier (Phoenix dactylifera L.): relation avec la résistance au bayoud.
Thèse Doctorat d’Etat, Université Tunis-El Manar, Fac. Sc. Tunis, 141.
[11] Nagaoka, T. and Ogihara, Y. 1997. Applicability of inter-simple sequence repeat
polymorphisms in wheat for use as DNA markers in comparison to RFLP and
RAPD markers. Theoretical and applied genetics 94: 597-602.
473
[12] Awasthi, A.k.; G.M. Nagaraja; G.V. Naik; S. Kanginakudru; K. Thangavelu and
J. Nagaraju (2004). Genetic diversity and relationships in mulberry (genus
Morus) as revealed by RAPD and ISSR marker assays. BMC Genet. 5(1):1.
[13] Saker M.M. and H.A. Moursy (1999). Molecular characterization of Egyptian
date palm:П RAPD fingerprints. Arab J. Biotechnology 2:71-78.
[14] Hussein Ebtissam H. A., Sami S. Adawy, Samer E. M. E. Ismail and Hanaiya A.
El-Itriby 2005. Molecular characterization of some Egyptian date palm
germplasm using RAPD and ISSR markers. Arab J. Biotech., 8: 83-98.
474
Table (1) Names and sequences of RAPD and ISSR primers
RAPD Primers
ISSR Primers
Name
Sequence
Name
Sequence
A10
5'-GTGATCGCAG-3'
HB 8
(GA)6GG
A12
5'-TCGGCGATAG-3'
HB 9
(GT)6GG
A13
5'-CAGCACCCAC-3'
HB 10
(GA)6CC
D05
5'-TGAGCGGACA-3'
HB 11
(GT)6CC
D07
5'-TTGGCACGGG-3'
HB 12
(CAC)3GC
D10
5'-GGTCTACACC-3'
814
(CT)8TG
D20
5'-ACCCGGTCAC-3'
844A
(CT)8AC
Table (2): Number of amplified fragments markers of seven palm cultivars based on
RAPD –PCR analysis.
RAPD Primers
Palm cultivars
A10
A12
A13
D5
D7
D10
D20
Total
Sakoty
AF
SM
9
2
7
1
9
2
18
1
21
0
6
0
3
0
73
6
Dagana male
AF
SM
7
0
8
0
10
1
13
4
21
0
5
0
4
1
68
6
Malkabi male
AF
SM
8
0
10
0
10
0
20
2
20
0
4
0
3
0
75
2
Sakoty male
AF
SM
8
1
8
0
8
0
19
0
21
0
5
0
4
0
73
1
Bertmoda
AF
SM
8
0
10
1
9
0
16
0
20
0
6
0
3
0
72
1
Malkabi
AF
SM
7
0
10
0
8
0
17
1
19
0
5
0
4
0
70
1
Dagana
AF
SM
8
0
11
0
7
2
15
1
15
3
6
1
4
0
66
7
TSM
3
3
5
9
3
1
1
25
TAF
PB
12
7
18
17
19
18
24
17
22
8
8
5
6
4
109
76
58.3
94.4
94.7
70.8
36.36
62.5
66.67
69.72
% polymorphism
TAF = Total number of amplified fragment, PB = Polymorphic bands, AF = Amplified fragment,
SM = marker, including either the presence or absence of a band in palm cultivar,
TSM = Total no. of specific markers across palm cultivars.
475
Table (3): Cultivar-specific markers resulting from RAPD- PCR analysis.
Palm cultivars
Positive marker
Negative marker
Total
Sakoty
5
1
6
Dagana male
3
3
6
Malkabi male
1
1
2
Sakoty male
1
0
1
Bertmoda
1
0
1
Malkabi
0
2
2
Dagana
2
5
7
Total
13
12
25
Table (4): Number of amplified fragments markers of seven palm cultivars based on ISSR analysis.
ISSR Primers
Palm cultivars
HB10
HB8
HB9
HB11
HB12
814
844A
Total
Sakoty
AF
SM
5
1
6
0
6
1
8
0
7
0
8
2
7
1
47
5
Dagana male
AF
SM
5
0
6
1
7
0
10
0
7
1
9
0
6
1
50
3
Malkabi male
AF
SM
5
0
5
0
6
2
7
2
10
2
6
0
5
0
44
6
Sakoty male
AF
SM
4
1
6
0
8
0
9
0
8
0
8
0
6
2
49
3
Bertmoda
AF
SM
2
0
5
0
4
1
7
0
8
0
6
1
3
0
35
2
Malkabi
AF
SM
2
0
6
1
7
0
6
2
9
0
5
0
4
1
39
4
Dagana
AF
SM
2
0
3
2
6
0
8
1
11
2
7
1
4
1
41
7
TSM
2
4
4
5
5
4
6
30
TAF
PB
8
7
10
8
13
12
16
14
16
14
12
9
14
13
89
77
87.5
80
92.3
87.5
87.5
75
92.85
86.1
% polymorphism
TAF = Total number of amplified fragment, PB = Polymorphic bands, AF = Amplified fragment,
SM = marker, including either the presence or absence of a band in palm cultivars,
TSM = Total no. of specific markers across palm cultivars.
476
Table (5): Cultivars-specific markers resulting from ISSR analysis.
Palm cultivars
Positive marker
Negative marker
Total
Sakoty
4
1
5
Dagana male
2
1
3
Malkabi male
4
2
6
Sakoty male
3
0
3
Bertmoda
1
1
2
Malkabi
3
1
4
Dagana
5
2
7
total
22
8
30
Table (6): Sex-specific markers in Date Palm based on RAPD and ISSR analysis
Male positive markers
No.
Female positive markers
No.
RAPD
A12-370, D10-675
2
A10-490, A12-750, D10-800
3
ISSR
HB10-1010, HB9-340, HB12-375, 814-590,
844A-920
5
477
0
M1
2
3
4
5
6
7
M
1
2
3
4
5
6
7
1500
1000
500
A-10
A-12
D-10
A-13
D-07
D-05
D-20
Figure (1) RAPD profile of the seven palm cultivars amplified with seven different RAPD primers. M: 100 bp
ladder marker. Lanes 1 through 7 refer to palm cultivars: Sakaote, Dagana male, Malkabi male, Sakoty male,
Bertamoda, Malakabi, Dajna.
478
M
1
2
3
4
5
6
M
7
1
2
3
4
5
6
7
1500
1000
500
HB-10
HB08
HB11
HB09
H44A
HB12
814
Figure (2) ISSR profile of the seven palm cultivars amplified with seven different ISSR primers. M: 100 bp
ladder marker. Lanes 1 through 7 refer to palm cultivars: Sakaote, Dagana male, Malkabi male, Sakoty male,
Bertamoda, Malakabi, Dajna.
479
Gآ Eت ورا ! Cا 3& B 6ا &'Hام 1 ,G
RAPD,ISSR
را -أ د ا
-ن
ا
ف ه:ا ا >5ا ; ل إ 7آZف ورا 5 dد ! i" jCأ;ف Kا!` ,ام )+ت
K RAPD,ISSR Wا ب و l; +ا:آ ا !)` ا * Cا ت ا )! ,ا k
ا!)` دة ا 65ل و mX6P i", G5+ا Cدة ! Nر .و*P >5,ل 2ا 8ا "! ت )
G+أن ه:ا ه أول l; + K!5 >5,ا ع ا را K dا!` ,ام !!5+آG K
>- ISSR , RAPDأ d*d 78آ_0ت 5دة oث وا:! Gdآ ر RAPD K!5+ه:ا
',إ j P 7آ_0ت :! Wآ ر . ISSR K!5+و - Gأ Pى ى
ا ت G,ا;ف آن %٧٠و % ٨٧ب 7!8 RAPD ,ISSRا ا.
480
OP 23
Genetic analysis of abnormalities in tissue culture-derived date
palm (Phoenix dactylifera L.), Barhi cultivars
Kh. A. Soliman1, R. M. Rizk2, and S. S. H. El-Sharabasy3
1. Genetics Dept, Fac. of Agri., Ain Shams Univ., Ain Shams Center for Genetic
Engineering and Biotechnology (ACGEB), Faculty of Agri. Branch. Cairo, Egypt, 2.
National Gene Bank, Agri. Research Center, 3. The Central Lab. of Date Palm
Research and Development, Agri. Research Center, Giza, Egypt
(khaled346@hotmail.com),(Sharabasydate@hotmail.com)
Abstract
Abnormalities and habituation as well as normal Barhi cultivars of tissue culture –
derived was collected and subjected to morphological and genetic comparison. The
level of abnormality was very low (dwarfism, 0.07%; Excessive vegetative growth,
0.25% and apical bent, 0.1% The dwarfism and excessive vegetative growth were
recovered and become a normal case within 4 years of field intensive care. Meanwhile
the apical bent was not recovered and in most cases die. The electrophoretic analysis
of leaf protein revealed that the abnormalities samples haven't two bands (76 and 27.5)
in the normal plants. The excessive of vegetative growth haven't the unique (positive
marker) band of molecular weight 18.5 KD which found in the normal plant.
Regarding ISSR analysis, bands of molecular weight 760 and 515bp of primer HB-12
were absent only from the apical pent abnormalities sample and present in all the rest
of studies samples. Bands of molecular weight 895 and 195bp of primer HB-15 were
absent from dwarfism sample, while present in all the rest of studied samples, which
found in the normal plant. The absence of this band could be taken as a positive
marker for excessive of dwarfism abnormalities. At RAPD level, a band of molecular
weight 245.5 bp of primer B-05 was specific to apical pent abnormality. A band of
molecular weight 177.5 bp was specific to excessive of vegetative growth. ISSR as
well as RAPD-PCR could be used to detect the abnormalities resulting in tissue culture
derived of Barhi cultivar.
Key words: DNA-RAPD, DNA-ISSR, Electrophoreses, Phoenix dactylifera L., protein
profile, Barhi
481
Introduction
Date palms (Phoenix dactylifera L., Phoeniceae: Caryhoideae) were amongst the
first crops domesticated in the Old World (Zohary & Speigel – Roy. (1975). The date
palm is an ancient plant with great diversity. Palms have been cultivated in the Middle
East and North Africa for at least 5000 years (Zohary & Hopf, 1988). It is among most
valuable species of the cultivated plants due to its ethnic, nutritional and medicinal as
well as traditional values. It was much revered and regarded as a symbol of fertility
and of horticultural and economic value in Egypt. From the ecological point of view,
date palm has wide ecological amplitude, ranging from desert oases, mangrove
margin, steep limestone cliffs and cultivated lands. Date palm is distributed in the
Northern subtropical zone between 10°-30° North (Al Baker, 1972 and Amer, 2000).
Protein electrophoresis profiles have been found to be valid evidence for taxonomic
treatment of several plant groups (Ladizinisky and Hymowitz, 1979, Welsh and
McClelland, 1990 and Badr, 1995). The development of so-called "DNA markers" which
are based on polymorphisms found in proteins or DNA has greatly facilitated research in
a variety of many biological branches such as taxonomy, phylogenetic relationships and
genetics (Abdelsalam et. al, 1998 Halward et al. and 1992; Carlson et al., 1991). The
ISSR strategy was therefore performed to access the DNA diversity among crop
genotypes (Zehdi et. al, 2004). Soliman et al. (2003) used RAPD markers to study 3
males and 4 females date accessions from Egypt. El-Assar et al. (2005) used AFLPmarkers to study genetic diversity of 21 named accessions and 9 unknown pedigrees of
date palm. Cleary, an integrated approach is needed incorporating morphological and
genetic studies to improve the knowledge of Date palm taxonomy and phylogeny.
Proteins or/ and DNA attributes can be used successfully for variety identification, source
of information of date palm gene bank and for studying the genetic diversity of cultivars.
Date palm can be promoted best through better characterization and evaluation.
Tissue culture techniques have been successfully employed in propagation and
adapted to be excellent tools to propagate and improve plants compared to the
conventional methods (Al-Ghamdi, 1989). The process of somatic embryogenesis
seems to be more attractive for on industrial production but the date palms derived
from somatic embryos must be true-to-type (Torres and Tisserat, 1990 and Bendiab et
482
al., 1998). Propagation by both offshoots and tissue culture generally results in true-totybe plants, but some off-types with abnormal phenotypes have developed in tissue
cultured plants. These abnormalities may be due to somaclonal variation. (Al-Khalifah
and Askari, 2007)
Genetic variations in tissue plantlets are commonly noted. Biochemical analysis has
been used to determine the genetic variations among plantlets (Tisserat, 1984 and AlGhamdi, 1993b). A technique based on the amplification of random DNA sequences
chain reaction (PCR) with arbitrary primers reported by Williams et al. (1990). RAPD
(Random Amplified polymorphic DNA) markers are now used for identification to
evaluate genetic integrity (Isabel etal.,1993) or analysis of somaclonal variants
(Hashmi etal.,1997).
Abnormalities and habituation as well as normal Barhi cultivars of tissue culture –
derived was collected from Samarina Farm, Ismalea governorate and was subjected to
morphological and genetic comparison.
Material and methods
Abnormalities and habituation as well as normal Barhi cultivars (Table 1) produced
by tissue culture techniques was collected from Samarina Farm, Ismalea governorate
and was subjected to genetic comparison. Barhi cultivar of tissue culture –derived has
been mass cultured since 2004 in Samarina Farm, Ismalea governorate. A survey of
morphological abnormalities has been done annually from 2006 to 2008.
Protein extraction:
Samples of squash leaves were taken from six date palm. Total soluble protein were
extracted by grounding 0.25g of each sample in 0.9 ml extraction buffer (10ml
0.5MTris pH6.8, 16ml 10%SDS, 30ml D.W) with shaking thoroughly. The extracts
were transferred to Eppendorf tubes and centrifuged for 10 min. at 10000rpm under
cooling. Supernatant were transferred by fresh tubes and used for SDS-PAGE analysis
and extraction of isozymes was used as described by Jonathan et al. (1990).
Protein related index:
Fractionation electrophoresis was performed under identical conditions on sodium
dedocyl sulphate polyacrylamide gel (SDS-PAGE) (12%W/V)vertical slab using
BIORAD Techware 1.5 mmm according to the method of Laemmli (1970) as modified
483
by Studier (1973). The molecular weights of proteins were estimated relative to
marker, a wide range molecular weight protein (Fermentas comp.).
Isozymes electrophoresis
Native–polacrylamide gel electrophoresis (Native-PAGE) was performed in 12%
(W/V) slab gel (Davis 1964). The gel was stained after run according to Tanksely and
Rick (1980) for Poly Phenyl Oxidase (PPO) isozymes and Grahan et al .(1964) for
peroxidase isozymes. The staining gel was incubated at 37 °C in dark for complete
staining after adding the appropriate substrates and staining solutions.
Gel documentation
Gels were photographed scanned, analyzed using Gel Doc VILBER LOURMAT
system to capture the image and to calculate band intensities.
RAPD-PCR Analysis
a. DNA Extraction
Young and fresh leaf samples were collected separately from six samples of date
palm, all the selected leaves were normal and free from any pathogenic symptoms and
all leave samples were saved in ice box and quickly transported to laboratory. Plant
tissues were ground under liquid nitrogen to a fine powder, then bulked DNA
extraction was performed using DNeasy plant Mini Kit (QIAGEN).
b. Polymerase Chain Reaction (PCR).
PCR amplification was performed using ten random 10 mer arbitrary primers synthesized
by (Operon biotechnologies, Inc.Germany) Table (2) with the following sequences:
Amplification was conducted in 25 µl reaction volume containing the following
reagents: 2.5 µl of dNTPs (2.5 mM), 2.5 µl Mgcl2 (2.5 mM), and 2.5 µl of 10 x buffer,
3.0 µl of primer (10 pmol), 3.0 µl of template DNA (25 ng / µl), 1 µl of Taq
polymerase (1U/ µl) and 10.5 µl of sterile dd H2O. The DNA amplifications were
performed in an automated thermal cycle (model Techno 512) programmed for one
cycle at 94º C for 4 min followed by 45 cycles of 1 min at 94º C, 1 min at 36º C, and 2
min at 72º C. the reaction was finally stored at 72º C for 10 min. Amplified products
were size-fractioned (using 1 Kbp ladder marker) by electrophoresis in 1.5 % agarose
gels in TBE buffer at 120 V for 1 h. the bands were visualized by ethidium bromide
under UV florescence and photographed.
484
ISSR-PCR Analysis
b. Polymerase Chain Reaction (PCR).
PCR amplification was performed using ten random 10 specific primers
Table (3) with the following sequences:
ISSR-PCR reactions were conducting using 10 primers. Amplification was
conducted in 25 µl reaction volume containing the following reagents: 2.5 µl of dNTPs
(2.5 mM), 2.5 µl Mgcl2 (2.5 mM), and 2.5 µl of 10 x buffer, 3.0 µl of Primer (10
pmol), 3.0 µl of template DNA (25 ng/ µl), 1 µl of Taq polymerase (1U/ µl) and 12.5
µl of sterile dd H2O. the PCRs were programmed for one cycle at 94º C for 4 min
followed by 45 cycles of 1 min at 94 ºC, 1 min at 57 ºC, and 2 min at 72 ºC. The
reaction was finally stored at 72 ºC for 10 min. the PCR products were separated on a
1.5 % agarose gels and fragments sizes were estimated with the 100bp ladder marker.
Data analysis
The similarity matrices were done using Gel works ID advanced software UVPEngland Program. The relationships among peach cultivars as revealed by
dendrograms were done using SPSS windows (Version 10) program
Results and discussion
Barhi cultivar of tissue culture –derived has been mass cultured since 2000 in
Samarina Farm, Ismalea governorate. A survey of morphological abnormalities has
been done annually from 2003 to 2007. The total number of Barhi plants is 425
individual derived by tissue culture.
Morphological abnormalities: the abnormalities observed in young tissue culturederived date palms were dwarfism, Excessive vegetative growth and apical bent.
Dwarf date palm plants are less than one meters high after four to five years in the
field in the normal conditions. While, the normal date palm plant with the same age is
3 meters. Excessive vegetative growth was appeared in broad leaves and large with
different spine structure. The apical pent was appeared by bent of growing apical tips.
The level of abnormality found in this survey was very low (dwarfism, 0.07%;
Excessive vegetative growth, 0.25 and apical bent, 0.1).
485
The dwarfism and excessive vegetative growth were recovered and become a
normal case within 4 years of field intensive care. Meanwhile the apical bent was not
recovered and in most cases die.
Protein and Isozymes: Summaries of total protein profiles are given in table 2. The
electrophoretic analysis of leaf protein extracts using discontinuous SDS-PAGE gel for
the studied sample of Barhi cultivar revealed the presence of eighteen bands of
molecular weight ranging from 15.5 to 185 KD (plate 1.a). The abnormalities samples
haven't the bands of molecular weight 76 and 27.5 which present in the normal plant
as well as the habituation sample. The excessive of vegetative growth haven't the
unique band of molecular weight 18.5 KD which found in the normal plant as well as
the habituation sample. The absence of this band could be taken as a positive marker
for excessive of vegetative growth abnormalities.
All isozyme system examined generated banding pattern that migrated nodally. The
electrophoretic pattern of peroxidase isozyme (Plate 1.b) revealed that five loci were
distinguished namely Per.1, Per.2, Per.3, Per.4 and Per.5 which differ in their relative
migration distance. The poly phenyl oxidase isozyme (Plate 1.c) revealed that two loci were
distinguished namely Poly.1 and Poly.2 which differs in their relative migration distance.
DNA profile: The results of DNA profiles using ISSR analysis for 5 primers (44a,
HB08, HB10, HB12 & HB15) and RAPD analysis for 6 primers (OP-A01, OP-A03,
OP-A05, OP-A17, OP-B05 & OP-B17) are illustrated in plate (2 & 3).
Regarding ISSR analysis, bands of molecular weight760 and 515bp of primer HB-12
were absent only from the apical pent abnormalities sample and present in all the rest of
studies samples. Bands of molecular weight 895 and 195bp of primer HB-15 were absent
from dwarfism abnormality sample, while present in all the rest of studied samples,
which found in the normal plant as well as the habituation sample. The absence of this
band could be taken as a positive marker for excessive of dwarfism abnormalities.
Bands of molecular weight 800 of the primer OP-A03 was recorded in all samples
except for the apical pent abnormality. Bands of molecular weight 220.5 and 148.5 bp
were not recorded in the abnormalities samples while, present in the normal as well as
the habitation samples.
486
Regarding RAPD analysis, band of molecular weight796 bp of primer OP-A01was
absent only from the excessive of vegetative growth abnormalities sample, while
present in all the rest of studies samples. The absence of this band could be taken as a
positive marker for excessive of dwarfism abnormalities. Bands of molecular weight
268.5 and 435 bp of primer OP-A01were absent from dwarfism and the excessive of
vegetative growth abnormality, while present in all the rest of studied samples, which
found in the normal plant as well as the habituation sample.
Bands of molecular weight 344.5 of the primer OP-A05 was recorded dwarfism
sample and not recorded in the rest of samples. A band of molecular weight 1608 bp
was recorded in the apical pent abnormality. A band of molecular weight 343.5bp was
recorded in both excessive of vegetative growth as well as dwarfism. These bands
could be taken as a positive marker for specific abnormalities.
Regarding primer OP-A17, bands of molecular weight 2002.5 bp was detected in both
excessive of vegetative growth and dwarfism abnormalities, while a band of molecular
weight 177.5 bp was specific to excessive of vegetative growth only. A band of
molecular weight 183.5 bp was recorded in normal and habituation samples only.
A band of molecular weight 245.5 bp of primer OP-B05 was specific to apical pent
abnormality. Bands of molecular weight 536 and 256.5 bp of the primer OP-B17 were
not recorded in dwarfism sample and recorded in the rest of samples. A band of
molecular weight 502 bp was not recorded in the excessive of vegetative growth
abnormality. These bands could be taken as a positive marker for specific abnormalities.
Similar results were reported in previous studies as dwarfism and abnormal floral
development as somaclonal variation in tissue culture derived date palm trees (Al-wasel,
2000). Several different DNA molecular methods have been used to screen date palms
derived from tissue culture for genetic stability e.g. RFLP, RAPD, AFLP and more advanced
ISSP ( Cormiquel and Mercier, 1994).RAPD techniques have been reported to be useful for
studying genetic variation in date palm ( Letouze et al. 1998; El-Hammady et al. 1999).
Acknowledgement
authors would like to acknowledge all Samarina Farm numbers especially to Mr.
Hassan Elgayar chairman of first company for live stoke of Agronomic Development ,
for his cooperation in success of this study.
487
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490
Table 1: the abnormalities and habituation in Barhi produced by tissue culture
ID
Items
4
Normality (Normal case)
3
Excessive of vegetative growth
12
Habituation Excessive of vegetative growth
1
Dwarfism
2
Habituation of dwarfism
11
Apical bent
Table (2): List of the primer names and their nucleotide sequences used in the study
No.
Name
Sequence
No.
Name
Sequence
1
OP-A01
5´ TCGGCCATAG 3`
6
OP-A17
5` GGG CGG TAC T 3`
2
OP-A03
5´ CCTTGACGCA 3`
7
OP-B05
5` GGACCCAACC 3`
3
OP-A05
5´ GTGACCCCTC 3`
8
OP-B17
5` AAGCCTCGTC 3`
Table (3): List of primer names and their nucleotide sequences used in the study.
No.
1
2
Primer
44 a
HB08
sequence
No.
5` GTGTGTGTGTGTGG 3`
5` GTGTGTGTGTGTGG 3`
5` GAGAGAGAGAGACC
3
HB10
3`
491
4
Prim
er
HB1
2
5
HB1
5
sequence
5` CACCACCACGC 3`
A
Table (2): Distribution of protein profile of the
leaf extract of the Barhi abnormalities, for
sample Id See table 1.
Sample ID
3
1
2
11
12
4
KD
1
185
0
1
1
0
1
0
2
160
1
1
1
0
1
1
3
142
1
1
1
0
1
1
4
125.5
1
1
1
0
1
1
5
115
1
1
1
0
1
1
6
95.5
1
1
1
1
1
1
7
76
0
0
1
0
1
1
8
61.5
1
1
1
1
1
1
9
54.5
0
1
1
0
1
1
10
47
1
1
1
1
1
1
11
40
1
1
1
1
1
1
12
30.5
0
1
0
0
0
1
13
27.5
0
0
1
0
1
1
14
25
0
1
0
0
1
0
15
22.5
0
0
0
0
0
1
16
20
1
1
1
1
1
1
17
18.5
0
1
1
1
1
1
18
15.5
1
0
0
1
1
0
SDS – PAGE
B
Peroxidase
C
Poly Phenyl Oxidase
Plate. (1): Peroxidase and poly phenyl
oxidase isosyme prophile of the studied
abnormalities of Barhi cultivars. for
sample Id See table 1.
492
44 a
HB 08
HB10
HB12
HB15
Plate. (2): DNA polymorphism based on ISSR-PCR analysis of the studied
abnormalities of Barhi Cultivars. for sample Id See table 1.
493
OP-A01
OP-A03
OP-A05
OP-A17
OP-B05
OP-B17
Plate. (3): DNA polymorphism based on RAPD-PCR analysis of the studied
abnormalities of Barhi Cultivars. For sample Id See table 1.
494
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496
OP 24
Physiological Modifications in Date Palm Seedlings in Response to X-Ray
Norah A. Al-Enezi1 and Jameel M. Al-Khayri2*
1
Department of Biology, Science College, Dammam University, P.O. Box 838, Dammam
31113, Saudi Arabia. 2Department of Agricultural Biotechnology, College of Agricultural and
Food Sciences, King Faisal University, P.O. Box 420, Al-Hassa 31982, Saudi Arabia. *Email:
jkhayri@kfu.edu.sa
Abstract
Ionizing radiation is known to adversely affect biological systems; however, low
doses may induce stimulation (i.e. hormesis). This study was conducted to determine
some physiological responses of date palm (Phoenix dactylifera L.) seedlings to Xirradiation. A therapeutic medical X-ray device was used to expose 15-day-old
seedlings to incremental increases of X-ray doses ranging from 0 to 1500 rad.
Seedlings were grown for 2 months, under greenhouse conditions, after which leaf
tissue was subjected to chemical analysis. The data indicated that water content and
proline accumulation significantly increased in response to stress induced by
increasing X-rays doses. The minimum dose of X-ray causing a significant stress
reaction in date palm seedling was 100 rad. The concentration of total photosynthetic
pigments showed inhibition at low X-ray dose, 5 rad, and continued to decrease
following dose increases reaching the lowest concentration at 1500 rad. Chlorophyll a
and carotenoids appeared to be more sensitive to X-irradiation than chlorophyll b. This
was illustrated by the significant reductions for chlorophyll a and carotenoids at 5 rad;
whereas, the content of chlorophyll b began to show significant decreases at a higher
dose, 10 rad. The DNA content was dose dependent showing a significant increase
with 5 rad, the lowest radiation dose tested. The highest level of DNA content was
observed at 25 rad; whereas, higher doses caused a reduction. This study contributes to
the basic knowledge required for the application of X-ray technology in date palm
genetic improvement.
Key words: Biotechnology, Chlorophyll, Date palm, DNA, Proline, X-ray.
497
Introduction
The biological effects of ionizing radiation range from stimulation by low doses, i.e.
hormesis, to inhibition and eventually death by high doses (Ling et al., 2008). Reports
on the biological modifications caused by X-rays irradiation included cellular
mechanisms and metabolism functions (Younis et al., 1962). In addition to reduced
seed viability, germination, and plant growth (Froier and Gustafsson, 1941), modified
transpiration rate and stomata opening resulting in decreased fresh and dry weights as
well as water content (Roy 1974) in response to irradiation with X-rays. Changes in
photosynthetic pigments were also reported in response to gamma radiation (Strid et
al., 1990; Marwood and Greenberg, 1996).
Moreover, X-radiation can cause cytological changes and induce mutations (Shima
and Ichikawa, 1994; El Araqi et al., 1997). The effect of ionizing radiation on the
genetic material is well documented in numerous plant species (Esnault et al., 2010).
Exposure to X-rays resulted in gene alterations (Kranz et al. 1994; Ken et al. 2005) as
well as chromosomal changes (El Araqi et al., 1997; Watanabe et al., 2009).
Furthermore, X-irradiation was shown to negatively influence synthesis of DNA
synthesis, RNA, and protein (Roy et al., 1972).
Mutations induced by gamma-radiation proved successful to enhance disease
resistance in date palm (Jain, 2011; Sedra and Lazrek, 2011). Other studies on date palm
reported the biological effects of non-ionizing radiation in the form of magnetic fields
(Dhawi and Al-Khayri, 2011). However, the literature lacked information related to the
impact of X-rays on date palm. The objective of this investigation was to assess the
effect of various doses of X-rays on date palm seedlings. Parameters studied included
water content, proline accumulation, DNA concentration, and photosynthetic pigments.
Materials and methods
Date palm seeds collected from female trees of cv. Khalas. The seeds were surface
sterilized with 1% sodium hypochlorite for 5 min, soaked in water for 24 h, and then
germinated on moist filter paper at 37 °C. Two-week-old seedlings were exposed to Xrays using a therapeutic medical X-ray device (Fig. 1, A). The seedlings were arranged
in Petri dishes (Fig. 1, B) prior to irradiating with different X-rays doses, from 0 to
498
1000 rad. After irradiation, the seedlings were planted individually in potting mix and
maintained in a greenhouse for further growth (Fig. 1, C).
To determine the effect on water content, fresh and dry weights of leaf samples was
recorded after 3 months of irradiation. The percentage of water content was calculated
according to the following equation: Water content % = fresh weight - dry weight /
fresh weight x 100.
Leaf sections from 3-month-old plants were isolated for proline content
determination. The procedures used for proline extraction were according to that
described by Bates et al. (1973) and proline concentration was calculated based on
spectrophotometer absorbance readings at 520 nm.
DNA isolation was performed using a commercial DNA isolation kit (DNeasy Plant
Mini Kit, Qiagen, Germany). Samples of 0.1 g leaf tissue were excised from 2-monthold date palm seedlings for DNA extraction and absorbance was measured at 260 nm
using UV/VIS spectrophotometer. The total DNA concentrations were calculated
according to the following equation: DNA concentration (µg/ml) = A260 x (dilution
factor x 50)/1000.
For photosynthetic pigments estimation, the procedures described by Lichtenthaler
and
Wellburn
(1983)
were
followed.
Extract
absorbance
was
measured
spectrophotometrically at 470, 646, and 663 nm. The concentrations of chlorophyll a,
chlorophyll b, and carotenoids were calculated according to the following equations:
Chlorophyll a = 12.21 (A663) - 2.81 (A646); Chlorophyll b = 20.13 (A646) - 5.03 (A663);
Carotenoids = (1000A470 - 3.27 (chlorophyll a) - 104 (chlorophyll b))/227.
The experiment was randomly designed with a single factor, X-rays dose at 11
levels (0, 5, 10, 25, 50,100, 250, 500, 750, 1250, and 1500 rad). Each treatment was
replicated 7 times. The replication consisted of a Petri dish containing 10 seedlings.
Data were subjected to analysis of variance (ANOVA) and the means were separated,
where appropriate, using the least significant difference (LSD) at 5%.
Results and Discussion
Water content in leaf tissue of irradiated seedlings increased significantly with
respect to the control. The data has shown that exposure to 5 rad caused a nonsignificant increase in water content; however, 10 rad resulted in a significant increase
499
as compared to the control. Increasing the radiation dose to 25 rad resulted in the
highest water content, 32.2%, as compared to 16.8% recorded for the control. Doses
higher than 25 rad caused no further significant changes in water content.
Proline accumulation was directly related to the dose increases. Incremental
increases of X-ray doses ranging from 5 to 50 rad caused increase in proline content
but the differences were not significant. In comparison to the control, proline
accumulation increased significantly starting at 100 rad reaching 3 times that of the
non-treated seedlings of the control. Increasing the dose to 250 rad, resulted in a
concentration of proline 4 times that of the control. At higher X-ray doses of 500, 750,
and 1250 rad significantly higher proline concentrations were observed reaching 6, 8,
and 12 times, respectively, as compared to the control. At 1500 rad, the highest
concentration of proline, 2.55 μmol/g was recorded.
The DNA content was positively influenced by 5 rad, the lowest radiation dose
tested, giving a 1.6-fold increase as compared to the control. Increasing the dose to 10
rad and then to 25 rad caused further significant increases in DNA content reaching 2.6
and 3.2 folds, respectively, as compared to the content of the control. The latter dose
resulted in the maximum DNA content, 16.22 µg/g. Contrarily, DNA concentration
decreased as the X-rays dose increased from 50 to 1500 rad. At 100 rad, however, the
DNA concentrations remained significantly higher than the non-treated seedlings.
Seedlings exposed to X-rays exhibited a significant reduction in the concentration of
photosynthetic pigments in comparison to the control. Generally, an inverse
relationship occurred between the X-rays dose and the amounts of the photosynthetic
pigments. The data have shown that as the X-rays dose increased, the content of
chlorophyll a, chlorophyll b, and carotenoids decreased. The reduction of chlorophyll a
commenced at 5 rad, 11.43 µg/g and reached its lowest level of at 1500 rad, 7.49 µg/g,
as compared to 12.35 µg/g observed in the control seedlings. The content of chlorophyll
b started to exhibit a significant decrease at 10 rad, 4.31 µg/g reaching its lowest level
of 2.55 µg/g at 1500 rad as compared to the control, 4.68 µg/g. Reduction in the
concentration of carotenoids occurred at 5 rad, 2.66 µg/g, reaching its lowest amount,
1.73 µg/g at 1500 rad as compared to the control, 2.94 µg/g. The total photosynthetic
pigment was negatively affected in response to X-rays. Reduction in the total
500
photosynthetic pigments was observed as the X-rays dose increased reaching its lowest
value, 14.79 µg/g, at 1500 rad as compared to the non-treated seedlings, 19.97 µg/g.
In response to stress induced by exposure to X-rays, this study has shown modified
biochemical and physiological processes in date palm seedlings. Modifications
included the content of water, proline, DNA, and photosynthetic pigments. The extent
of modifications of these biological processes appeared to be related to the radiation
dose. Moreover, the effect of ionizing radiation may be stimulatory or inhibitory
depending on the biological process being considered.
In a study involving non-ionizing radiation, Dhawi and Al-Khayri (2009a) observed
that leaf water content increased significantly in response to static magnetic fields. The
present study demonstrated that water content of leaf tissue was also modified in
repose to X-rays. Treatments with X-rays doses from 5 to 1500 rad induced proline
accumulation which became greater with dose increases. Proline accumulation in Xirradiated date palm seedlings may be due to the role of proline in the resistance of free
radicals that are expected to form in plants exposed to X-rays. The response of date
palm seedlings to X-ray-induced stress caused proline accumulation in resemblance to
proline accumulation found in date palm in vitro cultures in response to stress induced
by salt (Al-Khayri, 2002) and drought (Al-Khayri and Al-Bahrany, 2004). Date palm
seedlings were also found to accumulate proline in response to static magnetic field
but to a certain level (Dhawi and Al-Khayri, 2008a). It is evident that the amount of
accumulated proline differs according to plant species and the intensity of stress
factors (Ashraf and Orooj, 2006).
Exposure to X-rays was shown to negatively influence the synthesis of DNA, RNA
and protein (Roy et al., 1972). In date palm, metabolic processes involved in DNA
synthesis and degradation, as reflected by the total DNA content of, were influenced
by the X-rays dose. Cellular activities related to DNA and elements contents were
stimulated by low doses and inhibited in response to high doses. The present study
indicated that DNA content was significantly affected by X-rays dose. Low doses, up
to 25 rad, stimulated DNA accumulation, while higher doses ranging from 50 to 1500
rad were inhibitory. With these high doses; however, the DNA content remained
higher than the control. Macklis and Bresford (1991) showed that the abundance of
501
free radicals is correlated to the radiation dose. In response to stress induced by nonionizing radiation, Dhawi and Al-Khayri (2009b) reported reduction in DNA content
when date palm seedlings were exposed to magnetic fields. In pine (Pinus pinea), Roy
et al. (1972) has shown that exposure of seedlings to 15 kR X-rays resulted in
increased chromosomal aberrations and 40% reduction in protein content. Whereas,
Berkofsky and Roy (1977) demonstrated that exposure to 5 kR X-ray dose caused no
significant alterations in the relative amount of proteins, DNA, and RNA.
Increases in the content of chlorophyll due to low doses of gamma-irradiation and
reduction at higher doses were observed in different plant species (Ling et al., 2008;
Borzouei et al., 2010). The current study has revealed modifications of photosynthetic
pigments in date palm seedlings in response to X-rays radiations. The content of
chlorophyll a, chlorophyll b, and carotenoids decreased in response to X-rays. Low
doses, as low as 5 rad, induced a significant inhibition of photosynthetic pigments. In a
relevant study using magnetic fields, Dhawi and Al-Khayri (2008b) demonstrated that
low doses had a stimulatory effect on photosynthetic pigments of date palm seedlings;
whereas, high doses gave negative results. In concurrence to the data obtained in the
present study, others have demonstrated dose-dependent effect of X-rays on
photosynthetic pigments in different plant species (Kovács and Keresztes, 2002; Abu et
al., 2005; Ling et al., 2008). Moreover, the present data have shown that chlorophyll a
and carotenoids were more sensitive to X-irradiation than chlorophyll b. This confirms
observations made in previous studies using gamma-rays (Kim et al., 2005).
In conclusion, the current study has shown variable sensitivity of the various
cellular functions to low ionizing radiation. On the contrary to the DNA accumulation
mechanisms, processes involved in the accumulation of photosynthesis pigments were
inhibited at low radiation doses. Proline accumulation, on the other hand, showed
continuous increase within the range of radiation tested. The data presented provides
the required basic knowledge to utilize X-rays for mutation induction and selection for
the purpose of genetic improvement of date palm.
502
References
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505
Fig. 1. Date palm seedlings exposure to X-rays. (A) Therapeutic medical X-ray device
used to irradiate seedlings (Clinac 23EX Linear Accelerator, Varian Medical Systems,
USA); (B) Seedlings arranged in Petri dish prior to irradiation; (C) Seedling grown in
soil after exposure to X-rays.
506
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508
OP 25
First report on finger printing of famous date palm cultivars of Pakistan
using SSR markers for identification of their true to type suckers.
Muhammad Mansoor1, Michael Baum2 and Aladdin Hamwieh2
1
Arid Zone Research Institute, Pakistan Agricultural Research Council, Dera Ismail
Khan, KPK, Pakistan.
2
International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria.
joyadkpk@gmail.com
Abstract
Genetic information about the date palm is an asset for researchers working on its
quantitative and qualitative improvement. As the date palm plays a significant role in
economy of many countries and can give boost to economies of many countries like
Pakistan. As for as our knowledge is concerned the literature is deficient on size of
dates and we believe cv. Dhakki could be biggest date of the world sizing 6 cm length
and 4 cm girth and weighing up to 35 gm per fruit. Our study will play an important
role in finding out the gene responsible for the biggest size of Dhakki which could
then be incorporated in other high yielding date varieties of the world for increasing
their size. We ran 32 micro-satellite markers on DNA of four famous varieties of
Pakistan including Dhakki. It was observed that there are some specific polymorphic
bands which are possibly related to the huge size of Dhakki. Similarity coefficient
matrix was computed to cluster the data and to draw precise relationship among
genotypes studied. Thirty two (R&F) labeled primers were evaluated on three most
famous and physiologically identical cultivars of Pakistan including cv. Dhakki, the
jumbo size date. The results indicated that out of thirty two, only eight primers (25 %)
failed to amplify the expected PCR fragments, while seven primers (22 %) amplified
mono-morphic banding patterns and the remaining seventeen primers (53 %)
generated polymorphic banding patterns.
Keywords: Phoenix dactylifera L., Dhakki dates, SSR marker, Fingerprinting, Pakistan.
509
Introduction
Date palm (Phoenix dactylifera L.) has played a vital role in the history of mankind
by providing food and shelter to millions of people. It is a long-lived dioecious mono
cotyledon fruit plant (2n=36) belonging to the Arecaceae family [3]. It is believed to
have originated in Mesopotamia and thousands of cultivars have been reported [9]. It
is always been clonally propagated to ensure the identity and uniformity of the
cultivars. Discrimination among closely related cultivars and clones is often extremely
difficult. Identification of date palm cultivars is principally based on fruit morphology
[6]. However, morphological traits are often unreliable or imprecise indicators of plant
genotype, being influenced by environmental conditions or varying with the
developmental stage of plants.
Pakistan is the 4th largest date producing country, contributing 11% to world
production. A prominent local cultivar ‘Dhakki’ of Dera Ismail Khan, KPK, Pakistan
is economically very important as it has large fruit (6 cm long, 4 cm thick and 35
g/fruit) with a small pit, fine texture and delicious taste. Dhakki dates are used in cured
and chahrra form. Dhakki dates are used in cured form with a water activity close to
0.61 ±0.01 aw [14]. Since Dhakki date is a high quality fruit its demand for local
consumption as well as for the export is ever increasing. Due to expansion in the
cultivated area of this variety and heavy demand of offshoots/suckers have been
increased manifold and the price per sucker rose from 250 PKR to 2000 PKR.
However, there has been observed consistent complaint from the growers that the
offshoots sown were not true to type. Further they say they have wasted their resource
and after a lapse of 3-4 years they came to know that the suckers were off type.
The suckers of two closely identical varieties Gulistan and Zarin are sold on high
price instead of Dhakki suckers. DNA typing can be a convenient method for
accurately identifying date palm cultivars and analyzing their genetic diversity and
phylogenic relationships. Molecular markers can provide an effective tool for
efficient selection of desired agronomic traits because they are based on the
plant genotypes and thus, are independent of environmental variation. Several
molecular markers are being currently employed, of which simple sequence repeats
(SSRs) or micro-satellites are the most widely used types. SSRs are not only very
510
common, also hyper variable for numbers of repetitive DNA motifs in the genomes of
eukaryotes ([5]; [7]; [13]; [15], [16]). Recently, micro-satellite markers have been
developed and used to investigate genetic diversity in P. dactylifera [3]. To overcome
this problem it was unavoidable to finger print cv. Dhakki. In this study we have
attempted for the first time finger printing of Dhakki and its closely identical varieties,
Gulistan and Zarin. Such investigation would be helpful for growers to identify the
true to type suckers of Dhakki dates. This study was conducted at the molecular
technology laboratory of Biodiversity and Integrated Gene Management Program,
ICARDA, Aleppo, Syria.
Material and Methods
DNA Extraction
The frozen young leaf tissues of three date palm cultivars was collected and
were first cleaned carefully with distilled water to remove the waxy layer and
then one gram of leaf sample were cut into small pieces and grinded into fine powder
by using liquid nitrogen. DNeasy Plant Maxi kit protocol (QIAGEN) was used to
extract DNA by following the manual instructions of the kit (DNeasy Plant
Handbook). The obtained DNA were quantified and qualified by using Nanodrop
Spectrophotometer. For further estimation of the DNA quantity 2µl was loaded on
0.85% agarose gel at 100V for 30 min. The gels were stained in ethidium bromide and
visualized under UV light.
The length of the primers, sequence information, repeat motifs, allele size and
melting temperature (Tm) is shown in (Table-2). Polymerase Chain Reaction was
performed in a total reaction mixture of 25µl containing 2µl (20-30ng) of total
genomic DNA, 12.5µl of AmpliTaq Gold® 360 Master mix (Applied Bio-systems),
1µl (5pmol/µl) of primers each and 8.5µl of nuclease free water. Amplification was
carried out in a Veriti 96 Well Fast Thermal cycler (Applied Bio-systems) under the
following conditions: Initial denaturation 95oC for 10min, 35 Cycles (denaturation
95oC for 30sec., annealing oC depending on primer for 30sec., extension 72oC for
1min.), final extension 72˚C for 7 min. In order to achieve better resolution,
electrophoresis was performed using the Spreadex® EL 600 Mini gel (Elchrom
Scientific). These gels are manufactured from a novel monomer and have 3x higher
511
resolving power than any other synthetic gels with working separation range 40600 bp and optimal separation range 150-350 bp. The amplified DNA fragments 2µl
were diluted with 3µl distilled water and 2µl of loading dye making a total volume of
7µl were loaded on to the gel. Electrophoresis was carried out at 150 V for 150
minutes in 1X TAE buffer (30mM). The gel was stained with ethidium bromide (Et
Br) solution (1mg/ml) for 30min. at 350rpm and de-stained with double distilled water
for 45min. at 400rpm. The DNA banding patterns were visualized on an UV trans
illuminator and documented by using Gel Documentation System (Alpha Innotech).
Data
analysis:
Micro-satellite
bands
were
precisely
measured
by
gel
documentation system Alpha Imager EC by Alpha View software V.3.0.0.0 and
scored for each genotype. Each polymorphic DNA band at particular position on the
gel was treated as a separate character and scored as allele size. Data were then
computed with the Power Marker software V3.0 [12] to detect the percentage of
hetero-zygosity and the phylogenetic relationship among the genotypes on the basis
of the allele’s size.
Results and discussion
Genomic DNA micro-satellite markers were tested to assess the genetic diversity of
three closely resembling date palm genotypes (Table-1). The results indicated that out
of thirty two, only eight primers (25 %) failed to amplify the expected PCR fragments,
while seven primers (22 %) amplified mono-morphic banding patterns and the
remaining seventeen primers (53 %) generated polymorphic banding patterns which is
more than observed by [2] who selected seventeen markers (41%) as polymorphic,
after screening of forty one simple sequence repeats and (36%) observed by [8]. The
21 primers used in this study successfully produced clear amplified SSR band sizes
ranged from 109 bp (marker PDCAT18) to 301 bp (marker DP32), familiar to [1]
results which is ranged from 100-300 bp.
The phylogenetic diagram (Phylogram) illustrates the divergence between the
studied date palm cultivars and suggests their tree branching. Dhakki cultivar was
clustered separately from the rest of other cultivars. Mainly the phylogenetic tree
showed two major clusters, the first included two cultivars (Zarin and Gulistan)
and the second cluster contained Dhakki.
512
A dissimilarity matrix between date palm cultivars under study showed the cultivars
studied here were highly divergent at the DNA level. All the other cultivars displayed
different levels of dissimilarity but still were grouped with each other’s. It is suggested
that the variation or polymorphism of SSRs are result of polymerase slippage during
DNA replication or unequal crossing over [11].
Genetic diversity is desirable for long-term crop improvement and reduction of
vulnerability in plants to important crop diseases. Measurements of genetic diversity
can be used in breeding programs to increase the genetic variation in base populations
by crossing cultivars with a high level of genetic distance as well as for the
introgression of exotic germplasm. Molecular genetic diversity
extremely
useful
for
intellectual
property
protection,
estimates
particularly
in
are
the
determination of essential derivation. The genetic diversity estimates based on
molecular marker data may be compared to a minimum genetic distance which
indicates that two cultivars are not essentially derived [10].
513
References:
[1] Ahmed T. A. and Al-Qaradawi A. 2009. Molecular phylogeny of Qatari date palm
genotypes using simple sequence repeats markers. Biotechnology, 8: 126-131.
[2] Akkak, A., Scariot, V., Torello Marinoni, D., Boccacci, P., Beltramo, C. and Botta
R. 2009. Development and evaluation of microsatellite markers in Phoenix
dactylifera
L. and their
transferability to other Phoenix species. Biologia
Plantarum 53: 164-166.
[3] Barrow S. 1998. A monograph of Phoenix L. (Palmae: Cory-phoideae). Kew Bull.
53: 513–575
[4] Billotte, N., Marseilla, N., Brottier, P., Noyer, J. L., Jacquemoud-Collet, J. P.,
Moreau, C., Couvreur, T., Chevallier, M. H., Pintaud, J. C. and Risterucci, A. M.
2004. Nuclear micro-satellite markers for the date palm (Phoenix dactylifera
L.): characterization, utility across the genus Phoenix and in other palm genera.
Mol. Ecol. Notes, 4:256–258.
[5] Edwards, A., Civitello, A., Hammond, H. A., and Caskey, C. T. 1991. DNA typing
and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum.
Genet., 49: 746−756.
[6] Elhoumaizi, M. A., Saaidi, M., Oihabi, A. and Cilas, C. 2002. Phenotypic diversity
of date-palm cultivars (Phoenix dactylifera L.) from Morocco. Genet. Resour. Crop
Evol. 49: 483-490.
[7] Hamada, H., Petrino, M.G., Kakunaga, T., Seidman, M., and Stollar, B. D.
1984. Characterization of genomic poly (dT-dG) poly (dC-dA) sequences:
Structure, organization and conformation. Mol. Cell. Biol., 4: 2610−2621.
[8] Hamwieh, A., Farah, J., Moussally, S., Al-Shamaa, K., Elmeer, K., Khierallah, H.,
Udupa, S., Lababidi, S., Malek, J. A., Aaouine, M. and Baum, M. 2010.
Development of 1000 microsatellite markers across the date palm (phoenix
dactylifera L.) genome. Acta Hort. 882:269-277.
[9] Hanachi, S., Benkhalifa A., Khirtri, D. and Brac de la Perrière R. A. 1998.
Inventaire Varietal de la Palmeraie Algerienne. [Varietal inventory of the Algerian
palms.] CDARS, Anep Rouiba. [In French.].
514
[10] Lefebvre, V., Goffinet, B., Chauvet, J.C., Caromel, B., Signoret, P., Brand, R.,
and Palloix A., 2001. Evaluation of genetic distances between pepper inbred
lines
for
cultivar
protection purposes: comparison of AFLP, RAPD and
phenotypic data. Theor. Appl. Genet., 102, pp. 741-750.
[11] Levinson, G., and Gutman, G.A., 1987. Slipped-strand mispairing: A major
mechanism for DNA sequence evolution. Mol. Biol. Evol., 4, pp. 203−221.
[12] Liu, K. and Muse, S. V. 2005. PowerMarker: an integrated analysis environment
for genetic marker analysis. Bioinformatics, 21(9): 2128-2129.
[13] Rallo, P., Dorado, G., and Martin, A., 2000. Development of simple sequence
repeats (SSRs) in olive tree (Olea europaea L.). Theor. Appl. Genet., 101: 984−989.
[14] Saleem A. S., Ahmad K. Baloch, Musa Kaleem Baloch, Waqar A. Baloch and
Abdul Ghaffoor. (2005). Accelerated ripening of Dhakki dates by artificial
means: ripening by acetic acid and sodium chloride. J. of Food Engineering,
Vol:70(1):61-66.
[15] Vander Schoot, J., Pospiskova, M., and Vosman, B. 2000. Development and
characterization of micro-satellite markers in black poplar (Populus nigra L.).
Theor. Appl. Genet., 101: 317−322.
[16] Vosman, B., and Arens, P. 1997. Molecular characterization of GATA/GACA
micro-satellite repeats in tomato. Genome, 40: 25−33.
[17] Zehdi, S., Sakka, H., Rhouma, A., Salem, A. O. M., Marrakchi, M. and Trifi, M.
2004. Analysis of Tunisian date palm germplasm using simple sequence
repeat primers. Afr. J. Biotechnol. 3:215–219.
515
Table-1. Morphological characteristics of close resembling varieties of date palm under study.
Morphological Characteristics
Dhakki
Gulistan
Zareen
Tree height cm
800
850
820
Tree girth cm
195
184
150
Frond length cm
320
302
290
Leaf color
Dark green
Light green
Green
Leaf wax
Medium
Low-medium
Medium
Leaf flexibility
Medium
Medium
Medium
Number of pinnae per frond
175
163
155
Pinnae length cm
45
41
38
Pinnae breadth cm
2.50
2.34
2.20
Pinnae attitude
Erect
Semi erect
Erect
Number of spines per frond
20
18
16
Width and rachis cm
7.2
6.17
5.7
Dark green
Light green
Green
Number of peduncle cm
50
51
55
Length of peduncle cm
64
59
55
Number of nodes per peduncle
35
36
33
Yield per plant kg
120
113
110
Fruit length cm
6
4.22
3.9
Fruit diameter cm
4
2.41
2.25
Fruit weight gm
35
13
12
Volume of fruit
15
10
9
Fruit color at maturity Doka
stage
Yellow
Yellow
Yellow
Strand color
Yellow
Yellow
Yellow
Seed length cm
4
2.56
2.3
Seed diameter cm
1
0.97
1.23
2.5
1.7
2.4
87-88
85-86
75-77
30
29.75
32
47-50
59-60
52-55
Rachis color
Seed weight gm
Percent pulp
Moisture contents in fruit %
Total sugars in fruit %
516
Table 2: Forward and reverse primer sequences, and expected sizes of microsatellite
loci and its status of amplification of SSR loci of date palm.
Name of
Primers
Primer Sequences(5'-3')
Length
Label
Status of
amplification
10 F
ACCCCGGACGTGAGGTG
17
FAM
+
R
CGTCGATCTCCTCCTTTGTCTC
22
-
15 F
AGCTGGCTCCTCCCTTCTTA
20
NED
R
GCTCGGTTGGACTTGTTCT
19
-
16 F
AGCGGGAAATGAAAAGGTAT
20
VIC
R
ATGAAAACGTGCCAAATGTC
20
-
25 F
GCACGAGAAGGCTTATAGT
19
FAM
R
CCCCTCATTAGGATTCTAC
19
-
32 F
CAAATCTTTGCCGTGAG
17
NED
R
GGTGTGGAGTAATCATGTAGTAG
23
-
35 F
ACAAACGGCGATGGGATTAC
20
VIC
R
CCGCAGCTCACCTCTTCTAT
20
-
44 F
ATGCGGACTACACTATTCTAC
21
FAM
R
GGTGATTGACTTTCTTTGAG
20
-
48 F
CGAGACCTACCTTCAACAAA
20
NED
R
CCACCAACCAAATCAAAAAC
20
-
57 F
AAGCAGCAGCCCTTCCGTAG
20
FAM
R
GTTCTCACTCGCCCAAAAATAC
22
-
63 F
CTTTTATGTGGTCTGAGAGA
20
NED
R
TCTCTGATCTTGGGTTCTGT
20
-
70 F
CAAGACCCAAGGCTAAC
17
VIC
R
GGAGGTGGCTTTGTAT
16
-
78 F
TGGATTTCCATTGTGAG
17
FAM
R
CCCGAAGAGACGCTATT
17
-
85 F
GAGAGAGGGTGGTGTTATT
19
NED
R
TTCATCCAGAACCACAGTA
19
-
90 F
GCAGTCAGTCCCTCATA
17
VIC
R
TGCTTGTAGCCCTTCAG
17
-
93 F
CCATTTATCATTCCCTCTCTTG
22
FAM
R
CTTGGTAGCTGCGTTTCTTG
20
-
517
++
++
++
+
++
++
PDCAT10
F
CACTGCTCCTGTTGCCCTGT
20
FAM
R
TGTAGAAGGGCAGAGGACGG
20
-
PDCAT11
F
TTAGTAGACTCCCCACCGTCCT
22
NED
R
GTTTCATGGTGCTGGAGAATGAA
23
-
PDCAT12
F
CATCGTTGATTCCTAACCCCTC
22
VIC
R
GTTTAGATCTTGCATGGCAACGC
23
-
PDCAT13
F
TGTTGCCATTCACATGCTGC
20
VIC
R
GTTTGGACTAGTCCCTCCCTCCC
23
-
PDCAT14
F
TGCTGCAAATCTAGGTCACGA
21
FAM
R
GTTTACCCCTCGGCCAAATGTAA
23
-
PDCAT15
F
ACAGAGAGGTGGAGTTTTCGGAT
T
24
FAM
R
TCTTCCTTTCAAACCAGCAAGCT
23
-
PDCAT17
F
CAGCGGAGGGTGGGCCTC
18
FAM
R
GTTTCTCCATCTCCCTTTTTCTTCT
GCTACTC
32
-
PDCAT18
F
CCTAAACCTGAATGAATCAAAGC
A
24
VIC
R
ACTAACATAAGGACAGTGCTATG
TGATTG
29
-
PDCAT1
F
CTGAAATCTCTGTTCAAATCCA
22
FAM
R
GTTTGGATCTATTTGTGAGTTATT
TTCTTT
30
-
PDCAT20 TTTCAGACACATCAAGTAACGATG
F
A
25
NED
R
GTTTACGTCCACCCCAAGTTACGA
24
-
PDCAT21
F
GTGTTTGAAGATTGATTTTGTGTT
ATGAG
29
NED
R
GTTTCGAACTATAGGCATGCACAA
TAGTATATTG
34
-
PDCAT2
F
GGCCTTCTCTTCCCTAATGGGA
22
NED
518
++
++
+
++
++
+
++
++
++
++
+
++
R
GTTTCTTGCCCCTGTTCTTTCCCTC
25
-
PDCAT3
F
CAAGGATAGGTGTGATGACCACC
23
VIC
R
GTTTGTCCTTTTAACTTCTTGCTGG
AATT
29
-
PDCAT4
F
TAACGAGTCCACACAC
16
FAM
R
CTGGGTAAAGCTTATAAG
18
-
PDCAT5
F
GGCCCGTCCTTGGATTAGAG
20
VIC
R
CTACGTTGTCCCGTCAATTGG
21
-
PDCAT6
F
AATCAGGGAAACCACAGCCA
20
NED
R
GTTTAAAGCCTTCTCAAGATAGCC
TCAG
28
-
PDCAT8
F
GCTTAAGTGGTTAGTTGCCAA
21
NED
R
GTTTGGCAGAAGTATTGAAAAGTT
GA
26
-
+
++
+
++
++
(–) denotes non-amplification, (+) amplification of monomorphic band and (++) amplification
of polymorphic band of desired PCR product.
519
Table. 3: Microsatellite analysis among three famous date palm cultivars of Pakistan
Name of primer
Dhakki
Gulistan
Zerin
DP10
152
152
152
DP15
130
135
140
DP 16
-
-
-
DP 25
215/234
214
213
DP 32
288/300
294/301
293/301
DP 35
-
-
-
DP 44
-
-
-
DP 48
-
-
-
DP 57
-
-
-
DP 63
110
110
110
DP 70
-
-
-
DP 78
150
140
140
DP 85
-
-
-
DP 90
-
-
-
DP 93
256
250
257
PDCAT1
97
103
103
PDCAT2
187
193
193
PDCAT3
225
225
225
PDCAT4
155
130
103
PDCAT5
85
85
85
PDCAT6
161
155
155
PDCAT8
200
195
180
PDCAT10
132/120
132/115
133/123
PDCAT11
177
-
166
PDCAT12
144
144
144
PDCAT13
-
158
143
PDCAT14
118
122/136
111/133
PDCAT15
126
126
126
PDCAT17
130/136
124/136
134
PDCAT18
109/115
109/125
109
PDCAT20
110
-
-
PDCAT21
144
144
144
520
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٢
ا
ا&ت ارا ! " #ذ ا ن
آً وً () * .ة ا !
دورا ه& 32ا-/0د ا & ++ا+ان و 6أن 34د-/01 "0 "2د ا & ++ا+ان &
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ا 6و+!/EA UVEام ا Tا وي ا & X!/أر "Aأ Pف ) ة A 32آ /ن 32 AذW
.369 : Pو Z +0أن ه ك TAا 04ت ا " &+/دة ا6)Qل ا6 +0 3/ن ?(A "P
? .369 : - Rب && & "2]-ا "/6 \A=/ا ت واV!/Eص ا "0Vا A "; 0+
ا /اآ * ارا " #ا+رو "/E U 0 ."Eو/Eن واEت &دئ "#Vأ Pف & ا PQف اQآ ) ة
32اآ /ن و& & ";A4/ا " ا] 32 A " Kذ Wا 369 : -ذو ا(? ا 8/ +; . 6أزواج
ادئ ا 32 "&+!/ا+را cdA "Eر T ? !bا وي ?(Aا a0/آ ? ا :=6ا V/ف
Aا6)Qل ا+/دة 04ت ا" ت ا. ? 3/
521
522
OP 26
Genetic diversity analysis of the date palm (Phoenix dactylifera L.)
cultivars grown in Tunisian oasis: morphological and
molecular traits and their reports with agronomic performances
Ali FERCHICHI and Hammadi HAMZA
Institut des Régions Arides; 4119; Medenine; TUNISIE
Email : ferchichi.ali@ira.rnrt.tn
Abstract
In Tunisia, the date palm (Phoenix dactylifera L.) is the main component of oasis
agro ecosystems. In this area, the whole genetic material of date palm is exposed to
biotic and abiotic stress threatening genetic erosion. Moreover, the threat of extinction
of several cultivars is aggravated by monoclonal cultivation of major commercially
desirable cultivar especially Deglet Nour. By the present study, the genetic diversity of
date palm in the Tunisian continental oases was investigated in the objective to
elaborate and to establish an adequate methodology for cultivars identification. This
investigation is based on morphological and molecular markers. 72 morphological
characters are used; nine out of them were most stable and were selected. Their
applications on different cultivars of continental oasis (86 cultivars) showed great
phenotypic diversity. The studied cultivars are grouped into sub-populations according
to fruit characteristics: maturity period, consistency and harvest method. The cultivars
with soft fruit, early maturity and gradual harvesting are distinguished by their low
percentages of the spined midrib part and their high percentages of solitary spines.
These observations are confirmed by the Mantel test, which shows significant
correlations between selected morphological traits and fruit qualities.
The molecular analysis is conducted using seven ISSR and five SSR markers
revealing a high polymorphism. The used molecular markers corroborate the results
found by the morphological data. Globally, the phylogeny of the cultivars did not
show geographical gradient aggregation. Concerning ISSR primers, the AMOVA
showed that the subpopulation of soft cultivars and dried ones are significantly
523
differentiated. The SSR data differentiated the subpopulation of semi soft cultivars
with the others fruit consistency subpopulations.
This study shows that many morphological and molecular characters, especially the
percentage of the spined midrib part and their high percentages of solitary spines can
be used as parameter of differentiation. These characters can be also used as early
prediction of fruit consistency when palm trees are young or at the seedlings stage.
.Keywords: Phoenix dactylifera L., morphology, molecular, diversity.
Introduction
The date palm (Phoenix dactylifera L., 2n=36) is socioeconomically important in
the South of Tunisia where there is more than 4 million date palm trees speared on
40000 ha of oases. The specie dioecious nature causes a high genetic diversity
(Munier, 1981), actually we have more than 200 cultivars (Rhouma, 2005; Ferchichi
and Hamza, 2008) and the most cultivated is Deglet nour variety, the major
commercially desirable fruit.
In Tunisian oases, the high genetic diversity observed in date palm cultivars was
highlighted by many features such as morphologic and molecular tools. Hamza et al.
(2009) have selected steady morphological traits with a strong genetic control when
the environment component was discarded and can be used in several oases. Molecular
markers may provide a reliable tool for measuring genetic divergence. Several markers
have been used, random amplification polymorphism DNA (RAPD) (Sedra et al.,
1998; Trifi et al., 2000; Al-Khalifa and Askari, 2003), inter simple sequence repeats
(ISSR) (Zehdi et al., 2002), random amplified microsatellites polymorphism
(RAMPO) (Rhouma, 2008) and amplified fragment length polymorphism (AFLP)
(Rhouma, 2007). These researches revealed usually a high polymorphism among date
palm cultivars but it remains difficult to characterize cultivars. Simple sequences
repeat (SSR) was very useful to identify date palm cultivars and a high polymorphism
has been detected in date palm cultivars (Zehdi et al., 2004).
The purpose of the present study was to examine possible relations between
morphology and genetic structure (SSR markers) of Phoenix dactylifera L. of Tunisian
oases. The analyses were broken down into two parts. First, quantitative morphologic
variables and molecular markers were tested for their ability in subpopulations
524
discrimination. Second, hypotheses of correlations between genetic and morphological
distances were tested.
Materials and methods
The date palm materials were collected from the continental Tunisian oases. These
areas represent the major date palm oases of Tunisia. Cultivars were chosen for their
importance to farmers and for their good fruit quality (Ferchichi and Hamza, 2008)
and clustered into subpopulations referring to their maturity period (Early, Season and
Late) or to their fruit consistency (Soft, Semi-soft, Semi-dry, and Dry).
Morphological analysis
Nine characters were measured (Table 1), selected for their intra-genotype measure
reproducibility (Hamza et al., 2009; 2011), no significant environment plasticity and
with a strong genetic control.
Molecular analysis
Genomic DNA of each genotype was extracted from young leaves. Total nuclear
DNA was extracted according to Invisorb® Spin Plant Mini Kit (Invitek). DNA
polymorphism was detected by polymerase chain reaction (PCR) using SSR primers.
Five markers were used in this study being developed for Phoenix dactylifera L. by
Billotte et al. (2004) (Table 2) and chosen for their highly expected heterozygosis
values. The amplification products were detected using electrophoresis with 1%
agarose gels and by staining with ethidium bromide. For final analyses, 0.54µl of
amplified DNA and 5 µl of MagaBACE ET400-R DNA Size Standard were loaded.
Genotyping was carried out using an automatic DNA analyser, MegaBACE 1000.
Data analysis
The impact of selected morphological variables in subpopulations separation was
assessed by discriminant analysis (Sokal and Rohlf, 1995) and the statistical program
was SPSS 12.0 (SPSS, 2003). Molecular data were computed with Genalex program,
version 6 (Peakall and Smouse, 2006) to test the Analysis of Molecular Variance
(AMOVA) (Excoffier, 1992) using 999 re-sampled individuals. In addition, the
individual microsatellite genotypes scores were coordinated in a bi-dimensional space
by principal component analysis (PCA) by computing the genetic distance matrix. A
Mantel’s nonparametric test (Mantel, 1967) was performed to infer a possible
525
correlation between matrices of dissimilarity considering morphological variables,
fruit characteristics and genetic distances. The software package was Mantel (version
2.0) (Liedloff, 1999).
Results
In this study, we have used morphological traits and SSR markers to characterize
Tunisian date palm cultivars collected throughout continental oases. These cultivars
are clustered into subpopulations characterized by their fruit consistency or their
maturity period.
Morphological analysis
Concerning the maturity period groups, discriminant analysis showed that the two
functions represented 100% of the total variation in the data set. The first function
explained 80.6% of the total variance, and separated the early maturity cultivars from
the others (Figure 1). This function was highly correlated with PM9, EP3, EL3 and
RG3 parameters. The second discriminant analysis of fruit consistency subpopulations
showed that the first two functions explain 92.5% of total variation. The first function
accounted for 70.2% of the total variation and separated soft cultivars from the others
fruit consistency subpopulations (Figure 1). This function had a significant correlation
with PM9, EP3 and EL3 traits. The percentage of spinned midrib part (PM9) for early
and soft cultivars was significantly smaller than the other and at the same time with the
highest percentage of solitary spines (EP3). In addition, the percentage of bunch
without spikelet (RG3) and the one of the fructified spikelet (EL3) for cultivars of
early maturity subpopulation were, respectively, the lowest and the highest in
comparison with season and late maturity cultivars.
These Morphological variables have strong genetic control because they are steady
when edaphic or climatic factors change (Hamza et al., 2009). Interestingly that date
palm is characterized by these parameters because it's a genus with high phenotypic
plasticity, the reason that many farmers cannot recognize cultivars outside their oasis
and outside their restricted fruiting period (Munier, 1973; Sedra et al., 1993; 1996).
The distribution of maturity period groups or fruit consistency ones was linked to the
quantitative morphological variables. There may be several reasons for this harmony,
genetic relation and/or result of cultivar adaptation with local oases conditions.
526
Molecular analysis
The subpopulations affinities based on genetic distances were used for subsequent
principal component analysis (PCA). In the scattergram, the subpopulation separation
can be observed. Concerning the maturity period ones, earlier cultivars are also
associated and they are opposed to later maturity cultivars (Figure 2a). For the fruit
consistency, the semi soft cultivars are easily grouped (Figure 2b). AMOVA showed
no diversity among maturity period subpopulations, however significant genetic
diversity was observed among fruit consistency subpopulations (p<0.05). In fact, 7%
of the total genetic diversity was detected among fruit consistency subpopulations.
Pair-wise comparisons of populations showed that significant genetic differences
existed between semi-soft subpopulation and two others groups: semi-dry and soft
groups. This observation supports the morphologic distinction between fruit
consistency subpopulations. Another significant genetic difference was detected
between semi-soft groups and early cultivars groups.
All population differentiation detected in the field of date palm are the results of
geographic distance and the difficulty of exchanging vegetal materials (Elshibli and
Korpelainen, 2008; Zehdi et al., 2004). Another trend of differentiation was revealed
in this study, the FST values suggest significant genetic differentiation between
subpopulations. Semi-soft subpopulation was significantly differentiated with the other
fruit consistency subpopulations and with early maturity subpopulation. This genetic
segregation supposes that the Tunisian date palm may be not a unique population but a
set of populations with different origin. We cannot explain this result further because
of the lack of information about the history of Tunisian date palm culture.
The Mantel statistic tests look for relationships between distance matrices
representing the morphological variables, fruit characteristic and describing molecular
diversity. The null hypothesis of no correlation between different matrices was tested
(Table 3). Distances based on quantitative morphological traits were not correlated
with genetic distance. However, a significant positive correlation was found between
the molecular data and consistency matrix (r = 0.110; p = 0.029) but not with the
maturity matrix (r = -0.027; p = 0.382). All morphological data were correlated only
with maturity matrix (r = 0.1619; p = 0.020).
527
The no correlation between molecular and morphological variations can be
explained by the basic differences between morphologic and microsatellite markers.
The first tools are usually affected by environment when they are expressed, but
microsatellite variation is based directly on DNA sequence variation (Bruschi et al.,
2003). This hypothesis is rejected because morphologic parameters used in the setting
of this work have a strong genetic control (Hamza et al., 2009). This discrepancy
would be the result of mutation that can change a nucleotide repetition in
microsatellite pattern.
The present study reveals that Tunisian date palm population cultivated in the
continental oases are divided into subpopulations characterized by economic important
features such as fruit consistency and maturity period. Their determination is made
possible morphologically and genetically using reliable morphologic and microsatellite
markers. The findings could be utilized to improve management strategies in countries
where date palm is a significant culture. Additionally, the outcomes from this study
may assist in better conservation practices in the southern of Tunisia where many date
palm cultivars are endangered.
528
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Table 1. List of measured descriptors
Characters
Leaves
Spinned midrib part length (%)
Apical divergence angle (°)
Maximal pinnae width at the top leaf
Solitary spine number (%)
Spine length at the middle (cm)
Maximal spine angle (°)
Bunch length without spikelet (%)
Spikelet length without fruits (%)
Fruit internal cavity ratio (L/l)
Inflorescence and
fruit
Code
PM9
PM16
PN9
EP3
EP6
EP8
RG3
EL3
RB7
Table 2. The used SSR loci
Locus
Repetitive motif
He
mPdCIR010
(GA)22
0.85
mPdCIR015
(GA)15
0.76
mPdCIR032
(GA)19
0.85
mPdCIR070
(GA)17
0.78
mPdCIR093
(GA)16
0.86
He: Expected heterozygosis values according to Billote et al. (2004).
Table 3. Result of Mantel’s test of the pair-wise correlations between dissimilarity matrices.
First Matrix
Second matrix
Mantel’s r
Probability
All morphological
data
SSR
-0.007
0.496
Maturity
Fruit Consistency
Maturity period
SSR
SSR
All morphological
data
All morphological
data
-0.027
0.110
0.161
0.382
0.029*
0.020*
0.060
0.132
Consistency
* Rejection of the null hypothesis of no correlation within a 5% confidence interval
531
Maturity
Early
Late
Season
a
3
2
2
1
1
Discriminant Function 2
Discriminant Function 2
3
0
-1
-2
Fruit consistency groups
Dry
Semi soft
Semi dry Soft
b
0
-1
-2
-3
-3
-4
-2
0
2
-4
Discriminant Function 1
-3
-2
-1
0
1
2
3
Discriminant Function 1
Figure 1. Morphological differences illustrated through discriminant analysis. (a) The
scores of maturity period subpopulations. (b) The scores of fruit consistency subpopulations
Axis2 (20%)
Maturity period
Early groups
Late
Soft
Axis1 (29%)
Fruit Consistency
Dry
groups
Semi-dry
Semi-soft
a
Axis1 (29%)
Sea
son
b
Axis2 (20%)
Figure 2. Scattergram showing relative position of date palm cultivars defined by the
first two principal components based on the genetic distance of the five microsatellite loci.
532
:ص
533
534
PP 09
Protocol for in vitro regeneration of plantlets from root tip
explants differentiated from shoot tip explants and genetic
stability of date palm cultivars.
Essam Abdel-Rahman Madboly 1and Amina Hamed Gomaa٣
1 The Central Laboratory for Date Palm Researche, Agriculture Research Center, Giza, Egypt.
esammadboly@yahoo.com. or dresammadboly@gmail.com
3 Department of Pomology, Faculty of Agriculture, Cairo University,Giza, Egypt.
am.gomaa@gmail.com
Abstract
The present study was demonstrated a protocol for in vitro regeneration of plantlets
from root tip explants differentiated from shoot tip explants and genetic stability of
date palm (Phoenix dactylifera L.) cultivars Sewi and Zaghloul . Root tip explants
cultured on two callus induction and differentiation media M1 and M2 .The first culture
medium (M1) consists of modified MS basal nutrient medium supplemented with 100
mg/l 2, 4-D + 3 mg/l 2iP and the second (M2) consists of modified MS basal nutrient
medium supplemented with 10 mg/l 2, 4-D + 3mg/l 2iP. Explants cultured on medium
M2 showed the highest significant callus induction percentage, growth vigor of
embryogenic callus and number of individual somatic embryo. Increasing the
subculture number gradually to 7 increased callus induction percentage, value of
growth vigor of embryogenic callus, leaves number/explant, shoot length (cm) and
root length (cm). Increasing the subculture number from 7to 8 increased number of
normal individual somatic embryos and root number/explant. Sewi cultivar showed
higher value of swelling degree , callus induction percentage, value of growth vigor of
embryogenic callus and number of individual somatic embryo and root length(cm)
compared with those obtained from Zaghloul cultivar. Identification through RAPDPCR technique cleared that plantlets regenerated from normal somatic embryo
developed from root tip explants of date palm cultivars showed higher genetic stability
comparing with the mother plants
Key Words: Protocol ,Date Palm (Phoenix dactylifera L.), In vitro, Regeneration ,
Root tip explants, Somatic embryogenesis, Genetic stability
535
Introduction
Date Palm has been called the “tree of life “because of its indispensable utilization
in the economy and domestic life of inhabitants of palm – growing countries. Date
palm is considered one of the most important commercial crops in the Middle East and
Arab World. Moreover, the date palm tree successfully tolerates extremely adverse
environmental conditions, including drought, high temperature and salinity, which are
the peculiar criteria of desert lands (Barreveld, 1993).
Date palm is a monocotyledonous and dioecious fruit tree with a high degree of
heterozygosity. Conventional vegetative propagation, made through offshoots, is very
slow and laborious. In addition, the number of offshoots is complicated, time
consuming and the percentage of offshoots successfully established in soil is highly
variable (30 – 80 %) and cultivar dependent (Saaidi et al., 1979).
The development of micropropagation protocols is very important for this based on
organogenesis and somatic embryogenesis. Organogenesis in date palm has a low
efficiency due to the low number of explants that respond in vitro, the long time required
for the imitation phase, the low multiplication rate, and the strong influence of the variety
(Beauchesne, 1982). Shoot tip and bud excised from offshoots (Tisserat, 1979).
Unconventional vegetative micropropagation, made through callus formed at the
root tip region of young date palm seedlings produced leaves and shoots (Smith 1975).
On the other hand, Schroeder (1970) observed that root pieces of date palm cultured in
vitro developed secondary roots but did not produce shoots. Eeuwens and Blake
(1977) found that roots excised from in vitro cultured explant of date palms continued
to grow and produce lateral roots when subcultured on liquid media. Sharma et al.,
(1980) observed no growth in cultured date palm roots, and the explants experienced
severe browning and death within the first few weeks of culture. Zaid and Tisserat
(1983) obtained some callus from roots of date palm; such callus failed to exhibit any
morphogenic response .Micropropagation from root tip explants would be preferable
to the single shoot tip that is available for explants, high average from explants and
may be free from virus(Pierik, 1987).Madboly(2007)used New protocol for
regeneration of plantlets from root explants of date palm cultivars. The roots segment
explants were excised as two different explants, the first one root tip (RT) and the
536
second without root tip (RS) .Various molecular techniques are being practiced for
fingerprinting such as random amplified polymorphic DNA (RAPD). (Fukuoka et al.,
1992). Optimized conditions for regenerating random amplified polymorphic DNA
(RAPD) markers using the polymerase chain reaction (PCR) for date palm. They
discussed the importance of technique optimization to obtain reproducible and
applicable RAPD patterns. (Aitchitt et al., 1993) . Madboly(2007)cleared that plantlets
regenerated from somatic embryo developed obtained from root tip explants (RT) and
root segment without tip explants (RS) showed higher genetic stability comparing with
the mother plants.
The major objectives of the present study was demonstrated a protocol for in vitro
regeneration of plantlets from root tip explants differentiated from shoot tip explants
and genetic stability of date palm cultivars Sewi and Zaghloul .
Material and methods
This study was carried out during of 2008-2010 in Laboratory for Date Palm
Researches, Agriculture Research Center (ARC), Giza, Egypt.
A . Plant Material
In this experiment healthy offshoots of date palm (Phoenix dactylifera L.) cultivars
Sewi(semi-dry cultivar) grown at Al-Wahaat El-Baharia in Giza, Zaghloul (soft
cultivars) grown at Rashid in Bihara were selected from mother trees. The young
offshoots were of about 3-5 years; ranging in weight from 5 - 7 kg and about 50 - 80
cm in length.
B . Preparation of explants and surface sterilization :
The adventitious roots, fibrous sheath and leaves of selected young offshoot were
removed acropetally with the help of pruning scissors and serrated knife. The shoot tip
was cleaned by a brush, then soaked in 1: 1000 v/v benlate fungicide solution for 60
min and then rinsed by tap water. The shoot tip was handled very carefully for
removing soft inner leaves from the shoot terminal part after peeling away all the
mature leaves to obtain a shoot apex which was 2 – 4 cm in length and about 1 – 2 cm
in diameter as shown in Fig. (1). The shoot apex was immediately washed with
running tap water for 1 – 2 hours. All excised shoot apex’s were soaked temporarily in
537
an anti oxidant solution (150 mg/l ascorbic acid and 100 mg/l citric acid) for 30 min.
prior to surface sterilization.
Under aseptic conditions, shoot apex’s were soaked in 70% ethanol alcohol solution
for 30 seconds, followed by immersion in (1.0 g/l) of mercuric chloride for 5 min and
thoroughly washed with sterilized distilled water for one-time. After that additional
leaf primordial were removed from sterilized explants and then transferred to double
surface sterilization by commercial Clorox (5.25 % sodium hypochlorite NaOCl) plus
2 drop of Tween 20 per 100 ml solution ,the first one by 40% Clorox for 20 min with
rotary agitation, rinsed one times with sterilized distilled water and the second one by
60% Clorox for 20 min with rotary agitation, rinsed three times with sterilized distilled
water. Under aseptic conditions, outer soft leaves were removed to obtain a terminal
portion of the shoot. Two type of explants were excised in this experiment. The first
one was shoot tip explant (S T), shoot tip 5 – 10 mm in length (composed of apical
meristem, sub apical tissue) sliced longitudinally into 4 pieces, and the second explant
was leaf primordial explant (Lp) 5 – 10 mm in length(composed of several leaf
primordial) sliced longitudinally into 6-8 pieces .
C . Media preparation:
The basal medium used in this experiment was formula of Murashige and Skoog
(1962) basal nutrient medium with the following modification in mg/l: 170 NaH 2PO4,
2H2O; 200 glutamine; 40 adenine sulfate; 0.4 thiamine-HCl. The basal nutrient
medium was supplemented with mg/l: 3000 activated charcoal; 30000 sucrose; and
6000 agar as described by Tisserat (1984).
The sterilized explants of each explant type (ST and Lp) were cultured on nutrient
medium which consists of the previous modified MS basal nutrient medium
supplemented with 100 mg/l 2, 4-D + 3 mg/l 2iP as described by Matar (1986).
The pH of all culture media was adjusted to 5.8 ± 0.1 prior to the addition of agar,
and then 35 ml of medium was dispensed into small jars (150ml). The culture jars
were sealed with caps of polyvenylpropelin. The jars were autoclaved at 121 °C, and
1.1 kg/cm2 for 20min. One sterilized explant was cultured individually in each culture
jar. The explant was cultured horizontally with a good contact with the surface of the
culture medium . Each treatment consists of 3 replicates , each replicate consists of 6
538
culture jars , and each jar contained one explant ; culture jars were incubated in a
temperature controlled room at 27 °C ± 1 under complete darkness condition.
D . Callus induction
Uncontaminated and survived explants of each explant type (ST and Lp) were
differentiated to root organogenesis direct after 8 weeks in subculture media .this root
differentiated were excised to used explants type in this experiment. Root tip explants
were excised 1-2 cm in length including the root tip transferred and sub cultured into
corresponding fresh medium every 6-8 weeks interval for at least 8 subcultures.
Data of root tip explants type were calculated in every treatment at the end of each
subculture for at least 6 subcultures (6-8 weeks for each one) . Data were taken as the
average per explant for callus induction degree value. The average of swelling degree and
callus induction percentage were scored visually as follows: (according to Pottino, 1981).
Negative results (-) = 1
below average results (+) = 2
Average results (++) = 3
Good results (+++) = 4
E . Embryogenic callus and somatic embryo development:
White friable embryonic nodular callus developed from root tip explants type and
each subculture number (6,7 and 8) were transferred and cultured on differentiation
medium which consists of MS basal nutrient medium supplemented with 200 mg/l
glutamine, 0.4 mg/l thiamine-HCl mg/l and 0.1mg/l NAA (according to Mater, 1986).
Each treatment consists of 3 replicates and each replicate consists of 3 jars each jar
(150ml) each contained 20 ml of prepared medium. One pieces of white friable
embryogenic nodular callus (of about 0.1 g in weight and 1-2 mm in diameter ) was
cultured on each culture jar. All culture jars were incubated in growth room at 27 ±1°C
under 16 hrs daily exposure to low light intensity of 1000 lux illumination (Mater, 1986).
White friable embryogenic nodular callus developed from root tip explants type and each
subculture number (6,7 and 8) were transferred and sub cultured onto fresh differentiation
medium ( mentioned above) every 6-8 weeks intervals for at least two subcultures.
Data of root tip explants type and each subculture number (6,7 and 8) were collected
at the end of each subcultures every 6-8 weeks intervals for at least two subcultures.
The parameters were tabulated as average of the two subcultures as follows:
- growth vigor degree value of embryogenic callus . (according to Pottino, 1981).
539
- number of normal individual somatic embryo.
These shapes are described as follows:
Normal individual somatic embryo: (Individual embryo): Long of individual
embryoids were 5-10mm as described by Mater (1986). Small seedling with primary
root and shoot as described by George (1993). Mature embryos longer than 5mm as
described by Veramendi and Navarro (1997).
F . Germination of somatic embryos and development of plantlets:
The normal somatic embryos(Individual) formed from embryogenic callus
developed from white friable embryonic nodular callus which obtained from root tip
explants type and each subculture number (6,7 and 8) cultured onto differentiation
medium for at least two subcultures(6-8 weeks for each one) were transferred and
cultured onto germination and development medium which consists of MS basal
nutrient medium supplemented 200 mg/l glutamine, 0.4 mg/l thiamine-HCl and 0.1
mg/l NAA (according to Mater, 1986).
Each treatment included 3 replicates and each replicate included 3 jars (150ml) each
contained 20 ml of prepared medium. One normal somatic embryo (Individual or
multi-somatic embryo) was cultured on each culture jars. All culture jars were
incubated in growth room at 27±1°C under 16 hrs /day exposure to moderate light
intensity of 2000 lux illumination.
The normal somatic embryos(Individual) of root tip explants type and each
subculture number (6,7 and 8) were repeatedly subcultured onto fresh germination
and development medium every 4 weeks intervals for at least two subcultures. For
various shapes of embryos the following data were recorded in every treatment at the
end of the second subcultures. The parameters were presented as the average per
plantlet as follows: - leaves number of normal individual somatic embryo.
- shoot length (cm)of normal individual somatic embryo .
- root number of normal individual somatic embryo .
- root length (cm)of normal individual somatic embryo .
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G - Genetic stability of mother plant and plantlets regenerated from normal
somatic embryos developed from, subculture number 6 and 8 of date palm
cultivars Sewi and Zaghloul:
DNA-based tests for date palm identification include techniques such as RAPD
(Random Amplified Polymorphic DNA) technique .RAPD is possibly the simplest test
of all recently applied DNA-based tests for date palm identification. The RAPD
technique consists of the production of duplicate of segments of plant DNA and these
DNA segments are several million-fold amplified in a reaction called Polymerase
Chain Reaction (PCR). To duplicate the plant DNA, sets of very short nucleotides,
called primers that consist of the basic building blocks of DNA are required. These
primers, which bind selectively to the master-copy of plant DNA are commercially
available from several biotechnology companies. A thermo stable enzyme, called Taq
DNA polymerase, which carries out rapid temperature fluctuation cycles, is necessary
for the duplication process and consequently several million fold amplification of
DNA segments are obtained. Finally, amplified DNA segments are separated on either
agarose or poly acrylamide gels and visualized by staining. The detected
polymorphism between individual plants may result from either a DNA sequence
difference in the binding site for the primer on the plant DNA or a deletion of parts of
the plant DNA., both processes are visible as the absence of a particular RAPD band
after separation on a gel system, which allows the differentiation of cultivars.
The young leaves of mother plant, the young leaves of plantlets regenerated from
normal somatic embryos obtained from subculture number (6 and 8) in rooting stage
were collected randomly and used for DNA isolation from date palm cultivars Sewi
and Zaghloul. Genomic DNA extraction and the method of Gel electrophoresis as
described by Maniatis et al.(1982). RAPD-PCR reactions were conducted using thirtysix arbitrary 10-mer primers with the sequences indicated in Table (1).
H - Experimental design
The experiments were performed utilizing complete randomized block design with
factorial arrangement. The results were subject to analyzed for variance and the means
were compared using L.S.D at 5% level according to Snedecor and Cochran (1972).
541
Results and discussion
1. swelling degree value :
Data in Table (2) and Fig.(1) showed the effect of subculture number and culture
medium on swelling degree value of date palm cultivars Sewi and Zaghloul.
Concerning the effect of subculture number, data indicated that ,the highest significant
value of swelling degree (2.49 and 2.43, respectively)was noticed during subculture
number 6 and 7 respectively without significant differences in between .The lowest
significant value of swelling degree (1.45)was noticed during subculture number
1.Increasing the subculture number from 2 to 6 increased significantly degree value of
swelling(1.70,1.92,2.12 and 2.25,respectively ).The value of swelling degree decreased
significantly by increasing the subculture number to 8(2.14).
*Values determined as described by Pottino 1981
With regard to the effect of culture medium data revealed that, explant cultured on
M2 culture medium showed the higher value of swelling degree (2.31) compared with
the value of swelling degree (1.82) of explant cultured on
M1culture medium.
Concerning the effect of date palm cultivars , it could be noticed that, Sewi cultivar
resulted the higher significant value of swelling degree (2.27) compared with Zaghloul
cultivar which showed the lower significant value of swelling degree (1.82).
These findings are in agreement with those of Smith (1975) who reported that callus
was formed at the root region of young date palm seedlings, Zaid and Tisserat (1983)
who obtained some callus from roots of date palm Ong (1977) and Martin et al., (1972
a ,b) who observed root elongation and callus from root explant cultured in vitro in oil
palm Mandal et al., (1994) who mentioned that callus formation was highest, when
root explant of Carica paparya cultured in half MS medium and Handro et al., (1988)
who reported that callus was induced from root explant of Brazilian medicinal plant in
vitro. Zaid (2003) mentioned that increasing the number of subcultures increased the
values of callus initiation of all cultivars of date palm under investigation. Also,
Madboly (2007)who mentioned that increasing the subculture number gradually
from 3 to 7 increased the callus induction.
2. Callus induction percentage:Table (3) and Fig.(1) showed the effect of subculture number and culture medium
542
on percentage of callus induction percentage of date palm cultivars Sewi and Zaghloul.
Concerning the effect of subculture number ,data showed that, callus induction
percentage increased significantly by increasing the subculture number to7
(36.33%).While, increasing the subculture number from 7 to 8 decreased significantly
the callus induction percentage from 36.33% to 33.33%, respectively. The lowest
significant on callus induction percentage (8.50 %)was noticed during subculture
number 1.Increasing the subculture number from 2 to 6 increased significantly on
callus induction percentage (15.42%,18.17%,22.83%,27.58%and34.17%,respectively).
Regarding the influence of culture medium on callus induction percentage, explants
cultured on M2 culture medium showed the best significant results (26.79%) compared
to callus induction percentage (22.29%) showed from explants cultured on M 1 culture
medium. Concerning the effect of date palm cultivars, the best significant callus
induction percentage (28.35%) was obtained from Sewi cultivar compared with the
callus induction percentage obtained from Zaghloul cultivar (20.73%).
3. Growth vigor degree value of embryogenic callus:Data tabulated in Table(4) and Fig.(1) indicated the effect of subculture number
and culture medium of callus induction percentage of date palm cultivars Sewi and
Zaghloul of white friable embryonic nodular callus on values of growth vigor degree
value of embryogenic callus developed from white friable embryonic nodular callus
cultured on differentiation medium for2 subcultures 6-8 weeks for each. Regarding to
the effect of subculture number ,data indicated that, the highest significant value (1.50)
of growth vigor of embryogenic callus was observed when white friable embryonic
nodular callus developed during subculture number 7.This value decreased
significantly when white friable embryonic nodular callus developed in subculture
number 8 and 6 for each(1.42 and 1.43, respectively) without significant difference in
between. Concerning the effect of culture medium ,data showed that, explants cultured
on M2 culture medium showed the best significant results (1.62) compared to the
values of growth vigor of embryogenic callus (1.28) showed from explants cultured
on M1 culture medium.
Concerning the effect of date palm cultivars, Sewi cultivar was the best significant
value of growth vigor of embryogenic callus (1.58) compared with those developed
543
from Zaghloul cultivar (1.32)when transferred and cultured on differentiation medium
for 2 subcultures 6-8 weeks for each.
These findings are in agreement with those of Madboly (2007)who obtained that
Increasing the subculture number from 5 to 7 increased the normal somatic embryo
percentage of date palm cultivar Sewi.
4. Number of individual somatic embryo:Normal individual somatic embryo as described by Mater (1986) and George
(1993). Data tabulated in Table (5) and Fig.(1) showed the effect of subculture
number and culture medium of white friable embryonic nodular callus on number of
individual somatic embryo developed from embryogenic callus of date palm cultivars
Sewi and Zaghloul cultured on differentiation medium for 2 subcultures 6-8 weeks for
each. With regard to the effect of subculture number ,data indicated that, the number
of individual somatic embryo obtained from subcutlure number 7 and 8 cultured on
differentiation medium were the highest significantly (5.17 and 5.08 embryo/ explant,
respectively). This number reduced significantly when white friable embryonic
nodular callus obtained from subcutlure number7 were (5.17 embryo/ explant )
obtained from subcutlure number 6 (2.58 embryo/ explant ) with significant difference
inbtween. On the other hand, the effect of culture medium data revealed that, explant
cultured on M2 culture medium showed the higher number of individual somatic
embryo (5.22 embryo/ explant) compared with the explant cultured on M1culture
medium(3.33 embryo/ explant). Regarding, the influence of date palm cultivars, the
data indicated that, Sewi cultivar formed the higher significant number of individual
somatic embryo (6.17 embryo / explant) than those formed from Zaghloul cultivars
(2.39 embryo / explant) when transferred and cultured on differentiation medium for 2
subcultures 6-8 weeks for each.
5 . Shoot length(cm) of plantlet developed from normal individual somatic embryo:The results in Table (6) and Fig.(1) showed the effect of subculture number and
culture medium on shoot length(cm) of plantlet of date palm cultivars Sewi and
Zaghloul developed from normal individual somatic embryo developed from
embryogenic callus cultured on germination and development medium for 2
subcultures 4 weeks for each. Regarding to the effect of subculture number, data
544
showed that, the highest significant increments in shoot length/plantlet (7.21 cm)
developed from subculture number 7.This length was reduced significantly when
plantlet developed from normal individual somatic embryo developed from
embryogenic callus developed from white friable embryonic nodular callus obtained
from subculture number 6 and 8(4.13 and 4.54 cm, respectively) without significant
difference in between. Concerning the effect of culture medium, data showed that, the
shoot length of plantlet developed from normal individual somatic embryo was not
affected significantly by culture medium M1 and M2 were (4.64 and 4.61 cm,
respectively). Regarding to the effect of date palm cultivars, data showed that , the
shoot length of plantlet obtained from Sewi and Zaghloul cultivars were (4.67 and 4.58
cm, respectively) without significant difference in between when transferred and
cultured on germination and development medium for 2 subcultures 4 weeks for each.
6. Leaves number of plantlet developed from normal individual somatic embryo:The results in Table (7) and Fig.(1) showed the effect of subculture number and
culture medium of date palm cultivars Sewi and Zaghloul on leaves number of plantlet
developed from normal individual somatic embryo developed from embryogenic
callus cultured on germination and development medium for 2 subcultures 4 weeks for
each. Regarding the effect of subculture number, data showed that ,the highest leaves
number/plantlet (3.08 leaves/plantlet) developed from subculture number 7. No
significant difference noticed between the leaves number of plantlet developed from
subculture number 6 and 8(2.71 and 2.50 leaves/plantlet, respectively).With regard to
the effect of culture medium, data indicated that ,the leaves number of plantlet
obtained from explants cultured on M1 and M2 culture medium were (2.81 and 2.72
leaves/plantlet, respectively) without significant difference in between .With regard to
the effect of date palm cultivars ,data indicated that ,number of leaves/plantlet affected
by Sewi cultivars
(2.97 leaves/plantlet) was significantly higher as compared to
number of leaves/plantlet developed from Zaghloul cultivar (2.56 leaves/plantlet)
when transferred and cultured on germination and development medium for
2
subcultures 4 weeks for each.
7. Root length (cm) of plantlet developed from normal individual somatic embryos
The results in Table (8) and Fig.(1) showed the effect of subculture number and
545
culture medium of date palm cultivars Sewi and Zaghloul on root length(cm)of plantlet
developed from normal individual somatic embryos developed from embryogenic
callus cultured on germination and development medium for 2 subcultures 4 weeks for
each. Regarding to the effect of subculture number, data revealed that ,the highest
significant differences were observed in root length of plantlet were developed from the
subcultures number 7 (4.96 cm). This length was reduced significantly when plantlet
developed from subculture number 6 and 8(4.36 and 4.58 cm , respectively ) without
significant difference in between. Regarding the effect of culture medium, data showed
that, the root length of plantlet obtained from explants cultured on M1 and M2 culture
medium were (4.67 and 4.60 cm, respectively) without significant difference in
between. Concerning the effect of date palm cultivars, data clearly showed that, root
length of plantlet was affected by explant type since the root number of plantlet
developed obtained from Sewi cultivar (4.89 cm) was significantly higher as compared
with those obtained from Zaghloul cultivars (4.38) cm when transferred and cultured on
germination and development medium for 2 subcultures 4 weeks for each.
These findings are in agreement with those of Madboly (2007) who reported that
Plantlets regenerated from normal somatic embryos showed improvement in leaves
number, shoot length (cm), root number and root length (cm) by increasing subculture
number of embryogenic callus from 5 to 7 of date palm cultivars .
8 -Genetic stability of mother plant and plantlets regenerated from normal
somatic embryos developed from, subculture number 6 and 8 of date palm
cultivars Sewi and Zaghloul:
The young leaves of mother plant of date palm cultivars Sewi and Zaghloul and the
young leaves of plantlets regenerated from normal somatic embryos obtained from
subculture number 6 and 8 during rooting stage were collected randomly and used for
DNA isolation from date palm cultivar. Five arbitrary decamer oligonucleotide
primers were used; OP-B05, OP-B06, Op-B08, OP-B09 and OP-B10. As a general
molecular study on RAPD-PCR for mother plant and plantlets developed from normal
somatic embryos obtained from subculture number 6 and 8.
All these similarities showed in Table (9 and 10) and Fig.(2 and 3). In Sewi cultivar,
the higher similarity between mother plants , plantlet developed from normal somatic
546
embryos obtained from subculture number 6 was 98.00 % and between mother plants ,
plantlet developed from normal somatic embryos obtained from subculture number 8
was 93.00 %. Similarity decreased by increasing the subculture number from 6 to 8
was (90.00 %).
In Zaghloul cultivar, the higher similarity between mother plants , plantlet
developed from normal somatic embryos obtained from subculture number 6 was
94.00 % and between mother plants , plantlet developed from normal somatic embryos
obtained from subculture number 8 was 90.00 %. Similarity decreased by increasing
the subculture number from 6 to 8 was (87.00 %).
From all previous results, the entire similarity matrix of mother plants and plantlets
regenerated from normal somatic embryo developed from embryogenic callus of date
palm Sewi and Zaghloul cultivars obtained from mother plant and subculture number 6
and 8 showed higher genetic stability .
This is agree with Isabel et al., (1993) who reported that RAPD technique provides a
faster and easier approach to detect extensive polymorphism and needs very small amounts
of genomic DNA and this technique can be routinely applied for varietal identification.
Also, RAPD technology appears very effective for identifying accessions of date
palm, although the overall exhibited polymorphism cultivated species (He and Quiros,
1991; Yang and Quiros, 1993and Wolff and Van Run 1993).
Aitchitt et al., 1993 and Coriquel and Mercier, 1994 suggested that RAPD markers
should therefore be of high value for date palm germplasm characterization and
genetic maintenance. These results also are agree with Adawy et al., (2002) who used
RAPD technique to assay the genetic and found that all the tested primers exhibited
intervarietal polymorphism as well as inter varietal variation. El-Bahr (2007) who
showed shoot tip explants of date palm. cvs. Zaghloul, Samany Amhat the best results
of both in vitro and ex vitro results. The RAPD profiles of tissue culture derived
plantlets revealed high similarity to mother tree.
Madboly (2007)who obtained that entire similarity matrix higher between mother
plant (subculture number 0) and plantlets regenerated from normal somatic embryos
developed from root tip explants (RT) was 97.00 %. The lower similarity between
mother plant (subculture number 0) and plantlets regenerated from normal somatic
547
embryos developed from root segment without tip explants (RS) was 88.70 %. The
similarity between plantlets regenerated from normal somatic embryos developed from
root tip explants (RT) and plantlets regenerated from normal somatic embryos
developed from root segment without tip explants (RS) was 95.30 % .The genetic
stability between mother plant, plantlets developed from root tip explants (RT) and
plantlets developed from root segment without tip explants (RS) was higher .
548
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551
Table 1. Random primer codes and their sequences for RAPD-PCR analysis.
Primer code
Sequence
OP- B 05
OP- B 06
OP- B 08
5' - TGCGCCCTTC - 3'
5' - TGCTCTGCCC - 3'
5' - GTCCACACGG- 3'
OP- B 09
OP- B 10
5' - TGGGGGACTC- 3'
5' - CTGCTGGGAC -3'
Table 2. Effect of subculture number and culture medium on swelling degree
value of date palm cultivars Sewi (S)and Zaghloul(Z).
Medium (B)
M1
Subculture no.
(A)
M2
Cultivar (C )
Cultivar (C )
Mean
(A)
S
Z
Mean
S
Z
Mean
1
1.40
1.20
1.30
1.70
1.50
1.60
1.45
2
1.67
1.37
1.52
2.13
1.63
1.88
1.70
3
1.90
1.53
1.72
2.37
1.87
2.12
1.92
4
2.10
1.67
1.88
2.57
2.13
2.35
2.12
5
2.20
1.80
2.00
2.70
2.30
2.50
2.25
6
2.47
1.97
2.22
2.93
2.60
2.77
2.49
7
2.33
1.80
2.07
2.97
2.60
2.78
2.43
8
2.10
2.57
1.83
2.77
2.13
2.45
2.14
Mean (B)
Mean ( C)
Mean (B xC)
M1
M2
1.82
2.31
S
Z
2.27
1.85
M1 x S
M1 x Z
M2 x S
M2 x Z
2.02
1.61
2.52
2.10
Mean separation by L.S.D. at 0.05
A=0.11
B=0.05
C =0.05
AxB=0.16
AxC=0.16
BxC=0.08
AxBxC=0.22
552
Table 3. Effect of subculture number and culture medium on percentage of
callus induction percentage of date palm cultivars Sewi (S)and Zaghloul(Z).
Medium (B)
Subculture
no. (A)
M1
M2
Cultivar (C )
Cultivar (C )
Mean
(A)
S
Z
Mean
S
Z
Mean
1
8.33
5.33
6.83
11.67
8.67
10.17
8.50
2
16.00
11.33
13.67
21.33
13.00
17.17
15.42
3
18.33
13.00
15.67
23.33
18.00
20.67
18.17
4
22.33
16.00
19.17
29.67
23.33
26.50
22.83
5
27.00
22.67
24.83
32.00
28.67
30.33
27.58
6
37.67
26.67
32.17
40.00
32.33
36.17
34.17
7
41.33
27.67
34.50
43.67
32.67
38.17
36.33
8
40.00
23.00
31.50
41.00
29.33
35.17
33.33
Mean (B)
Mean ( C)
Mean (B xC)
M1
M2
22.29
26.79
S
Z
28.35
20.73
M1 x S
M1 x Z
M2 x S
M2 x Z
26.38
18.21
30.33
23.25
Mean separation by L.S.D. at 0.05
A=1.46
B=0.73
C =0.73
AxB=2.06
AxC=2.06
BxC=1.03
AxBxC=2.91
553
Table 4. Effect of subculture number and culture medium on growth vigor degree
value of embryogenic callus cultured on differentiation medium of date palm
cultivars Sewi (S)and Zaghloul(Z).
Medium (B)
M1
Cultivar (C )
S
Z
Mean
S
1.33
1.33
1.33
1.70
6
1.33
1.20
1.27
2.00
7
1.30
1.17
1.23
1.83
8
M1
Mean (B)
1.28
S
Mean ( C)
1.58
M1 x S
M1 x Z
Mean (B xC)
1.32
1.23
Subculture
no. (A)
Mean separation by L.S.D. at 0.05
A=0.06
B=0.04
AxB=0.12
AxC=0.12
AxBxC=0.18
*Values determined as described by Pottino 1981
M2
Mean
(A)
Cultivar (C )
Z
Mean
1.37
1.53
1.43
1.47
1.73
1.50
1.42
1.37
1.60
M2
1.62
Z
1.32
M2 x S
M2 x Z
1.84
1.40
C =0.04
BxC=0.09
Table 5. Effect of subculture number and culture medium on number of individual
normal somatic embryo of date palm cultivars Sewi (S)and Zaghloul(Z).
Medium (B)
M1
Cultivar (C )
S
Z
Mean
S
2.33
1.00
1.67
4.67
6
6.67
1.67
4.17
9.00
7
6.33
2.00
4.17
8.00
8
M1
Mean (B)
3.33
S
Mean ( C)
6.17
M1 x S
M1 x Z
Mean (B xC)
5.11
1.56
Subculture
no. (A)
Mean separation by L.S.D. at 0.05
A=0.75
B=0.61
AxB=1.06
AxC=1.06
AxBxC=1.50
M2
Mean
(A)
Cultivar (C )
Z
Mean
2.58
2.33
3.50
5.17
3.33
6.17
4.00
6.00
5.08
M2
5.22
Z
2.39
M2 x S
M2 x Z
7.22
3.22
C =0.61
BxC=0.87
554
Table 6. Effect of subculture number and culture medium on number of shoot length (cm)
of individual normal somatic embryo of date palm cultivar Sewi (S)and Zaghloul(Z).
Medium (B)
M1
M2
Mean
(A)
Cultivar (C )
Cultivar (C )
S
Z
Mean
S
Z
Mean
4.00
4.17
4.08
4.17
4.17
4.17
4.13
6
5.17
5.17
5.17
5.17
533
5.25
5.21
7
4.83
4.50
4.67
4.67
4.17
4.42
4.54
8
M1
M2
Mean (B)
4.64
4.61
S
Z
Mean ( C)
4.67
4.58
M1 x Z
M2 x S
M2 x Z
M1 x S
Mean (B xC)
4.67
4.61
4.67
4.56
Subculture
no. (A)
Mean separation by L.S.D. at 0.05
A=0.80
B=N.S
AxB=0.30
AxC=0.30
AxBxC=0.52
C = N.S
BxC= N.S
Table 7. Effect of subculture number and culture medium on number of leaves of
individual normal somatic embryo of date palm cultivars Sewi(S)and Zaghloul(Z).
Medium (B)
M1
Subculture
no. (A)
M2
Cultivar (C )
Cultivar (C )
Mean
(A)
S
Z
Mean
S
Z
Mean
6
2.67
2.83
2.75
3.00
2.33
2.67
2.71
7
3.33
2.83
3.08
3.50
2.67
3.08
3.08
8
2.67
2.50
2.58
2.67
2.17
2.42
2.50
Mean (B)
Mean ( C)
Mean (B xC)
M1
M2
2.81
2.72
S
Z
2.97
2.56
M1 x S
M1 x Z
M2 x S
M2 x Z
2.89
2.72
3.06
2.39
Mean separation by L.S.D. at 0.05
A=0.34
B=N.S
AxB=0.48
AxC=0.48
AxBxC=0.68
C =0.28
BxC=0.39
555
Table 8. Effect of subculture number and culture medium on roots length of
individual normal somatic embryo of date palm cultivar Sewi (S)and Zaghloul(Z).
Medium (B)
M1
M2
Mean
(A)
Cultivar (C )
Cultivar (C )
S
Z
Mean
S
Z
Mean
4.50
4.17
4.33
4.33
4.43
4.38
4.36
6
5.33
4.50
4.92
5.17
4.83
5.00
4.96
7
5.17
4.33
4.75
4.83
4.00
4.42
4.58
8
M1
M2
Mean (B)
4.67
4.60
S
Z
Mean ( C)
4.89
4.38
M1 x S
M1 x Z
M2 x S
M2 x Z
Mean (B xC)
5.00
4.33
4.78
4.42
Subculture
no. (A)
Mean separation by L.S.D. at 0.05
A=0.31
B= N.S
C =0.25
AxB=0.43
AxC=0.43
BxC=0.35
AxBxC=0.61
Table 9. Similarity matrix between mother plant, plantlet from subculture
number 6 and plantlet from subculture number 8 of date palm cv. Sewi.
Similarity
matrix
mother plant
Plantlet from
subculture
number 6
plantlet from
subculture
number 8
mother
plant
100.00
98.00
93.00
plantlet
from
subculture
number 6
98.00
100.00
90.00
plantlet
from
subculture
number 8
93.00
90.00
100.00
556
Table 10. Similarity matrix between mother plant, plantlet from subculture
number 6 and plantlet from subculture number 8 of date palm cv. Sewi.
Similarity
matrix
mother plant
Plantlet from
subculture
number 6
plantlet from
subculture
number 8
mother
plant
100.00
94.00
90.00
plantlet
from
subculture
number 6
94.00
100.00
87.00
plantlet
from
subculture
number 8
90.00
87.00
100.00
ϱ
Ϯ
ϯ
ϰ
Fig .1. Protocol for in vitro regeneration of plantlets from root tip explants differentiated
from shoot tip explants of date palm (Phoenix dactylifera L.) cultivars Sewi and Zaghloul.
1- Shoot tip explants of date palm cultivars .
2- root tip explants differentiated from shoot tip explants.
3- Embryogenic callus .
4- Normal somatic embryo development.
5- Shoot length (cm) ,Leaves number - root number and root length (cm)of plantlet
regenerated from normal individual somatic embryo.
557
M
1
2
3
4
5
6
Figure (2): RAPD-banding pattern of date palm(Phoenix dactylifera L.); Sewi cultivar ;
(lanes; 1, 2and 3) and; Zaghloul cultivar (lanes; 4, 5 and 6) amplified with primer; OPB08, M= one K bp ladder,( Lanes; 1,4 mother plant ;2,5 plantlet from subculture
number 6 and 3,6 plantlet from subculture number 8) .
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
M
Figure (3): RAPD-banding pattern of date palm(Phoenix dactylifera L.); Sewi cultivar ;
(lanes; 1,2,3,4,5,6,7,8 and 9) and; Zaghloul cultivar (lanes; 10,11,12,13,14,15,16,17 and
18) amplified with primers; OP- B06, OP- B09 and OP- B 10 respectively, M= one K bp
ladder,( Lanes; 1,4,7,10,13 and 16 mother plant ;2,5,8,11,14 and 17 plantlet from
subculture number 6 and 3,6,9,12,15 and 18 plantlet from subculture number 8) .
558
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E-mail: esammadboly@yahoo.com. or dresammadboly@gmail.com
559
560
PP 10
Molecular and Morphological Identification of Some Elite
Varieties of Date Palms in Saudi Arabia.
Nasser S. Al-Khalifah, Ejaz Askari and Shanavas Khan A.E.
King Abdulaziz City for Science and Technology (KACST), P.O.Box 6086, Riyadh, Saudi Arabia.
Abstract
Date palm (Phoenix dactylifera L.), a highly out breeding, dioecious plant species
has enormous amount of genetic diversity. Genotype identification of date palm is an
intricate empirical exercise based on morphological characters. In date palms most of
the female cultivars are recognised by their fruit characteristics such as size, shape,
colour and taste. Morphologic characters of the tree are also taken into consideration
for the cultivar identification. However some date palms have similar or narrow
distinguishing morphological characters that complicate cultivar identification and
demand genetic evidence to prove phylogenetic relationships. Random Amplified
Polymorphic DNA (RAPD) analysis is a comparatively simple, quick and less
expensive procedure for generating genomic markers. This technique has been
successfully applied for cultivar identification of date palms. The objectives of the
present study were to characterize some elite cultivars of date palms using
morphological characters of fruits and to correlate these results with RAPD markers.
Fourteen well known cultivars of date palm ('Barhy', 'Deglet Noor', 'Hilaliah',
'Hilwa', 'Khalas', 'Makhtomi', 'Moneifi', 'Nabtet Ali', 'Omal Khashab', 'Rothana',
'Sabbaka', 'Shagra', 'Sukkary', 'Wannanah') were selected from Saudi Arabia. Analysis
of the morphological data of fruits showed high level of diversity in length-width ratio,
colour, shape of the fruit, fruit-base and in the percentage of area covered by the fruit
cap. The length-width ratio of these fourteen cultivars ranged from 1.1 to 2.62,
indicating a great variation in their shape.
Correlation of morphologic characters with genomic similarity using RAPD
markers showed that the fruit shape is one of the characteristics mostly influenced by
genetic variation. Where ever there was insignificant length-width ratio between
cultivars, more genomic similarity was observed. Genetic variations at the molecular
561
level have resulted in the production of many elite date palm cultivars which are
highly variable in fruit size, shape, colour, texture, sugar and protein content. The
methods followed in this study can be extended to other cultivars also, which may
ultimately result in the making of an authenticated manual describing the diagnostic
characters of date palm cultivars with their available synonyms. The RAPD analysis
will help to solve the ambiguity regarding the identity of narrowly distinguishable
cultivars and to assess genetic diversity for the conservation of date palm germplasm
in Saudi Arabia.
Key words: Morphology, fruit shape, length-width ratio, genetic diversity, RAPD.
Introduction
Date palm (Phoenix dactylifera L.) cultivation is the main source of agricultural
income in many countries of arid regions of West Asia and North Africa. With its
ability to accumulate exceptionally high level of metabolites under extreme arid
conditions, it is a unique physiological entity (Al-Khalifah et al. 2006). Being a key
species, adapted to the harsh environmental conditions of arid zones date palms are
regarded as one of the important components of biodiversity in the inhospitable areas
of deserts. Phoenix dactylifera L. is inter fertile with its allied species (Muirhead,
1961) and are successfully pollinated with P.rectinata and P.atlantica in Africa. In
India and Pakistan it is pollinated with P.sylvestris and in Spain with P.canariensis
(Oudejans, 1979; Benbades, 1992). This highly out breeding behavior has brought
about immense genetic diversity in this species. Zaid and de Wet (1999) reported the
occurrence of 3000 cultivars all around the world. There are about 450 cultivars in
Saudi Arabia (Bashah, 1996), 400 in Iran (FAO, 1996), 370 in Iraq (Dowson, 1923),
250 in Tunisia (Kearney, 1906), 244 in Morocco (Saaidi, 1979) as well as many others
in other date growing countries (Zaid and de Wet, 1999).
Most of these cultivar identification works are of enumerative type based on local
names which varies from place to place. These cultivars are location specific, known
by different names at different places or one name is assigned to different cultivars at
different places. This has created lot of ambiguity in enlisting the cultivars based on
local names. A scientific approach of characterizing cultivars and assigning a more
562
acceptable legitimate name to the cultivars was seldom attempted in this species,
especially in Saudi Arabia.
Genotype identification of date palm is an intricate empirical exercise based on
morphological characters (Sedra et al., 1998). In date palms most of the female
cultivars are recognised by their fruit characteristics such as size, shape, colour and
taste. Morphologic characters of the tree are also taken into consideration for the
cultivar identification. During the ripening process, the date fruits pass through 4
distinct stages of maturity viz. 'Kemri', 'Beser', 'Rutab' and 'Tamar' (Al-Ghamdi,1993).
When the fruits are young they are green in colour (varies in different cultivars) and
are termed 'Kemri'. Beginning of ripening marks the 'beser' stage, half ripened stage is
called 'rutab' and fully ripened, soft textured stage is called 'Tamar'. These colour
variations during the ripening of fruits are important morphological markers for the
cultivar identification.
However some date palms have similar or narrow distinguishing morphological
characters that complicate cultivar identification and demand genetic evidence to
prove phylogenetic relationships at the inter specific level. Random Amplified
Polymorphic DNA (RAPD) analysis is a comparatively simple, quick and less
expensive procedure for generating genomic markers (Welsh and Mc Clelland, 1990;
Williams et al., 1990). This technique has been successfully applied for cultivar
identification of date palms (Saker et al., 2000; Al-Khalifah and Askari, 2003; Askari
et al., 2003; Al-Khalifah, 2006).
The objectives of this study were to characterize some elite cultivars of date palms
using morphological characters of fruits and to correlate these results with RAPD markers.
Materials and methods
Fourteen well known cultivars of date palm ('Barhy', 'Deglet Noor', 'Hilaliah',
'Hilwa', 'Khalas', 'Makhtomi', 'Moneifi', 'Nabtet Ali', 'Omal Khashab', 'Rothana',
'Sabbaka', 'Shagra', 'Sukkary', 'Wannanah') trees were tagged in two orchards of AlQassim area, Saudi Arabia. Colour variations during the three fruit ripening stages
('beser', 'rutab' and 'tamar') were recorded directly from the tagged trees. Hundred fruits
from each cultivar were collected during 'rutab' stage and their length and width were
measured using Vernier Calipers. Shape and colour of the fruits was documented using
563
digital camera. Base and apex of the fruits were also noted carefully and the diameter of
the fruit cap (persistent calyx) was measured using a millimeter scale. Based on this
data the total area of the fruit base covered by the fruit cap was calculated.
For Random Amplified Polymorphic DNA (RAPD) analysis young sprouting
leaves from each cultivar were collected. Total genomic DNA was extracted using the
protocol of Dellaporta et al. (1983). After determining the quality and quantity of
extracted DNAs with a UV Spectrophotometer, the stock DNA samples were diluted
in distilled water to make a working solution of 10 ηg/μl.
Polymerase Chain Reaction (PCR) was performed as described by Al-Khalifah and
Askari, (2003) using 130 random 10-mer RAPD primers (OPERON Tech.) of A to G
series. PCR products of each primer were separated by electrophoresis according to
their molecular weight on 1.4% (w/w) agarose gels. The profiles of each primer were
then documented by Gel Documentation System of Bio Rad. (Hercules, Calif.). The
length of the amplified RAPD fragments was estimated by running Kilo Base DNA
marker (Amersham Pharmacia Biotech.) in the gel as standard size marker.
Amplification profiles of all the cultivars were compared with each other using the
Diversity Data Base software package (Bio-Rad).
Results and Discussion
Analysis of the morphological data of fruits showed high level of diversity in
length-width ratio, colour, shape of the fruit, fruit-base and in the percentage of area
covered by the fruit cap (Table1). Their shape varied from globular, elliptic, ovate,
oblong, and to linear oblong as in 'Deglet Noor' (Figs 1&2). Many intermediary forms
or combination of one or two forms were also observed. The length-width ratio of
these fourteen cultivars ranged from 1.1 to 2.62, indicating a great variation in their
shape. Even within the cultivars having same or insignificantly different length-width
ratio there was variation in shape mainly due to the position of the widest portion, i.e.
widest near the base in 'Shagra' and 'Wannanah' and widest near the middle as in
'Moneifi'. Fruit base varied from truncate to cordate or sometimes oblique. During
'Kemri' stage all these cultivars had green coloured fruits which turned to yellow or red
or various degrees of combination of red and yellow in 'beser' stage. During 'rutab'
stage ripening process usually starts from the tip of the fruit which brought different
564
colouration to the fruits (Table1). 'Tamar' is the harvesting stage in which they showed
colour variation from amber, golden brown, reddish brown to chocolate brown. The
size of the fruit cap and percentage of the fruit-base covered by the fruit cap are
important morphological markers to distinguish between cultivars. This marker
showed variations from 25 % coverage to 90% in different cultivars (Table1).
Random Amplified Fragment DNA (RAPD) markers were also produced for the
identification of these cultivars. Out of 130 primers screened for reproducible and
polymorphic DNA amplification patterns 42 were selected for DNA fingerprinting.
The DNA profiles produced by 14 cultivars with OPERON A06 primer are presented
in Figs 1&2 along with their fruit morphology. The analysis of pair-wise genetic
distance and similarity matrix based on Nei and Li's (1979) similarity coefficient
showed an average of more than 50 % similarity among the cultivars (Table-2, from
Al-Khalifah,2006). Cluster analysis using unweighted pair group method of arithmetic
means (UPGMA) and the dendrogram (Fig.3 from Al-Khalifah,2006) showed
maximum similarity between Makhtomi and Nabtet Ali (0.70) followed by Barhy and
Hilaliah (0.65). Out of the 19 cultivars screened by Al-Khalifah, (2006) 12 formed
couples and the rest showed various percentages of similarity with either to one of the
couples or to more than one couples.
Correlation of morphologic characters with genomic similarity showed that the fruit
shape is one of the characteristics mostly influenced by genetic variation. Where ever
there was insignificant length-width ratio between cultivars, more genomic similarity
was observed. In the case of Makhtomi and Nabtet Ali, where the maximum genomic
similarity was observed, their length-width ratios were only different by 1.46 and 1.44
respectively. The second genomically similar couplet (Hilaliah-Barhy) also showed a
very narrow variation in their length-width ratio (1.1-1.2). The other pairs that
followed the same rule were Khalas-Makhtomi (1.46-1.46), Sabakka-Rothana (1.51.4), and Shagra-Wannanah (1.32-1.44). But there was an exception exhibited by a
pair Nabtet Ali-Wannanah, where their length-width ratio was similar (1.44) to each
other but their genomic similarity was the least (44.1%). But in this case, irrespective
to their similar length-width ratio they were very distinct in their fruit morphology, i.e.
their shape (Elliptic-oblong and ovate), colour and fruit base.
565
The present data generated by using different primers suggests genetic diversity
among date palm cultivars. Molecular phylogeny of 13 date palm cultivars studied by
Al-Khalifah and Askari (2003) and 7 cultivars by Askari et al. (2003) also showed the
same tendency of genetic diversity. These genetic variations at the molecular level
have resulted in the production of many elite cultivars which are highly variable in
fruit size, shape, colour, texture, sugar and protein content. The methods followed in
this study can be extended to other cultivars also, which may ultimately result in the
making of an authenticated manual describing the diagnostic characters of date palm
cultivars with their available synonyms. Addition of tree characteristics, protein and
sugar content of each cultivar, to this data in future will make a perfect manual that
can be used as reference book to identify the presently known cultivars of date palms.
The RAPD analysis will help to solve the ambiguity regarding the identity of narrowly
distinguishable cultivars and to assess genetic diversity for the conservation of date
palm germplasm in Saudi Arabia.
Acknowledgement
Authors are thankful to the King Abdulaziz City for Science and Technology,
Riyadh for providing technical and financial support to this study.
566
References
[1] Al-Ghamdi, A.S. (1993).True-to-type date palm (Phoenix dactylifera L.) produced
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Theor. Appl. Genet. 107: 1266-1270.
[3] Al-Khalifah, N.S. (2006). Micro propagation and DNA fingerprinting of date palm
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[4] Al-Khalifah, N.S., F.A.Khan, E.Askari and S.Hadi (2006). In Vitro Culture and
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M.G., I. Holb and Gy.D.Bisztray (Eds). Proc.Vth International Symposium on In
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[5] Askari, E., Al-Khalifah, N.S., Ohmura, T., Al-Hafidh, Y.S., Khan, F.A., Al-Hindi,
A., and Okawara, R. (2003). Molecular Phylogeny of seven date palm (Phoenix
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pp 1225-1319.
[7] Benbades, A.K. (1992).Coconut and Date palm. In: Benbades, A.K. and
F.E.Hammerschlag (Eds.), Biotechnology of Perennial fruit crops. pp 383-400.
[8] Dellaporta, S.L., Wood, J. and Hicks, J.B. (1983). A plant DNA minipreparation:
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[9] Dowson, V.H.W. (1923). Dates and date cultivation of Iraq. Part III. The varieties
of date palms in the Shalt-Al Arab. Agr. Directorate of Mesopotamia, Memoir III,
Heffer, Cambridge.
[10] FAO (1996). Agro-statistics DatabaKearney, T.H (1906). Date varieties and date
culture in Tunis. US. Dept.Agr.Bur.Plant Industry Bull. 92:112.
[11] Muirhead, D. (1961). Palms. Dale Stuart King Publishers, Arizona
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[12] Nei, M. and Li, W. (1979). Mathematical model for studying genetic variation in
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[13] Oudejans, J.H.M. (1979). Date Palm (Phoenix dactylifera, Palmae) In:
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568
Table 1. Comparative fruit morphology of fourteen date palm cultivars.
Cultivar
Shape
Colour variation during
ripening
'Beser'
'Rutab'
'Tamar'
Length- Fruit
width
Cap
ratio
(%)
Base
Barhy
GlobularBroadly
elliptic
Lemon
yellow
Amber
Golden
brown
1.21
40
Truncate
Deglet
Noor
Linearoblong
Lemon
yellow
Reddish
brown
Amber
2.62
90
Truncate
Hilaliah
Globular
Yellow
with rose
tinge
Amber
Reddish
brown
1.1
90
Truncate
Hilwa
Oblong
Scarlet
red
Dark red
Chocolate
brown
1.5
50
Shallowly
cordate
Khalas
Ovate
Light
yellow
Amber
Amber
1.46
30
Oblique
Makhtomi
Ovateoblong
Greenishyellow
Amber
Amber
1.46
60
Shallowly
cordate
Moneifi
Elliptic
Yellow
Amber
Reddish
brown
1.51
50
Truncate
Nabtet Ali
Elliptic
oblong
Light
yellow
Reddish
brown
Reddish
brown
1.44
25
Shallowly
cordate
Omal
Khashab
Oblong
Reddishyellow
Amber
Amber
1.84
60
Truncate
Rothana
Ellipticoblong
Lemon
yellow
Amber
Reddish
brown
1.4
30
Deeply
cordate
Sabbaka
Oblong
Light
yellow
Reddish
brown
Light
brown
1.5
50
Shallowly
cordate
Shagra
Ovateoblong
Yellow
with red
dots
Brown
Reddishbrown
1.32
33
Cordate
Sukkary
Ovate
Reddish
yellow
Reddish
brown
Reddish
brown
1.43
60
Cordate
Wannanah
Ovate
Yellow
with red
dots
1.44
30
Oblique
Chocolate Chocolate
brown
brown
569
Table 2. Similarity matrix based on Nei and Li's coefficients of 19 date
palm cultivars obtained from RAPD markers (Al-Khalifah, 2006).
570
Rothana
Nabtet Ali
Sukkary
Hilwa
Omal Khashab
Khalas
Deglet Noor
Moneifi
Sabbaka
Barhy
Hilaliah
Makhtomi
Fig.1. Fruit morphology and DNA profiles of twelve cultivars produced by A-06 OPERON primer.
571
Wannanah
Shagra
Fig.2. Fruit morphology and DNA profiles of two morphologically
similar cultivars produced by A-06 OPERON primer.
Fig. 3: A dendrogram of phylogenetic relationships among 19 cultivars of
date palm based on the RAPD analysis using 42 primers (Al-Khalifah, 2006).
572
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574
OP 27
Bt: A new strategy towards date palm orchards resistant to Red Palm Weevil
S.A. Bekheet
Plant Biotechnology Dept., National Research Center, El-Tahrir Str., Dokki, Cairo, Egypt
e-mail: shawky005@yahoo.com
Abstract
The Red Palm Weevil is considered one of the major pests of the date palm
orchards where whole plantations had to be cut down due to palm weevil infestation.
Current systems of the weevil control are largely based on insecticide and pheromone
traps applications. In addition to the ineffectiveness of these methods, there are now
deep concerns about environmental pollution. The excessive use of insecticides is also
likely to limit the activity of natural enemies of Red Palm Weevil. Therefore,
development of new breeds resistance to insects notably Red Palm Weevil is necessary
for date palm plantains. Genetic improvement of date palm for insect resistance by
conventional breeding has been faced with certain limitations. In view of the fact that
date palm is a perennial and highly heterozygous and to the long generation period, its
traditional breeding is a very slow and not effective process. Biotechnology tools of
tissue culture can now effectively speed up all of the above processes. Moreover,
genetic engineering has made it possible to use Bt proteins more effectively to control
insects that are harmful to crops. Genes from Bacillus thuringiensis (Bt) are inserted
into cells of crop plants to make them capable of producing an insecticidal toxin and
therefore resistant to certain pests. Bt and its delta endo-toxin are considered to be
much more selective and safer for humans and non-target organisms than most
conventional insecticides. This article presents the advantages of using Bt-toxins in
controlling the Red Palm Weevil and possibility of transfer its genes into date palm
genome using biotechnology techniques.
Introduction
The date palm is an important horticultural crop grown mainly in Middle East and
Arabian region. Palm tree is a target for several diseases and pests. Red Palm Weevil
(Rhynchophorus ferrugineus) considered one of highest destructive pests in date palm
575
plantation. It is rapidly spread throughout the Middle East, North Africa and
Mediterranean areas [12]. Whether these insects become a problem depend on the
level of damages of the special ecosystem. Transfer of date palm offshoots as a
planting material has played a major role in rapid proliferation of the pest. Damage to
date palm is mainly caused by the larval stage feeding within the trunk of palms. In
this respect, Red Palm Weevil is reported to mostly attack young palms less than 20
years old [2]. This concealed feeding habit of larvae makes it more difficult to detect
infestation at an early stage. To control and eradicate the Red Palm Weevil it is
fundamental to detect the infested trees before they constitute new focus of dispersion
of the pest. Unfortunately, the larvae that cause the damages to the palms live inside
the stems and at the base of the leaves. Their presence in these locations does not
create usually any visible symptoms till the infestation is already serious. Current
tactics to manage the Weevil are largely based on insecticide applications although
there are now deep concerns about environmental pollution. The excessive use of
insecticides is also likely to limit the activity of natural enemies in plantations. Much
research has been conducted on other techniques, notably pheromone traps. Thus, the
control of the Red Palm Weevil needs the adoption of an integrated control
management strategy. In this connection, development of new breeds of date palm
resistance to insects notably Red Palm Weevil is in demand.
.
In
view of the fact that date palm is a perennial and highly heterozygous, its breeding is a
very slow process. In addition to the long generation period, breeding of date palm
requires large areas for breeding trials and extensive series of backcrossing which is
time consuming. It usually takes more than 30 years to complete three backcrosses and
to obtain the first offshoots from an intervarietal cross. To produce sufficient offshoots
for testing in the field, other generations are required and if the breeding target is yield
or fruit quality even more time will be needed as a date palm does not reach full
commercial production for 10 years. It is therefore not surprising that little date palm
breeding has been achieved using traditional approaches.
.
Therefore, innovative methods are needed to rapidly enhance the incorporation of new
genetic breeds into date palm in order to speed up the progress of its breeding
programs, particularly in those cases where date palms are being threatened by
576
devastating pests like Red Palm Weevil. Biotechnology tools of tissue culture and
genetic engineering can now effectively speed up all of the above processes and in
addition genetic fingerprinting can be used to generate quicker and more precise
analyses of elite clones, thus taking years off the traditional methods. The new
technology of genetic manipulations, allows the transfer of selected gene(s) to a
specific genotype in only a single generation that would not be possible by
conventional breeding [34]. Genetically engineering inherent crop resistance to insect
pests offers the potential of a user-friendly, environment-friendly and consumerfriendly method of crop protection. Genes from Bt are inserted into crop plants to
make them capable of producing an insecticidal toxin and therefore resistant to certain
pests. Because control of Red Palm Weevil has not been successful by traditional
methods, a genetic engineering strategy therefore would expect to aid the breeder in
introducing such traits directly into elite or commercially valuable genotypes of date
palm. For transgenic resistance to insects the expression of Bacillus thuringiensis (Bt)
toxins is the most well-known approach, which is also used commercially on
significant acreages [41, 25]. The main insecticidal activity of B. thuringiensis is due
to insecticidal crystalline (Cry protein) inclusions formed during sporulation. In this
respect, Bt were expressed in large number of different crop species (in field trials)
and have been shown to confer resistance to various pests, mainly of the orders
Lepidoptera and Coleopteran [28, 40, 22]. This article discusses different applications
of Bt (Bacillus thuringiensis) in Red Palm Weevil control in date palm orchards with
special emphasis on transformation.
Threats of Red palm weevil to date palm plantations
Red Palm Weevil (Rhynchophorus ferrugineus Olivier) is the most important pest
of coconut palm, date palm, oil palm and sago palm. It also attacks a wide range of
ornamental palms. All stages (egg, larva, pupa and adult) are spent inside the palm
itself and the life cycle can't be completed elsewhere. Moreover, many generations can
be passed in the same palm tree. The damage is due entirely to the larvae, which feed
on the trunk and also the growing point viz., the heart or the cabbage of the crown of
the palm and once they have gained access, death of the palm generally ensues. The
symptoms of infestation show the presence of small holes at the leaf scars and oozing
577
out of a reddish-brown fluid and extrusion of fibers from these holes and slightly
audible sound of the feeding activity of the grubs within the stem attacked trees.
Unfortunately, the attack is discernible unless extensive damage is happened. The
controlling possibilities are very limited in due to the hidden lifecycle of the weevil.
The common and practical curative measure is through the use of insecticides. The
chemical treatments against the Red Palm Weevil have to be considered as an element
of a global strategy because, to date, no other treatment especially the biological ones
has demonstrated a sufficient efficiency in the field [6]. Although infested palms in the
early stage of attack recover with insecticide (trunk injection), palms in the late stage
of attack have to be eradicated. Now there is also a strong emphasis on the
development of integrated pest management based on pheromone traps and biological
control rather than insecticides. In this respect, although various mites have been
reported in India as parasites of R. ferrugineus [29], their impact on the population
needs to be ascertained. Otherwise, pheromones are increasingly being used as a
management tool against R. ferrugineus. Detailed protocols for pheromone-based
mass trapping of the weevil have been described [18]. Moreover, evaluation of
pheromone lures for the weevil in date plantations in Saudi Arabia have been achieved
and it was found that high release lures (Ferrolure and Ferrolure+) obtained from
Chem Tica Natural, Costa Rica, attracted twice as many weevils as low release
formulations [11].
.
Biotechnology of date palm
1. Tissue cultures and molecular analysis
Current conventional breeding programs for date palm are supported by modern
biotechnology approaches such as the use of tissue culture, genetic transformation and
marker-aided selection, to further improve of commercial value and productivity.
Biotechnology was introduced to the date palm industry almost four decades ago.
Initially, tissue culture techniques were used to propagate superior date palm cultivars.
In this respect, plantlets have been successfully regenerated from various explants of
date palm. The cultured explants included mature and immature embryos [36, 31],
apical meristems [43, 39, 3], leaf primordia [24] and inflorescences [19]. Now, in vitro
production of hundred thousands of date palm plants has become almost routine in
578
many tissue culture laboratories. On the other hand, the excessive use of growth
regulators particularly auxins to speed up embryoids multiplication resulted in the
production of clonal date palm with abnormal growth [42]. In this respect, continuous
researches are going on to minimize abnormalities of date palm plants derived from
tissue cultures. However, the ability to regenerate complete plants from all the above
explants has made date palm amenable to genetic manipulation for the incorporation of
foreign gene. Molecular and biochemical analysis have been used for characterization,
indexing and detection of genetic stability of the propagated and conserved plant
species. In this connection, various techniques such as isozymes, restriction fragment
length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPD) and
AFLPs have been tested for their application in date palms [5, 8, 35, 4]. In this
connection, AFLP markers were used affectively for the identification and comparison
of different cultivars and also tissue cultured plants. This technique provided useful
information regarding genetic relationships and genetic diversity [9].
.
2. Transformation
Genetic improvement of date palm by conventional breeding has certain limitations.
For instance it is heterozygous, has a long juvenile phase and exhibit nucellar
embryony, thereby leading to delay and difficulty in the selection of new genotypes
with improved characteristics. Taking into account the requirement for back-crossing
in conventional breeding, genetic engineering could save 80–90% of the time required
for introducing a new gene / trait into date palm. About 4–5 years are required to
produce transgenic plantlets carrying a new trait. The commonly used vector
(Agrobacterium) and vectorless (biolistics) methods of gene transfer have lead to
production of transgenics in a range of fruit crops including tropical, sub-tropical and
temperate species [13, 38].
.
However, to produce
transgenics, there are certain pre-requisites such as availability of efficient plant
regeneration, gene transfer and selection systems which allow the production of
transgenic with stable inheritance of traits in further generations. Moreover, the
condition of the explants, such as the age or condition of the culture, prior to selecting
explants, influences gene delivery. The explants from in vitro seedlings or actively
growing plant parts/meristem show a better response to in vitro manipulation
579
with/without growth hormones and thus are amenable to modification by recombinant
technology for producing transgenics on a commercial scale [14]. Moreover, it was
found that promoter plays a major role for successful gene transfer into plant cells. The
promoter is the on/off switch that controls when and where in the plant the gene will
be expressed.
The most commonly used promoters for genetic transformation is
CaMV35S. Moreover, development of transgenic plant requires the use of suitable
selectable marker genes. The marker genes that were suitable for dicots may not be
suitable for monocots. By following advancement in genetic transformation studies, it
is possible to transfer foreign genes into date palm genome. One routine technique is
by using Agrobacterium spp. True (not chimearic) transgenic plants are easily obtained
from plants that are very responsive in vitro, with short period for callus initiation,
multiplication and subsequent plant regeneration. Therefore, the long culture period
together with possible deterioration on the fidelity of the produced clones makes rapid
production of transgenic date palm plant relatively difficult. Thus the choice of target
tissue becomes critical in date palm transformation using Agrobacterium. However, it
is now possible to produce transgenic plants from bombarded embryo without going
through callusing, multiplication and plant regeneration, but the success rate still very
low. Plant transformation mediated by biolistic is tissue and variety independent.
However, Agrobacterium infection depends mainly on pretreatment of the target
tissues prior to infection. Over the last few years, few researches developed conditions
to make date palm tissues become susceptible to Agrobacterium infection [32].
The amenability of oil palm (Elaeis guineensis Jacq.) tissues to gene transfer was
first demonstrated since several techniques, both direct and vector mediated were
developed to introduce useful genes into different target tissues [1]. The genes
introduced include those conferring resistance to insect pests, diseases, herbicide,
antibiotics. The Agrobacterium-mediated gene transfer system for oil palm was further
improved with the possibility of co-transforming two Agrobacterium strains with two
different demonstrated plasmids. This was done by infecting longitudinally-cut in vitro
plantlets with exposed apical meristem for infection by Agrobacterium carrying the
desirable genes in their plasmids. The advantage of this approach is that, the cocultivated apical meristems were able to regenerate into complete plants within 3 to 4
580
months. Thus the total time required to produce complete plants from explant
preparation, co-cultivation and regeneration is only about 3.5 to 4.5 months. It is very
much shorter compared to when undifferentiated tissues such as immature embryos
and other embryogenic cultures derived from other explants were used as initial target
tissues. Identifying the major parameters controlling DNA delivery by particle
bombardment to date palm embryogenic calli and somatic embryos has been
investigated [17]. The main factors studied were osmotic conditioning of explants
before and after bombardment, type of explants and different bombardment parameters
like acceleration pressure, bombardment distance and gold particle size. Efficiency of
DNA (gus gene) delivery was assessed by scoring transient GUS expression in
bombarded tissues. Moreover, establishment of an efficient genetic transformation
system in date palm (Phoenix dactylifera L.) using particle bombardment has been
reported [17]. Somatic tissues derived from offshoots' meristem cultures were
bombarded with genetic constructs harboring the uidA gene under control of the
CaMV 35S or Act1 promoter. To develop date palm plants encoding insecticidal
toxins through transfer of Bacillus thuringiensis (Bt) the following stage should be
followed: a) establishment of in vitro regeneration protocols using different explants,
b) optimization of the transformation procedures, c) construction of expression vectors
and cloning of the amplicons of Bt gene fragments, d) transformation of Bt into date
palm explants, e) selection and regeneration using selective agents, f) confirmation of
transformation using histochemical assay and molecular analysis and g) determination
of gene expression and bioassay tests.
In this respect, expression of Bacillus
thuringiensis insecticidal protein gene in transgenic oil palm was studied [23].
4. Bacillus thuringiensis (Bt)
Bacillus thuringiensis (Bt) is a soil bacterium that produces insecticidal toxins.
There are thousands of different Bt strains, producing over 200 cry proteins that are
active against an extensive range of insects and some other invertebrates. The Cry
proteins are classified into 24 major groups and are usually specific for a limited range
of species within certain insect order, mainly Lepidoptera, Coleoptera and Diptera. In
general Cry1, Cry2 and Cry9 proteins affect Lepidoptera, Cry3, Cry7, and Cry8
proteins are active against Coleoptera, and Cry4, Cry10, and Cry11 proteins affect
581
Diptera [7]. This group of toxins is considered relatively harmless to humans and most
non-pest species. Delta endo-toxins are stomach poisons that must be eaten by the
insect in order to be effective. The mechanism of Bt toxicity has been reviewed [21].
Essentially, the active toxin binds to glycoprotein receptors in the brush border
membrane of susceptible insects midgut epithelium. Electro-physiological evidence
suggested that crystal proteins are dissolved in the larval insect midgut (pH 9-10) and
are proteolytically converted into toxic core fragments. Delta endo-toxins rapidly
paralyze the insect’s digestive system, so damage to the plant stops soon after the
insect is exposed to the crystals. Mortality may take several days, so the effects of
delta endo-toxins are very different from what we expect from conventional
insecticides. Bt is largely used in agriculture, especially organic farming. It is also used
in urban aerial spraying programs, and in transgenic crops. Genes from Bt can be
inserted into crop plants to make them capable of producing an insecticidal toxin and
therefore resistant to certain pests. Bt products are found to be safe for use in the
environment and with mammals. The Environmental Protection Agency (EPA) has not
found any human health hazards related to using Bt. In fact the EPA has found Bt safe
enough that it has exempted Bt from food residue tolerances, groundwater restrictions,
endangered species labeling and special review requirements. It is often used near
lakes, rivers and dwellings, and has no known effect on wildlife such as mammals,
birds, and fish. Bt also breaks down under the ultraviolet (UV) light of the sun [15].
.
4.1. Bt as bio pesticide agent
Bio pesticides represent approximately 1% of the world pesticide market, and Bt
products represent 80% of all bio pesticides sold. Bt is really an insecticide, used like
an insecticide, and regulated like an insecticide, just that it isn’t produced from
synthetic chemicals. Bt insecticides, consisting of dormant Bt and delta endo-toxin,
have been available commercially and used in agriculture for more than 30 years (e.g.,
Bactimos, Biobit, Dipel, Javelin, Teknar, Vectobac). The delta endo-toxins are
considered to be much more selective and safer for humans and non-target organisms
than most conventional insecticides because they attack sites that are found only in a
few groups of insects. Commercial Bt insecticides are classified as Generally
Regarded As Safe (GRAS) by the EPA, and are approved for most organic
582
certification programs. The crystal proteins are insect stomach poisons that must be
eaten to kill the insect. Once eaten, an insect's own digestive enzymes activate the
toxic form of the protein. The Cry proteins bind to specific "receptors" on the
intestinal lining and rupture the cells. Insects stop feeding within two hours of a first
bite and, if enough toxin is eaten, die within two or three days.
Farmers started to
use Bt as a pesticide in 1920. France soon started to make commercialized spore based
formulations called Sporine in 1938. Sporine, at the time was used primarily to kill
flour moths. In the US, Bt was used commercially starting in 1958. By 1961, Bt was
registered as a pesticide to the EPA. Up until 1977, only thirteen Bt strains had been
described. All thirteen subspecies were toxic only to certain species of lepidopteran
larvae. In 1977 the first subspecies toxic to dipteran (flies) species was found, and the
first discovery of strains toxic to species of coleopteran (beetles) followed in 1983. In
the 1980's use of Bt increased when insects became increasingly resistant to the
synthetic insecticides and scientists and environmentalists became aware that the
chemicals were harming the environment. More products containing Bt were
marketed, but many of these products had limitations. Bt products such as sprays are
rapidly washed away by rain, and degrade under the sun's UV rays. Also, there were
many insects that are not susceptible to any of the limited number of Bt strains known
at the time. All the Bt strains known at the time were toxic to lepidopteran (moth)
larvae only and it is ineffective against most adult insects. There were also some
insects that live within the plant or underground where the Bt sprays could not reach.
Since Bt is applied topically, insects that attack the roots or the insides of a plant will
not be affected.
4. 2. Bt as transgenes
Biotechnology has made it possible to use Bt proteins more effectively to control
insects that are harmful to crops. The genes coding for these proteins are actually
introduced into the plant, enabling it to produce the protein continuously and so protect
itself against attacks from insect pests. Today, there are thousands of strains of Bt.
Many of them have genes that encode unique toxic crystals in their DNA. With the
advancement in molecular biology, it soon became feasible to move the gene that
encodes the toxic crystals into a plant. Insect resistance, based on Bacillus
583
thuringiensis (Bt) endotoxins, is the second most widely used trait (after herbicide
resistance) in commercial genetically modified (GM) crops. Other modifications for
insect resistance, such as proteinase inhibitors and lectins, are also being used in many
experimental crops [27]. As a result of consistent and substantial benefits during the
first dozen years of the commercialization of GM crops (from 1996 to 2007), farmers
have continued to plant more every year. In 2007, 114.3 million hectares of GM crops
were grown in twelve developing and eleven industrial countries and this number is
constantly increasing [20]. Currently, the only insect-resistant GM crops that are
grown commercially are Bt crops. Bt genes have been transferred to a large number of
plant species, such as cotton, pigeon pea, potato, rice, eggplant, oilseed rape and
chickpea [10, 37, 33]. However, in 2007, only Bt-transgenic cotton and maize varieties
expressing either lepidopteran or coleopteran specific Cry proteins were grown on 42.1
million hectares worldwide [20]. A successful application of this technology is applied
in cotton plants to protect them from damage by the budworm / bollworm complex
(Helicoverpa/Heliothis spp., Pectinophora gossypiella). These Bt cotton plants,
expressing either the gene cry1Ac alone or in combination with cry2Ab, are highly
resistant to damage by lepidopteran pests and consequently, the application of
chemical insecticides has been greatly reduced [30].
.
4.3. Bt safety
Bt proteins are allowed in organic farming as an insecticide because Bt is a natural,
non-pathogenic bacterium that is found naturally in the soil. It has also been found to
be safe to all higher tested animals. Because Bt is species specific, beneficial and nontarget insects are usually not harmed. Bt has been found through rigorous testing to be
harmless to humans, other mammals, fish, birds, or basically all vertebrates. It has not
been shown to have any chronic toxicity or any carcinogenic effects. Moreover, Bt
breaks down readily in the environment. Because of this Bt poses no threat to
groundwater. Bt is not known to cause injury to plants and is not considered harmful to
the environment [16].
Conclusion
The dramatic extension of Red Palm Weevil in date palm cultivations demonstrates
that the present control measures are quite insufficient in the Arab countries. New
584
strategy and regulations must be adopted urgently. This strategy should include in a
complementary and inseparable systems. Biotechnology techniques can play an
important role in the field of Red palm weevil control. Many advances have been
made in the frontier areas of biotechnology, tissue culture, genetic transformation and
genomics of plants can be useful in this manner. Genetic transformation has made it
possible to use Bt proteins more effectively to control insects that are harmful to crops
such as Red palm weevil. Economic return (increasing date productivity) can be
gained through application of this technology. Moreover, environmental impacts can
be attended by using Bt date palm such as reducing chemical insecticides.
585
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Disease and Pest Management
593
594
OP 28
Dubas Bug (Ommatissus lybicus Aschc & Wilson), A New Pest of
Date Palm and Canary Palm in Helwan Governorate – Egypt
Abd Rabou Eid Hussain
Plant Protection Department Faculty of Agriculture Al Azhar University Nasr City
Cairo Egypt
Abdraboueid@hotmail.com
Abstract
Dubas bug was recorded for the first time as a pest attacking young Date palm and
Canary palm in Helwan governorate in April 2007, it was found to attack green fronds
of the palm. Field studies were carried out during 2007 – 2008 by fortnightly visits to
examine the fronds of the selected palms for studying population dynamics of Dubas
bug and associated natural enemies. The data showed that there are two generations
per year (spring and autumn generations) on both of Date palm and Canary palm Date
palm harboured higher numbers of Dubas bug more than canary palm. There was a
negative correlation between (Temp &.R.H) and Dubas numbers. The most wide
spread predators were Chrysoperla carnnea Steph., Mantids and Coccinellids , no
chemical measures were applied during the investigation period.
Key words: Dubas – Predators - Date Palm – Canary Palm
Introduction
Dubas bug Ommatissus lybicus DeBerg.( Homoptera :Tropiduchidae ). Is a serious
pest of date palm in Iraq (Dawson,1936; Hussain,1963 ), in Libya ( Lal &Naji, 1979;
in Bahrin ( El – Haidari , 1981);in Sudan ( El Haidari, 1982 ); in Israel . ( Klien
&Venezian,1985 );in Qatar, (Al – Azawi, 1986) ; in Egypt (Hussein&Ali,1996);
Dawson ( 1936) reported that Dubas Bug cause extensive damage to date palm in
Basra area of Iraq .Hussein (1996) stated that Dubas Bug has two generations per year
in Egypt.The present study was carried out to shed light on the occurrence, population
dynamics and bionomic observation of Dubas Bug and associated natural enemies on
date palm & canary palm in Helwan Governorate
595
Materials and methods
To study the population dynamics of Dubas bug Ommatissus lybicus , three date
palm trees & three canary palm trees infested by this insect, were selected in Helwan
university court, the numbers of eggs ,nymphs and adults were counted fortnightly on
10 random pinnae of four fronds for each palm tree (one frond from each cardinal
direction of the palm tree ), the number of each stage at each count were recorded.
Also the numbers of predators were recorded, these counts continued from April 2007
till Mar.2009,no control measures were applied for the inspected palm. Data of the
climatic factors was taken from Central Laboratory of Agricultural Climate.Statistical
analysis were carried out (correlation coefficient and T values).
Results and discussion
Eggs, nymphs and adults were observed on green leaflets, females laid eggs on the
midrib of the leaves and on the blade and the vein of the leaflets.
Population dynamics of Dubas bug in the first season 2007/ 2008
Data presented in table 1, it is illustrated by figure 1,2 show that eggs were found
during two periods ; the first period occurred through July 2007 with a maximum
density 76 egg/ 40 leaflet and 166.6 egg/40 leaflet on Canary palm &date palm
respectively ,the second one recorded through the last months of.2007 with a max.
density 103 egg/40 leaflet on Canary palm and149 egg/40 leaflet on date palm during
Dec. (table 1 and fig. 1 & 2) .
Nymphs was recorded on canary palm, in considerable numbers 9 and 23.3
nymphs/40 leaflet through April and May respectively and decreased gradually to
small numbers through July and August 2007, while it was not observed during
Jul.2007 on date palm ( table 1 & fig. 1 ).Nymph numbers fluctuated to give a peak
10.6 nmph/40 leaflet in second week of Oct. 2007 on canary palm, these numbers
decreased gradually and the nymphs were not observed in the second week of
December 2007, and through the period from fourth week of Jan. till second week of
Mar. 2008, they were recorded in the fourth week of Mar.2008, with a max. density
41 nymph /40 leaflet. Nymph numbers were recorded on date palm in higher numbers
(83.3 nymphs /40 leaflet ) than that of canary palm, in the fourth week of April
2007,these numbers decreased gradually to disappear in July 2007,the nymphs
596
appeared in Aug. ( 5.3 nymph /40 leaflet ) and increased gradually to give peak in
Sept.2007, ( 41.3 nymph /40 leaflet ), then decreased gradually to disappear through
the period extended from fourth week of Jan.2008 till fourth week of feb.2008. they
appeared again in Mar 2008.with gradual increase recording 104 nymphs /40 leaflet
during April 2008 ( table 1 & fig.2 ).
As regard to adult stage on canary palm, it was recorded in fourth week of May
2007 and the first peak of adult numbers ( 8.7 adult /40 leaflet ) occurred in the fourth
week of Jun. 2007,another peak ( 13 adult / 40 leaflet ) was recorded in the fourth
week of Nov.2007.On date palm the first peak of adult numbers ( 12.6 adult /40
leaflet ) were recorded during Jun.2007 and the second one ( 18 adult /40 leaflet )
occurred in Oct. 2007 .Generally the adult stage observed in the most year months
approximately ( table 1 & and fig. 2 )
Population dynamics of Dubas bug in the second season 2008/ 2009
Data is presented in table ( 2 ), and illustrated by figures 3 & 4 show that the density
of the different stages of Dubas bug during 2008 / 2009 has the same trend of 2007
/2008 approximately.The first period of egg laying extended from 4th week of June to
4th week of July 2008 with maximum density 1098.3 egg /40 leaflet during July and
105.3 egg /40 leaflet during June on each of canary and date palm respectively, while
the second period extended from Dec.2008 till Feb.2009 on the palm trees with a
maximum density146.3 egg /40 leaflet during Jan. 2009 on canary palm and 93.3 egg
/40 leaflet during Feb. 2009 on date palm.
Also, there are two peaks of nymph numbers were recorded on canary and date
palm, the first, 49.3 nymph / 40 leaflet during April 2008 on canary palm and 130.6
nymph / 40 leaflet during May 2008 on date palm , the second one ( 42 nymph / 40
leaflet on canary palm and 48.3 nymph / 40 leaflet on date palm during Sept.2008.As
respect to adult stage it observed around the year approximately and there are two
peaks recorded, the first (15.3 adult /40 leaflet on canary palm & 29.6 adult /40 leaflet
on date palm occurred during Jun. 2008 and the second one (42 adult/40 leaflet on
canary palm and 49.6 adult/40 leaflet on date palm) during Dec. 2008 (table 2 & fig. 3
& 4).From these data it is clear that Dubas bug has two generations per year, these
results agree with Hussein et Ali 1996 and Hussain, 1963.
597
Susceptibility of palm species to infestation by Dubas bug
Data existed in tables (1, 2 and 3) show that date palm harboured number of
nymphs & adults more than Canary palm without significant differences.
Correlation between some climatic factors (temperature &R.H. %) and Dubas numbers
Tables (1, 2 and 4) show that there was negative insignificant correlation between
temperature & Dubas bug numbers (nymphs and adults), also there was negative
insignificant correlation between R.H.% and Dubas bug numbers ( nymphs and adults )
on canary & date palm.
Associated predators
The number of the recorded predators during the present study was eight larvae and
six adults of Chrysoperla carnnea on canary palm and seventeen larvae and thirteen
adults of Chrysoperla carnnea on date palm. One and forty nine Cybocephalus
flavipes fam.: nitidulidae
on canary and date palm resp. Also eleven nymphs and
three adults of Mantids were recorded on canary palm and three adults of Mantids plus
one Coccinella undecimpunctata adult on date palm
598
References
[1] Al - Azawi, A. F. 1986. A survey of insect pests of date palm in Qatar, Date palm
Journal, 4, (2) 247 – 266
[2] Dawson,V.H.W. ( 1963 ) A serious pest of date palm, O.binotatus, Fieb., Trop.
Agric., Trin., 13 ( 7 ) : 180 – 181.
[3] El – Haidari, H. S. 1981. Ommatissus binotatus De Bergevin ( Homoptera
:Tropiduchidae ) .Date Palm Journal. P. 133
[4] El- Haidari, H.S. ( 1982 ).New records of Dubas bug Ommatissus binotatus lybicus
( De Bergevin ) on date palms in Sudan. Date palm Journal, 1 ( 2 ) : 308.
[5] Hussain,A.A. 1963 biology & control of the Dubas bug Ommatissus binotatus
lybicus De Berg. (Homoptera :Tropiduchidae ) infesting date palm in Iraq. Bull.
Entomol. Res., 53 :737 – 745
[6] Hussein, A. E. and M. A. Ali 1996 New record of the Dubas bug, Ommatissus
binotatus lybicus De Berg., a threat to date palms in Bahria oases, Egypt Bull. Ent.
Soc. Egypt. 74, 1996 ( 1 ) : 1 - 8
[7] Klein,M. and Veneztan,A. ( 1985 ).The Dubas date tropiduchid. Ommatissus
binotatus lybicus ,a threat to date palms in Israel. Phytoparasitica, 13 ( 2 ) : 95 – 101.
[8] Lal. O. P., and A. H. Naji.1997 Observation on some new insect pests and
parasites from the Socialist People’s Libyan Arab Gamahiriya. Rivista
diAgricoltura subtropicale e Tropicale. 73 ( 3 – 4) : 219 – 232
599
Table (1) population density of different stages of Dubas bug Ommatissus lybicus
on palm trees, mean temp. & R.H% during 2007 /2008
Date
Unknown date palm
cultivar
Canary palm
Egg*
Nymph* Adult*
Egg*
Nymph*
Adult*
Temp.
R.H.
%
0.0
9
0.0
0.0
83.3
0.0
22.23
71.7
0.0
23.3
0.0
0.0
30.0
0.0
13.79
53.6
4 th w.of May 2007
0.0
15
6.7
0.0
9.3
0.6
23.85
45.7
2 nd w.of Jun.2007
0.0
8.5
7.7
0.0
7.3
12.6
25.07
38.8
4 th w.of Jun.2007
0.0
4
8.7
0.0
3.0
5.3
28.07
40.4
2 nd w.of Jul.2007
76
0.3
3.0
14.3
0.0
2.3
28.14
39.7
4 th w.of Jul.2007
32.3
0.5
1.0
166.6
0.0
1.0
31.69
39.8
2 nd w.of Aug.2007
0.0
0.6
0.3
0.0
5.3
1.0
30.35
41.3
4 th w.of Aug.2007
0.0
2.0
0.0
0.0
14.6
2.3
32.77
43.5
2 nd w. of Sept.2007
0.0
4.6
1.5
0.0
41.3
2.0
31.00
45.5
4 th w. of Sept.2007
0.0
7.5
3.5
0.0
18.3
1.6
30.49
46.9
2 nd w. of Oct.2007
0.0
10.6
9.0
0.0
21.6
13.0
23.00
53.7
4 th w. of Oct.2007
0.0
7.3
8.6
0.0
12
18.0
22.30
45.7
2 nd w. of Nov.2007
0.0
2.6
10.6
0.0
4.6
7.0
21.00
45.0
4 th w. of Nov.2007
71
1.0
13.0
0.0
10.6
4.3
18.00
43.2
2 nd w. of Dec.2007
0.0
0.0
3.6
0.0
3.0
1.3
18.00
44.3
4 th w. of Dec.2007
103
0.3
4.0
149
0.6
12.6
15.00
48.0
2 nd w. of Jan.2008
75
0.3
3.3
0.0
1.0
4.0
12.80
50.0
4 th w. of Jan.2008
0.0
0.0
3.0
0.0
0.0
13.6
12.80
52.3
2 nd w. of Feb.2008
0.0
0.0
2.3
0.0
0.0
9.0
13.20
51.2
4 th w. of Feb.2008
0.0
0.0
1.6
0.0
0.0
5.3
13.10
48.8
2 nd w. of Mar.2008
0.0
0.0
1.3
0.0
0.3
3.0
17.20
51.4
4 th w. of Mar.2008
0.0
41
0.6
0.0
91
0.6
19.53
38.9
2 nd w.of Abril.2008
0.0
30
0.0
0.0
104
0.0
19.00
43.7
Mean
14. 89
7.02
3.89
13.75
19.21
5.02
4 th w.of Abril 2007
2 nd w.of May 2007
* The mean numbers / 40 leaflets per palm
600
Table (2) population density of different stages of Dubas bug Ommatissus
lybicus on palm trees, temp. & R.H. % during 2008/ 2009
Unknown date palm
cultivar
Canary palm
Date
Egg*
Temp.
R.H.
%
Nymph* Adult* Egg* Nymph* Adult*
4 th w.of
Abril 2008
0.0
49.3
0.0
0.0
126.6
0.3
21.00
40.8
2 nd w.of
May 2008
0.0
38.0
0.0
0.0
130.6
0.0
20.70
42.0
4 th w.of May
2008
0.0
36.0
8.0
0.0
50.3
4.3
25.20
42.0
2 nd w.of
Jun.2008
0.0
7.3
14.6
0.0
3.3
29.6
24.90
43.8
4 th w.of
Jun.2008
144.6
2.6
15.3
105.3
1.3
13.6
26.50
48.9
2 nd w.of
Jul.2008
691.6
1.6
7.0
98.6
1.0
3.6
27.50
50.9
4 th w.of
Jul.2008
1098.3
2.3
4.0
43.6
0.0
0.0
27.50
53.3
2 nd w.of
Aug.2008
0.0
0.0
1.3
0.0
0.0
0.0
27.00
57.3
4 th w.of
Aug.2008
0.0
22.0
1.3
0.0
19.3
0.0
26.80
51.1
2 nd w. of
Sept.2008
0.0
41.0
1.3
0.0
48.3
0.3
27.00
56.2
4 th w. of
Sept.2008
0.0
42.0
1.6
0.0
33.3
1.3
26.00
43.0
2 nd w. of
Oct.2008
0.0
20.6
2.0
0.0
41.3
10.0
25.00
47.0
4 th w. of
Oct.2008
3.6
15.6
10.3
0.0
25.6
15.0
23.00
53.7
2 nd w. of
Nov.2008
6.0
18.0
16.3
0.0
8.6
15.6
22.30
54.7
4 th w. of
Nov.2008
0.0
4.0
16.3
31.0
4.0
21.6
21.00
54.0
2 nd w. of
Dec.2008
0.0
3.0
42.0
0.0
1.0
16.3
18.00
43.2
4 th w. of
Dec.2008
17.6
0.0
6.3
67.3
0.0
49.6
15.00
44.3
2 nd w. of
Jan.2009
120.6
0.0
7.0
90.0
0.0
9.3
12.00
48.0
4 th w. of
Jan.2009
146.3
0.0
1.6
34.6
0.0
5.0
15.30
52.0
2 nd w. of
Feb.2009
56.6
0.6
2.0
93.3
0.0
0.3
12.30
51.0
4 th w. of
Feb.2009
35.3
1.3
1.0
0.0
0.0
0.0
13.00
48.2
2 nd w. of
Mar.2009
0.0
0.0
1.0
0.0
0.3
0.0
13.00
51.1
Mean
105.22
14.65
7.28
25 61
22 49
8.7
* The mean numbers / 40 leaflets per palm tree.
601
Table (3) Statistical variance between different stages on each of date palm & canary palm
Comparison
Mean Difference
T value
P value
1.142
0.1535
ns P>0.05
Canary nymph vs. Cultivar nymph
-12.196
1.640
ns P>0.05
Canary adult vs. cultivar adult
-1.129
0.1519
ns P>0.05
Canary egg vs. Cultivar egg
Table (4): correlation coefficient between mean numbers ofDubas
bug (egg, nymph and adult) and both of Temp. & R.H.
Insect stage
Temp.
R.H.%
Dubas egg on canary palm
0.1442
0.1534
Dubas nymph on canary palm
-0.1626
-0.1609
Dubas adult on canary palm
-0.0125
-0.0303
Dubas egg on date palm
-0.0210
-0.0849
Dubas nymph on date palm
0.0932
-0.0474
Dubas adult on date palm
-0.1662
-0.2033
Egg
Nymph
Adult
120
100
80
60
40
20
th
4
4
th
w
.o
f
M
ar
w
.2
.o
00
f
4
8
F
th
e
b
w
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606
OP 29
Acremonium as an endophytic bioagent against date palm
Fusarium wilt
Hatem M. El-Deeb*(1) and Youssef A . Arab **
*National Research Center, Plant Pathology Dept., Dokki, Egypt.
**Al-Azhar Univ., Fac. of Agric., Botany Dept., Egypt.
(1) Correspondence to: H.M.El-Deeb E-mail: israa_el_deeb@yahoo.com
Abstract
A total of 250 endophytic fungal isolates representing 30 morphotaxa were isolated
and characterized, collected from the different living symptomless parts of date palm
trees of orchards of 6 Egyptian governorates. Colonization was greater in samples
from the midrib than in those from laminar tissue and slightly greater at the tip of the
lamina compared with the base of the leaf. Acremonium spp. were frequently isolated
as date palm root endophytes. Acremonium isolates were screened in Petri dishes to
select the highest antagonistic one against an Algerian isolate of Fusarium oxysporum
f.sp. albedinis. Two-week-old axenically reared date palm seedlings grown in Petri
dishes were direct injected with spore suspension (1.5 x 107 spores / ml ) of a pure
culture of the virulent antagonistic isolate of Acremonium sp. One week after
endophytic colonization , date palm seedlings were then challenged with the
pathogen, F. albedinis. The challenged seedlings exhibited a significant reduction in
wilt symptom percentage by 87.0 % , while the seedlings exposed to Fusarial toxin
without pathogen exhibited the wilt disease symptoms. This indicate that the
endophyte ably depress any toxic action of F. albedinis. The endophytic fungus was
recovered from sites distant from the point of inoculation after 6 months from
application , indicate that the Acremonium sp. has the potential to move throughout
the tissue plant , even the end time of trial. The Acremonium mode of action as a bio
control agent was discussed.
Key words : Endophyes , wilt of date-palm , Acremonium sp. , Fusarium oxysporum
f.sp. albedinis , disease resistance inducing .
607
Introduction
Bayoud ( date-palm wilt ) is one of the most destructive Phoenix dactylifera
diseases in many countries , especially in hot inland valleys and desert regions ( Djerbi
, 1983 ). Bayoud causes considerable damages when the disease presents its violent
epidemic aspect (Feather et al.,1989). The causal fungus ( Fusarium oxysporum f.sp.
albedinis ) belongs to the mycoflora of the soil. It is preserved in the form of
clamydospores in the dead tissues of infested palm especially in the roots. The only
productive means of controlling date - palm wilt disease lies in continued research
into resistant varieties ( Djerbi and Sedra , 1986 ).
Recent researches reported that endophytes ( any microorganisms inhabit plants
without causing visible disease symptoms) may exudate chemicals inhibit the growth
of competitors, including harmful insects and mammalian herbivores ( Carroll, 1998
and Azevedo ,2000 ) , produce some plant-growth-promoters ( Varma et al., 1999 and
Adeline et al.,2008) and induce plant resistance against different pathogens ( Redman
et al.,1999, Narisawa et al.,2000, Arnold et al., 2003 and Henson et al., 2007 ). There
is always a degree of antagonistic balance between endophytes and their plant hosts (
Schulz, Barbara and Christina Boyle , 2005 ) except when the host is under stress
conditions. So, fungal endophytes appear to be associated with initial degradation of
the plant tissues following senescence ( Amico, Margherita et al., 2007 ).
Many successful trials including artificially injected of antagonistic fungi to induce
plant disease resistance , i.e. Acremonium sp. against Fusarium wilt of linseed and
tomato ( Grunewaldt-Stoecker et al., 1998 ) , a mutant strain of Colletotrichum sp.
against cucurbits canker
( Redman et al., 1999) and Heteroconium sp. against
Verticillium wilt of Chinese cabbage ( Narisawa et al., 2000 ). Grunewaldt-Stoecker
and Alten ( 2003 ) stated that the injected endophyte must be permanently present
within plant tissues before pathogenic infection, to successfully maintain systemic
disease protection.
In the present study we isolated some endophytic fungi from healthy leaf and root
tissues of Egyptian date-palm trees to further identify possible bio control agents for
Fusarium albedinis and to use them for inducing disease resistance against datepalm wilt disease.
608
Materials and methods
Date-palm sample collection
One hundred and twenty healthy living symptomless leaf and root samples of 60
mature date-palm trees were collected during the ripeness stage from six Egyptian
governorates, i.e. Damietta ( Snannia ) , Sharkia ( Zagazig ) , Fayoum ( Sannoris ) ,
New Vally ( Kharga Oasis ) and Aswan ( Kom Ompo ). Two orchards in each
governorate were chosen. The samples were transferred to the laboratory for
processing as soon as possible.
Isolation and identification of date-palm endophytic fungi :
The samples were washed in running tap water for one hour. Two pieces ( approx.
10 x 5 mm in size were excised from each leaflet lamina , one from near the tip and the
other from near the base. Each of these pieces was then divided into 10 subsamples (
approx. 2 x 2.5 mm in size ). Twenty samples approx. 10 x 5 mm in size were also
taken from the midrib pith of each leaf, and divided laterally into 5 subsamples
approx. 5 x 2 mm in size. Each healthy root was divided onto three sections : root
apex, medium zone and mature zone. Each section ( 5 mm long ) was then sliced into
1 mm thick discs.
All the subsamples were then treated by triple surface sterilization technique as
described by Lodge et al., 1996 by immersion in sequence of 75 % alcohol for 1 min.
and 75 % alcohol for 30 sec., before being rinsed in sterile distilled water. Each set of
subsamples were placed onto malt extract agar ( Theantana et al., 2007 ) which
containing 20 g / l malt extract , 0.033 g / l rose Bengal, 50 mg / l chloramphencol, 15
g / l agar. Each sampled plate ( containing 5 fragments ) in darkness at 27 ºC until
fungal mycelium developed. Then the plates were examined at regular intervals during
eight days. The hyphal tips were cut and transferred to potato dextrose agar ( PDA)
plates. Half strength PDA was used for subculture and stock culture. Due to the large
number of generated colonies and the need to generate rapid results , it was not
possible to grow up every colony for identification. Therefore, strains were selected
for subculture from each primary inoculation plate and then identified using the
morphology of fructiferous bodies and spores.
609
In most cases , fungal strains were identified to genus level., and the nonsporulating colonies and sterile mycelium were designated as morphotypes. The total
number of subcultures isolates was calculated.
Fungal inocula preparations
An Algerian isolate of Fusarium oxysporum f.sp. albedinis was friendly obtained from
Laboratory of Plant Pathology,N.R.C.,Dokki, Giza, Egypt. For rapid characterization of
the Fusarium albedinis isolate , random amplified polymorphic DNA ( RAPD) analysis
was applied by the staff of Biotechnol. Dept., Fac. Agric., Al-Azhar Univ. according to
the method described by Fernandez and Tantaoui ( 1994 ). Fusarial spore suspension ( 10
6
spores / ml ) was obtained through 8 days shaking culture ( Potatoes : 200 g, Sucrose :
20 g , Water : 1000 ml ) ( Feather et al., 1989 ). On the other hand, the spore suspension
of Acremonium antagonistic strain ( 1.5 x 107 spores / ml ) was obtained from bio malt
liquid culture according to Theantana et al., 2007.
Selection of antagonistic endophytic strain under In vitro conditions
The antagonistic effect of the isolated endophytes against the date-palm wilt
pathogen, Fusarium oxysporum f.sp. albedinis was studied by using a mycelial-disc
assay on agar plates. Mycelial discs ( 5 mm in diameter ) were cut from 10-day-old
agar cultures. Two different discs were transferred to each experimental plate : One
from an endophyte fungus and the other from Fusarium oxysporum f.sp. albedinis
culture. Inocula of the two fungal groups in all possible combinations, were posed side
by side at a standard distance of 40 mm onto PDA plates. Four replicates per each
combination were incubated at 25 ± 2 ºC to obtain constant types of interactions (Sabet
and Khan, 1969). Inhibition zone diameters ( in mm ) were determined after 7 days
incubation period.
Endophytically colonization of date-palm seedlings :
One hundred 2-week-old axenically reared date-palm seedlings cv. Medjhool grown
in Petri-dishes were inoculated with the Acremonium spore suspension by using of
direct injection method according to Usuki et al., 2002. One week after endophytic
colonization, date-palm seedlings were transplanted into ( 5 ml spore suspension / Kg
soil ). The treated date-palm seedlings ( Endophytically colonized and Fusariumchallenged ) were compared with another not endophytically colonized date-palm
610
seedlings grown in similar potted infested soil , and with endophytically colonized
date-palm seedlings grown in potted not infested soil.
All the seedlings were left up to 6 months under greenhouse conditions of Fac.
Agric., Al-Azhar Univ., Cairo for the appearance of first signs of date-palm wilt
disease as described by Djerbi and Sedra, 1986 , then the wilted seedlings was
calculated. The causal fungus was reisolated from the wilted leaves and reidentified by
the same previously mentioned procedure of PCR technique. Also, the recovery of
Acremonium sp. was made in sites distant from the point of inoculum and detected by
the PCR procedure of Grunewaldt-Stoecker et al., 1998.
Date-palm leaf cuttings bioassay
Uniform discs from 7-day-old Fusarium oxysporum f.sp. albedinis culture were
transferred into 500 ml-milk pottles , each containing 150 ml of potato dextrose liquid
medium, then incubated without shaking at 25 ± 2 ºC for 15 days. After incubation
period , mycelia were collected and the culture filtrates were centrifuged at 6000 g for
10 min. and the residues were discarded.
The supernatant was evaporated to one-tenth its original volume on water bath. Leaf
cuttings ( 10 cm long ) were taken from 6-month-old endophytically colonized datepalm seedlings and immerged in test tubes containing 5 ml of the concentrated culture
fluid according to the procedure of Hartman et al., 2004. The same procedure was
adopted for the control treatment by using similar date-palm leaf cuttings taken from
not endophytically colonized seedlings and placed in distilled water. The tubes were
kept at room temperature under a continuous light source. Distilled water was daily
added to the tubes to maintain the original water level throughout the incubation
period. Chlorosis was visually detected during 4 days incubation period.
Results and discussion
Isolation and identification of date-palm endophytic fungi
Endophytic fungi were isolated from living symptomless Egyptian date-palm trees.
A total of 250 endophytic fungal isolates representing 30 morphotaxa were derived
from a total of 2400 sample units. Analysis per leaf zone revealed that the midrib was
greater in endophytically colonization ( 73 fungal isolates ) than the lamina ( 41 fungal
isolates ) , and slightly greater at the lamina tip ( 22 fungal isolates ) than the base ones
611
( 19 fungal isolates ). Sixty isolates representing three morphotaxa of mycelia sterilia
and twenty five isolates representing three morphotaxa of yeasts were also isolated.
On the other hand, analysis per root zone revealed that the root apex is not preferred
as a host to endophytic fungi where it supported only light infections ( 31 fungal
isolates ) , whereas 46 and 77 of the fungi were isolated from the root medium zone
and mature zone , respectively. Similar results were obtained in the investigation of
Cannon and Simmons, 2002 on leaf zones of forestry trees and Ovando et al., 2005 on
root zones of some tropical orchards.
Different endophytes were previously isolated from tissues of some palm species by
Rodrigues, 1994, Taylor et al., 1999, Arnold et al., 2001 and Gomez-Vidal et al., 2006
and Lopez-Jimenez and Llorca , 2007. This indicate that no distinct fungal
communities were identified for individual plant species , suggesting that degree of
host preference is low. Acremonium spp. were most frequently endophytes isolated
( 30 isolates ) from Egyptian date-palm tissues followed by Aspergillus ,
Paecilomyces , Penicillium , Alternaria and Cladosporium ( 25,20,20,19 and 19
isolates , respectively ) Species of Fusarium , Gloeosporium , Phomopsis and
Pistalotiopsis were also isolated with low frequencies ( 11, 8, 6 and 5 isolates ,
respectively ),while the lowest frequency was recorded with Myxosporium (2 isolate ).
The fungus Acremonium ( Fam. Moniliaceae ) is an anamorph stage of the
Ascomycetous
fungi
Chaetomium,
Necteripsis
or
Emericellopsis.
Most
of
Acremonium spp. are saprophytic , being often isolated from soil and dead plant
samples ( Sarookhni and Moazzami, 2007 ).
The Acremonium spp. prevalence inside Egyptian date-palms may attributed to soil
alkalinity of Egypt. This explanation was agreed with the result of Specht and Murray
( 1989 ) who reported that Acremonium conidial survival was greater at soil pH 7.5
than 5.7 or 6.7.
Selection of antagonistic endophytic strain under In vitro conditions
All the thirty Acremonium isolates exhibited inhibition effect with different degrees
against the Fusarium oxysporum f.sp. albedinis growth on PDA plates. The aggressive
Acremonium isolate of 95-mm inhibition zone in diameter was chosen for use later as a
tool to induce disease resistance in tissues of date-palm seedlings. This result agreed
612
with using of Acremonium ( Cephalosporium ) spp. As a source of the antibiotic,
Cephalosporin C ( Nash and Huber, 1971 and Matsumura et al., 1980). In this respect,
Acremonium spp. Can produce the toxin, Trichothecene ( Pathre and Mirocha, 1979 ).
Endophytically colonization of date-palm seedlings:
The application of Acremonium spore suspension to date-palm seedlings prior to
Fusarium oxysporum f.sp. albedinis infection achieved many effects :
-No significant damage was detected on endophytically colonized seedlings, which
grown in not infested soil ( control treatment ). This is in agreement with the
meaning of the term " endophyte " of Carroll , 1998. There was a profuse proliferation
of endophytically colonized roots, which was not observed in control treatment (
endophyte- free roots). The fresh weight of endophytically colonized roots increased
with time from 20 gm. At 1 month-old to 75 gm. At 6-month-old. So, there was a
remarkable enhancement to the growth of the endophytically colonized date-palm
seedlings. This result is in harmony with the findings of Verma, 1999 and Redman et
al., 2001. They noted that root colonization with endophytic fungi increased with time.
After 6 months keep under greenhouse conditions , the endophytically colonized and
challenged with Fusarium albedinis date-palm seedlings exhibited a significant
reduction in wilt symptom percentage by 87 %. The precolonized with undesirable
endophytic fungi which transmitted vertically through seed may be reason of the
wilted portion of seedlings ( 13 % ).
This result agreed with those reported by Bargmann and Schonbeck, 1992,
Grunewaldt-Stoecker and Alten,2003. They used Acremonium spp. to induce
resistance against Fusarium wilt of different plants.
To discover the mechanisms behind induced systemic effects of delayed and
reduced Fusarium-wilt symptoms and reduce pathogen spread, Grunewaldt-Stoecker et
al., 1998 carried out histological and physiological investigation on tomato stems
protected by Acremonium endophytes. They found in the based tomato stem , tyloses
occurred early in the period of secondary xylem differentiation, serving as
morphological barriers for subsequent spread of Fusarium. This is contrast to the
behaviour of Acremonium spp. of turf grasses which are distributed mainly by seeds
and synthesize antibiotic or alkaloid substances.
613
The endophytic fungus was recovered from sites distant from the point of inoculum
injection after 6 months from application, indicate that the Acremonium sp. has the
potential to move throughout the tissue plant. This result is agreed with the findings of
Grunewaldt-Stoecker et al., 1998 who stated that endophytic fungi are easily detected
inside the target plant tissues in the second week after injection.
Date-palm leaf cuttings bioassay
All the immerged date-palm cuttings exhibited chlorosis symptoms during the
endophytically colonized tissues and the free endophyte-colonized ones. This indicate
that the endophytically colonized tissues are still able to exhibit the phytotoxic effect
in the presence of any sufficient amount of Fusarium oxysporum f.sp. albedinis toxins
, but the injection of the endophyte fungus to date-palm tissues depressed the toxic
production of the pathogen.
614
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mutualism to parasitism , who control the outcome , host or invader ?.New
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795-804.
[29] Rodrigues, K.F., 1994. The foliar fungal endophytes of the Amazonian palm,
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[32] Schulz, Barbara and Christin Boyle, 2005. The endophytic continuum.
Mycological Research of Cambridge Univ., 109 ( 9 ) : 661-686.
617
[33] Specht, L. P. and T.D. Murray , 1989. Sporulation and survival of conidia of
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the temperate palm, Trachycarpus fortunei within and outside its natural
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Piriformospora indica , a cultivable plant- growth- promoting root endophyte.
App. Environ. Microbiol., 65 : 2741-2744.
618
Table ( 1 ) : Endophytic fungal genera and morphotaxa isolated from leaf and
root zones of Egyptian symptomless date-palm trees.
Endophytic
genera
Morpho
taxa
Lamina
Tip
Base
Acremonium
Alternaria
Aspergillus
Cladosporium
Fusarium
Gloeosporium
Myxosporium
Paecilomyces
Penicillium
Pestalotiopsis
Phomopsis
Sterile
mycelium
Yeast
3
3
3
2
4
1
1
2
3
1
1
3
3
3
3
2
2
2
2
1
4
3
3
3
1
2
1
1
1
4
3
Total
30
Midrib
pith
8
13
8
15
6
6
3
10
4
41
Root zones
Total
Apix
Midium
Mature
1
1
1
6
4
5
4
4
3
4
4
2
15
5
10
9
6
5
2
7
7
4
21
6
73
30
19
25
19
11
8
2
20
20
5
6
60
25
136
250
Table ( 2 ) : Wilted date-palm seedlings % after Acremonium application inside
plant tissues and root fresh weight / seedling ( in gm. ) during 6 months keeping
under greenhouse conditions.
Root fresh weight / seedling ( in gm. )
Treatment
Wilted seedlings
%
2-month-old
3-month-old
6-month-old
Protected seedlings
13
20
43
75
Control I
100
10
25
46
Control II
00
22
47
79
* Control I = Free endophye and challenged seedlings.
** Control II = Endophytically colonized and not challenged seedlings
619
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ا Aا 5ا N Vا" .
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ا!درات ` K^%وف ا ة ٦أ. D
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0ع اF0ري آ K 7%ا!درات 5ا 7ر ]mه ااة Vا^ 7آ م .
"%إ!Mت رة 45أآ م 5 45 Xزار م 3ا5از = 9دا )1أ 0#ا3 )/
o1ل ا!A1ر Pي ?#ت ^ 4? 7أآ م و nر أSا 5 5أ Nا!A1ر A^%ي
را -Dارع ا? 4اض ) 5زار م ا! ( ً ١٥آ?= Kت ا7ر nر
أSا 5 5أ Nء m5 "7 47إ\?ار ا?#ت ا nرة 5اا -Dا?4ي وذp
o1ل ٤أ م 3ا 3Y^Aدل أن ا? 4أآ م [ 7م د = م ا? زار م دا)1
ا ^ ) ، 0#ل دون ا Aأ\ ًoن ا 0#ا^ 7آ م زا Kـ # MAم
ا!P %ل %اه.
"%ا 3 ErCو د 45اآ م 5ا Xfة Xf 3ا^ 37دل رة ا?4
ا^ 7ن ا^Aك دا )1أ 0#ا )/و 9+PإPاث 7و ز fا? 4اض.
620
OP 30
Effect of Some Streptomyces Isolated from Date Palm
Rhizosphere on Some Toxigenic Fungi in Saudi Arabia in Vitro
M.A. El-NAGGAR1, MUNEERA D.F. ALKAHTANI2 , T. M. THABT1, E.A.
SARHAN3 and K.MOSTAFA3
1
National Research Center Lab, GSFMO, KSA; 2Fac. of Sci., Prince Nora Univ., KSA
and
3
Plant Pathology Res. Institute, ARC,EGYPT
Corresponding Author: Medhat A. El-Naggar National Research Center Lab,
GSFMO, KSA
E-mail: medhat14@yahoo.com
Abstract
Date palm (Phoenix dactylifera L.) was considered the main crop in deserts and arid
areas as Saudi Arabia. Some streptomyces was isolated from date palm rhizosphere of
a number of cultivars Barhi, Khalas, Sullaj and Sukkari as well as different fungi
species in Ghat and El-Gouf region. The isolation of streptomyces was performed on
Universal Growth Agar (BUG) while, the identification was conducted by BIOLOG
technique. The major streptomyces in survey were Streptomyces plicatus followed by
St .rimosus, St .rutgersensus and St. Griseus. The soil borne fungi count was dropped
in the tested soil with increased numbers of streptomyces. There was a positive
significant correlation between cultivar date and the population of isolated
streptomyces. In vivo, the effect of isolated streptomyces has been studied on more
toxigenic fungi. The Evaluation of the different mycotoxins has been using ELISA,
Immuno-affinity column and HPLC. The most of tested mycotoxins concentration
were reduced significantly. It is concluded from this study Some isolates of
streptomyces that establish on the soil of palm trees have the ability to reduce
mycotoxins produced by fungi and also reduce the occurrence therefore can be applied
in wide range the field of mycotoxins bio-control.
Keywords: Phoenix dactylifera, Aflatoxins, Zearalenone, Fumonisin,T-2, AOH,
AME , Streptomyces
621
Introduction
Date palm (Phoenix dactylifera L.) is one of the staple crops in desert areas where it
is of nutritional value and high economic. However, one of the oldest cultivated crops
in arid areas, which bear the harsh environmental conditions. The number of the date
palms is about 100 million worldwide, of which 62 million palms can be found in the
Arab world. The Date Palm is a palm extensively cultivated for its edible fruit. Due to
its long history of cultivation for fruit, its exact native distribution is unknown, but
probably originated somewhere in the desert oases of northern Africa, and perhaps
also southwest Asia. On a commercial scale, the Middle East and North Africa are the
major date palm producing areas in the world. Normally, this palm is cultivated for
subsistence/local markets on small holder farms besides other crops. Because of its
high nutritional value, great yields and its long life (yielding up to 100 years). The
fruits of date palm is one of the most food-rich integrated nutrients as carbohydrates,
amino acids, fatty acids, salts, vitamins, minerals, and dietary fibre. The world
production of dates has increased by 2.9% over the past 40 years.(Barreveld, 1993; AlShahib and Marshall, 2003; Erskine et.al, 2003 ).
The Kingdom of Saudi Arabia with an estimated 25 million date palms produces
nearly a million tons of dates annually accounting for about 15% of the global date
production. The date palm covering approximately 72% of the total area under
permanent crops. More than 400 different date palm cultivars are reported to exist in
Saudi Arabia (Anonymous, 2006a; Anonymous, 2009; Al-Abbad, 2011).
Root of the palm may be exposed to attack from many pathogenic micro-organisms,
whether bacteria, fungi or actinomyces. The rhizosphere area is rich in diverse and
complex interactions between organisms and each other. Rooted date palm are infected
with F. solani and F. moniliforme and necrotic date palm tops are infected with
Botryodiplodia theobromae .Declined date palm roots are infected with Fusarium
oxysporum, F. solani, F. moniliforme (and with Thielaviopsis paradoxa (Djerbi et al.,
1986; Al-Roubaie et al., 1987; Abbas et al., 1989). Several soilborne fungi attach date
palm were causing root rot, wilt and decline diseases. The dominant fungi associated
with date palm, death and decline were Fusarium oxysporum, Diplodia phoenicum,
622
Ceratocystis radicicola, and Phomopsis phoenicola (Rattan and Al-Dboon, 1980;
Mousiri et al., 2000).
Actinomyces considered one of micro-organisms that may be present in high
density in the rhizosphere. The studies indicated that 89% of the actinomyces isolates
belonged to the genus Streptomyces, 11% were non-Streptomycetes: Actinomadura
sp., Microbispora sp., Micromonospora sp., Nocardia sp, Nonomurea sp. and three
isolates were unclassified. The highest number and diversity of actinomycetes were
isolated from rhizosphere soil. Twenty-three Streptomyces isolates showed activity
against at least one of the five phytopathogenic fungi: Alternaria brassicicola,
Collectotrichum gloeosporioides, Fusarium oxysporum, Penicillium digitatum and
Sclerotium rolfsi (Khamna et al., 2009).
Since the discovery of mycotoxins in the sixties of last century and is still ongoing
research to find the most appropriate ways to reduce the risk of these toxins. There are
many ways to reduce the risk of mycotoxins by detoxification in contaminated
agriculture materials through various physical, chemical and biological treatments.
There was many strains of Streptomyces, previously isolated from soil, presented a
strong and specific antagonistic effect against toxigenic fungi. This action was
attributed to a proteinaceous compound (molecular weight estimated to be 14 kDa)
present in the supernatant of the culture. This compound showed non-chitinolytic
fungicidal activity (Fulgueira et al., 2004).
The study aims to isolate some streptomyces strains from date palm rhizosphere in
different area of Saudi Arabia. Also, study their impact on soil bone fungi enumeration
and type as well as their effect on fungal ability to excrete different mycotoxins in vitro.
Materials and methods
Sampling procedure:
Date palm farming soil samples obtained from 20-25 cm depth and then, collected
in sterile containers from different areas (Ghat and El-Gouf) in Saudi Arabia. The
samples will be taken
randomly over the year 2010 from rhizosphere of many
cultivars as Barhi, Khalas, Sullaj and Sukkari.
623
Isolation of Streptomyces and fungi from the farming soil samples:
The samples were dried for 3 days at room temperature to reduce the bacterial flora
with no harms to the growth of Streptomyces (Goodfellow and Minnikin, 1985). One
gram of air dried soil was shaken in a flask containing 100 ml of distilled water, and
serial dilutions were placed on starch casein solid media (Kuster and Williams, 1964).
The plates were incubated at 29 ± 2 until the sporulation of Streptomyces colonies
occurred. The colonies (where the mycelia remained intact and the aerial mycelia and
long spore chain were abundant) were picked up and transferred to a starch nitrate
medium (Lechevalier, 1970). The isolation of fungal isolation as mention with
streptomyces except the isolations trial were conducted on fresh sample and the
dilution were placed on PDA media plates.
Identification of Streptomyces and fungal isolates:
Pure cultures of streptomyces were obtained from selected colonies for repeated
sub-culturing. An agar disk of a grown Streptomyces culture was removed aseptically
and placed on BUG media plate to identify. the incubation extended for 48 hours at
29ºC and then identified by BIOLOG system according to Smalla et al., 1998.
After 3 days incubation for plates at 30°C, fungi developed from, then hyphal tips
or single spores were transferred to test tubes containing slant PDA medium. The
purified fungi were verified and identified by the author according to Barnett (1960),
Subramanian (1971) and Tousson and Nelson (1976).
Mycotoxins determination:
Mycotoxins have been estimated for more frequent fungi which isolated from Ghat
and El-Gouf areas. The isolates of Ghat were 6 isolates Apergillus parasiticus and
other six isolates of Fusarium solani. While, El-Gouf area was represented by six
isolates of Alternaria alternata and other six of Fusarium oxysporum. All selected
isolates were grown on SMKY liquid media for 7 days at 27±2°C. The different
concentration of toxins is considered the control.
Aspergillus toxin:
5 ml of homogenized sample was mixed with 70% methanol (1:5 ratios) and was
infiltrated. 5 ml suspension diluted to 1:3 ratio. A total of 20 ml solution was placed
into ELISA colon by injector. Immuno affinity colon (IAC) was used for detection of
624
aflatoxin. This colon is composed of gel suspension which covalent binding with
monoclonal antibodies and these antibodies are specific for aflatoxin B1, B2, G1 and
G2. Standard preparing procedure for aflatoxin types was applied also for aflatoxin
B1, B2, G1 and G2. However incubation duration was extended to 2 h from 30 min.
Ridasoft win programme was used for data obtained from reading results of aflatoxins.
Theses protocol according to Ozaslan et al., 2011.
Fusarium toxins:
Fumonisin toxins were determined according to the method described by Mazzani
et al. (2001) as follows: fifty grams of ground sample of each isolate were blended
with 5 g sodium chloride and 100 ml methanol: water (80:20) at a high speed for one
min., then filtered through glass micro-fiber filter papers. Ten ml of the filtrate were
diluted with 40 ml of wash buffer and filtered again through 1.0 μm micro-fiber filter.
Ten ml of the diluted extract were passed through fumontest column (Vicam
Company) and then, the column was washed by 10 ml of the same dilute solution. The
fumonisin was eluted by passing one ml of HPLC grade methanol through the column
and the elutes were re-collected again. One ml of each of developer A (Vicam product
No. G5005 ) and developer B (Vicam product No. G5004) were added to the elute and
placed in calibrated fluorometer (Series-4 / Vicam). The zearalenone and T-2
estimated by same protocol as mention by method of Martins et al. (2003).
Alternaria toxins:
50 ml of crude extract transferred to blender cup with the help of 150 ml methanol.
It was blended at low speed for 3 minutes and transferred to a glass funnel fitted with a
fluted filter paper. An additional 50 ml methanol was used for washing the residues
left in the blender cup into the filter paper. An aliquot of 200 ml of the filtrate was
collected into a beaker and 60 ml of a 10% ammonium sulfate solution was added. The
mixture was filtered through fluted filter paper. An aliquot of 200 ml of the filtrate, or
less, was then transferred to a separating funnel and 50 ml of water at 8°C or below
were added. Two extractions with 40 ml chloroform, shaking for 2 minutes each time,
were conducted. All the chloroform was collected in a separating funnel and washed
with 30 ml ultra-pure water at 5 - 8°C. The chloroform was then transferred to a
graduated cylinder and the volume noted for future calculations. The chloroform
625
extract was evaporated in a rotary evaporator at 35°C. The residue was dissolved in
2 ml methanol and filtered through anhydrous sodium sulfate. The HPLC system
consisted of a HP 1050 liquid chromatography equipped. The analytical column was
Spherisorb ODS-2, 5 mm, 250 mm. The mobile phase was methanol/water (80:20)
containing 300 mg ZnSO4. H2O/L, 0.7 ml/min. The wavelength for recording
chromatograms was 250 nm. A calibration curve was constructed for quantification
purposes using the toxin standards according to Silvana (2000).
Mycotoxins detoxification in vitro:
Under same condition, the selected isolates (Alternaria alternate, Aspergillus
parasiticus, Fusarium solani and Fusarium oxysporum) were grown on liquid SMKY
media with streptomyces plicatus, St. rimosu, St. rutgersensus and St. griseus
individually and once again a mixture. Each treatment was incubated at 27±2°C for 7
days in three replicates. Then different mycotoxins were determined as mention before.
Statistical analysis:
The obtained data were statistically analysed using the analysis of variance
(ANOVA) with the MSTAT-C statistical package The least significant difference
procedure (LSD) was used at 0.05 level of probability (Fisher, 1948).
Results and discussion
The obtained data in Table (1) showed that, 141 fungal isolates belong on 28
species and 14 genera were isolated from soil of date palm from Ghat and El-Gouf.
Seventy five fungal isolates were isolated from the Ghat area of rhizosphere soil of
date palm Barhi, Khalas, Sullaj and Sukkari cultivars. Aspergillus parasiticus and
Fusarium solani were the highest frequencies about 8.0 %, while isolates Alternaria
sp., Chaetosphaeropsis sp., Fusarium equiseti, Mycosphaerella sp., Penicillium
chrysogenum, and Phoma sp. were lowest frequent 1.3%. The rhizosphere soil of
Sullaj date palm cultivar was more contain of fungal isolates (31) than other cultivars
Barhi, Khalas, and Sukkari being 19, 10 and 15, respectively. In El-Gouf area the total
isolated fungi were 66 isolates, while the most frequencies were Alternaria alternate
and Fusarium oxysporum (9.1%). The lowest frequencies were concentrated with
Chaetomium spp., Phoma spp., Gliocladium spp. (15%). The rhizosphere soil of
Khalas cultivar was less infestations followed by Sukkari, Barhi and Sullaj. The
626
differences in type and number of fungi isolated from rhizosphere soil from both Ghat
and El-Gouf may be due to difference in characteristics of soil such as pH,
fertilization, humidity percentage, root secretions, microbial competition and climatic
conditions as well as the different of date palm cultivars, tree age and plant density per
the unit of area. These results was in a harmony with the findings of Albiach et al.
2000, Broeckling et al., 2008 and Ndubuisi-Nnaji,2011. The most common fungal
isolates were Aspergillus parasiticus, Fusarium solani, Aspergillus flavus, Fusarium
oxysporum, penicillium expansum , Aspergillus niger and Botryodiplodia theobromae
in both different locations Ghat and El-Gouf. These results was largely similar with
recorded by Mousiri et al., 2000 and Abdullahi et al.,2010
In the Ghat area was isolated 14 streptomyces strains. Streptomyces plicatus was the
most frequent 38.7% followed by St. rimosu 21.4%, while the lower frequency was in
St. rutgersensus7.1% and St. griseus7.1%. Streptomyces spp. presented with 28.6% of
frequencies. The most number of streptomyces was isolated from Khalas cultivars soil
while; the lower one was from Sullaj cultivars, (Table 2). All streptomyces isolated
from El-Gouf area was 18 strains as 7 stains of St. plicatus, 4 strain of St. rimosu, 2
strains of St. rutgersensus and 1 strains of St. griseus while 4 strains of Streptomyces
spp. The most stains of streptomyces concentrated in Khalas rhizosphere followed by
Sukkari, Barhi and Sullaj as 8, 5, 3 and 2, respectively.
Through the isolation trials; 75 of fungal isolates and 14 of streptomyces were isolated
from Ghat area, while from El-Gouf were 66 fungal isolates and 18 streptomyces. The
isolation was conducted for fungi and actinomyces from same samples of cultivars
Khalas, Sukkari, Barhi and Sullaj under same conditions but each one had different
media and special incubation conditions. An inverse association was found between the
number and the species of isolated fungi and streptomyces in both regions of Ghat and
El-Gouf. These results may be return to antibiosis of streptomyces against a wide range
of fungi which inhibit in date palm soil rhizosphere and this findings agreement with
recorded by Haskell et al., 1958; Lockwood, 1964; Errakhi et al., 2007 . The most
common streptomyces strains of soil samples in both regions was St. plicatus, St.
Streptomyces rimosu, Streptomyces rutgersensus and Streptomyces griseus. This result
was in parallel with Carvajal,1946; Abd-Allah,2001and Hozzein et al., 2011.
627
Is clear from Table 3 that the isolation No. As. parasiticus 6 was a more excrete to
total aflatoxins while, isolates No. As. parasiticus1, As. parasiticus4 As. parasiticus5
were equal the amount of aflatoxin B1. Streptomyces plicatus was the most effective in
aflatoxins detoxification especially with As. parasiticus4 while, St. griseus was the
lowest effective. The result was in parallel with recorded by Hassan et al., 2011.
Table 4 revealed that, F. solani 2 was more producing of Fusarium toxins
(fumonisin, zearalenone and T-2 toxins) than other isolates. The most detoxification
was occurred with St. plicatus with F. solani 6 while the lowest effective was St.
griseus with F. solani 4. This findings was similar with Alkahtani,2011. Isolate No. Alt
alternate 3 produced a higher concentration of AOH toxins (15 ppb) while, both
isolate Alt alternate 3 and Alt alternate 6 excreted the higher concentration of AME
toxin (9 ppb) than other tested isolates. Alternaria toxins were decreased with
inoculation with tested streptomyces strains especially St. plicatus and the reduction of
toxins reached to 69.6% with Alternaria alternate No.1. The detoxification ratio was
decreased to 22.1% with Alternaria alternate isolate No 4 when inoculated with
streptomyces griseus, (Table 5 and Fig 1). This results was largely matching with
recorded by Karlovsky,1999
Fusarium oxysporum 3 excreted the highest concentration of total Fusarium toxin
and zearalenone (1709 and 353 ppb), respectively but the highest concentration of
fumonisin and T-2 toxin were produced by F. oxysporum 5and F. oxysporum 4 as
1413 and 98 ppb, respectively. The highest reduction of total toxins was 70.5% and
observed when St. plicatus inoculated with isolate F. oxysporum2. On contrast the
lowest reduction was occurred by St. griseus when inoculated with tested isolate of F.
oxysporum 1. In vitro streptomyces strains had a clear role in reducing aflatoxin B1,
and total aflatoxin, fumonisin, zearalenone, T-2 toxin, AOH and AME toxins with all
tested isolates of fungi while the St. plicatus strain was more efficient than other
strains and these findings was in a harmony with Nakayama et al., 1999; Zucchi et
al.,2008 and; . This efficiency may be return to genetic structure or may be due to the
use of fungi secondary metabolites in their metabolisms and/or antagonistic behaviour
of these strains.
628
Conclusion
Through this study fungal diversity exists in the soil surrounding the root of some
palm cultivars. Khalas was less frequent varieties of these fungi followed by, Barhi
and Sullaj so that can be recommended for planting Khalas and Sukkari to avoid any
fungal infection. In addition there were some strains of streptomyces which has been
associated with some types of some tested palm cultivars. Laboratory studies showed
the ability of
streptomyces to reduce the amount of mycotoxins produced as
aflatoxins, fumonisin, zearalenone,T-2, AOH and AME toxin.
There was
differentiation in the ability of these streptomyces strains to toxins detoxify since the
streptomyces plicatus had the most efficiency, the St. griseus had a weak ability. The
active strains can be used as bio-agent to mycotoxins detoxification in animal and
poultry feed and/or used on different crops to reduce the mycotoxins contamination
ratio. Although these trend needs to be complementary studies
Acknowledgment
The authors wish to thank Dr. Eman M. Abdelkareem (Plant Pathology Institute, Egypt)
and Dr. Guy mozolowski (University of Nottingham, England) for thoughtful criticisms on
earlier versions of this manuscript, which substantially improved this manuscript.
629
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633
Table 1: The occurrence and frequency percentage of fungi isolated from rhizosphere
soil of different date palm cultivars, on PDA media after 7days incubation at 27 ±2°C
Fungal
isolates
Ghat
El-Gouf
No. of
isolates
% of
freque.
Barhi
Khalas
Sullaj
Sukkari
No. of
isolates
% of
freque.
1
4
5.3
2
1
2
1
6
9.1
1
0
1
1.3
1
0
1
0
2
3.0
0
1
1
3
4.0
1
0
2
1
4
6.1
1
1
2
1
5
6.7
1
0
1
1
3
4.5
Aspergillus fumigatus
2
0
1
0
3
4.0
0
1
2
0
3
4.5
Aspergillus parasiticus
1
1
2
2
6
8.0
1
0
3
0
4
6.1
Aspergillus spp.
0
0
2
1
3
4.0
0
1
0
0
1
1.5
Botryodiplodia
theobromae
1
0
1
0
2
2.7
1
0
2
1
4
Chaetomium sp.
1
0
1
0
2
2.7
0
0
1
0
1
1.5
Chaetosphaeropsis sp.
0
1
0
0
1
1.3
0
0
0
0
0
0.0
Diplodia sp
1
0
1
1
3
4.0
1
0
1
0
2
3.0
Fusarium sp.
1
0
2
1
4
5.3
0
1
1
0
2
3.0
Fusarium equiseti
1
0
0
0
1
1.3
1
0
1
1
3
4.5
Fusarium moniliforme
1
2
0
1
4
5.3
1
0
1
0
2
3.0
Fusarium semitectum
0
0
2
1
3
4.0
1
1
1
0
3
4.5
Fusarium solani
1
1
2
2
6
8.0
1
0
0
1
2
3.0
Fusarium oxysporum
1
0
3
1
5
6.7
2
1
2
1
6
9.1
Gliocladium sp.
0
0
0
0
0
0.0
0
0
1
0
1
1.5
Graphiola phoenicis
0
0
0
0
0
0.0
0
0
0
0
0
0.0
Mycosphaerella sp.
0
0
1
0
1
1.3
0
0
0
0
0
0.0
Pencillium spp.
2
0
2
0
4
5.3
0
1
1
0
2
3.0
Penicillium notatum
0
1
0
0
1
1.3
1
0
1
1
3
4.5
Penicillium italicum
0
1
1
1
3
4.0
0
0
2
0
2
3.0
Penicillium
chrysogenum
0
0
1
0
1
1.3
1
0
0
1
2
3.0
Penicillium expansum
1
0
3
1
5
6.7
0
0
2
0
2
3.0
Phoma sp.
0
0
1
0
1
1.3
1
0
0
0
1
1.5
Phomopsis sp.
1
0
0
0
1
1.3
0
0
1
1
2
3.0
Thielaviopsis paradoxa
1
1
0
0
2
2.7
1
0
1
1
3
4.5
Total
19
10
31
15
75
18
7
30
11
66
Barhi
Khalas
Sullaj
Sukkari
Alternaria alternata
1
1
1
Alternaria sp.
0
0
Aspergillus flavus
1
Aspergillus niger
634
6.1
Table.2: The occurrence and frequency percentage of streptomyces strains from rhizosphere soil of
different date palm cultivars, on BUG media after 2days incubation at 29 ±2°C
Streptomyces
strains
Ghat
El-Gouf
Barhi
Khalas
Sullaj
Sukkari
No. of
isolates
% of
freque.
Barhi
Khalas
Sullaj
Sukkari
No. of
isolates
% of
freque.
Streptomyces
plicatus
1
3
0
1
5
35.7
1
3
1
2
7
38.9
Streptomyces
rimosu
1
1
0
1
3
21.4
1
2
0
1
4
22.2
Streptomyces
rutgersensus
0
1
0
0
1
7.1
0
1
0
1
2
11.1
Streptomyces
griseus
0
1
0
0
1
7.1
0
1
0
0
1
5.6
Streptomyces sp.
1
1
1
1
4
28.6
1
1
1
1
4
22.2
Total
3
7
1
3
14
99.9
3
8
2
5
18
100
Table 3: Determination of total aflatoxins and aflatoxin B1produced by six
isolates of A. parasiticus isolated from Ghat area, grown on SMKY media for 7
days at 27 ±2°C and detoxification effect of some different streptomyces strains.
Treatments
As. Parasiticus1
As. Parasiticus2
As. Parasiticus3
As. Parasiticus4
As. Parasiticus5
As. Parasiticus6
Toxins
measurement
B1
Total
% of
reduction
B1
Total
% of
reduction
B1
Total
% of
reduction
B1
Total
% of
reduction
B1
Total
% of
reduction
B1
Total
% of
reduction
Total aflatoxins and B1 toxins (ppb) excreted by 6 isolates of A.
parasiticus from Ghat and effect of different streptomyces strains on
toxin concentration
St.
St.
Contro
St.
St.
Mixture
rutgersensu
griseus
l
plicatus
rimosu
s
11
20
0
4
7
65.0
6
10
50.0
5
11
45.0
5
13
35.0
3
6
70.0
10
21
0
3
8
61.9
5
9
57.1
7
12
42.9
6
14
33.3
3
9
57.1
10
19
0
5
7
63.2
7
9
52.6
7
8
57.9
8
10
47.4
6
7
63.2
11
20
0
2
6
70.0
6
9
55.0
6
11
45.0
7
12
40.0
3
6
70.0
11
19
0
4
8
57.9
5
10
47.4
5
12
36.8
4
11
42.1
3
7
63.2
10
22
0
3
7
68.2
3
9
59.1
6
10
54.5
8
12
45.5
4
7
68.2
LSD at 5%: 2.78
635
Table 4: Determination of Fumonisin, Zearalenone and T-2 toxin produced by six
isolates of F. solani isolated from Ghat area, grown on SMKY media for 7 days at
27 ±2°C and detoxification effect of some different streptomyces strains.
Treatments
F. solani1
F. solani2
F. solani3
F. solani4
F. solani5
F. solani16
Toxins
measurement
Fumonisin, Zearalenone and T-2 toxin (ppb) excreted by 6 Isolates
of F. solani isolated from Ghat area and effect of different
streptomyces strains on toxin concentration
Contro
l
St.
plicatus
St.
rimosu
Fumonisin
1050
260
280
Zearalenone
150
85
T-2
50
Total
St.
griseus
Mixture
355
325
255
100
120
135
83
20
27
35
30
19
1250
365
407
510
490
357
% of reduction
0.0
70.8
67.4
59.2
60.8
71.4
Fumonisin
1200
340
390
425
440
320
Zearalenone
325
115
135
200
185
110
T-2
70
25
35
40
38
23
Total
1595
480
560
665
663
453
% of reduction
0.0
69.9
64.9
58.3
58.4
71.6
Fumonisin
950
285
310
315
325
283
Zearalenone
190
75
89
110
98
72
T-2
90
42
49
57
60
40
Total
1230
402
448
482
483
395
% of reduction
0.0
67.3
63.6
60.8
60.7
67.9
Fumonisin
1300
410
460
485
530
408
Zearalenone
220
70
69
84
90
66
T-2
40
14
18
24
21
14
Total
1560
494
547
593
641
488
% of reduction
0.0
68.3
64.9
62.0
58.9
68.7
Fumonisin
650
205
215
227
238
200
Zearalenone
250
88
89
125
120
86
T-2
50
18
21
29
27
14
Total
950
311
325
381
385
300
% of reduction
0.0
67.3
65.8
59.9
59.5
68.4
Fumonisin
1100
198
212
240
261
195
Zearalenone
240
55
61
66
78
55
T-2
30
11
18
19
19
11
Total
1370
264
291
325
358
261
% of reduction
0.0
80.7
78.8
76.3
73.9
80.9
LSD at 5%: 3.36
636
St.
rutgersensus
Table 5: Determination of Alternariol and Alternariol monomethyl ether
produced by six isolates of Alternaria alternate isolated from El-Gouf area,
grown on SMKY media for 7 days at 27 ±2°C and detoxification effect of some
different streptomyces strains.
Treatments
Alt. alternate1
Alt. alternate2
Alt. alternate3
Alt. alternate4
Alt. alternate5
Toxins
measuremen
t
Alternariol and Alternariol monomethyl ether (ppb) excreted by
6 isolates of Alternaria alternate isolated from El-Gouf area and
effect of different streptomyces strains on toxin concentration
Control
St.
plicatus
St.
rimosu
St.
rutgersens
us
St.
griseus
Mixture
AOH
14.5
4
4.5
5.8
5.6
3.5
AME
8.5
3
3.5
4.2
4
2.5
Total toxins
23
7
8
10
9.6
6
% of
reduction
0.0
69.6
65.2
56.5
58.3
73.9
AOH
10.5
3.5
4.1
6.2
6
3.5
AME
6.5
2.5
3
3.7
3.5
1.9
Total toxins
17
6
7.1
9.9
9.5
5.4
% of
reduction
0.0
64.7
58.2
41.8
44.1
68.2
AOH
15
6.5
6.5
8.4
8.8
4.5
AME
9
5.5
6
6.9
6.7
3.5
Total toxins
24
12
12.5
15.3
15.5
8
% of
reduction
0.0
50.0
47.9
36.3
35.4
66.7
AOH
8
5
6.8
7.8
7.6
4
AME
6
2
2.6
3.5
3.3
1.6
Total toxins
14
7
9.4
11.3
10.9
5.6
% of
reduction
0.0
50.0
32.9
19.3
22.1
60.0
AOH
11
5
5.9
6.9
7.1
3.8
LSD at 5%: 0.97
637
Table. 6: Determination of Fumonisin, Zearalenone and T-2 toxin produced by six
isolates of F. oxysprum isolated from El-Ghouf area, grown on SMKY media for 7
days at 27 ±2°C and detoxification effect of some different streptomyces strains.
Treatments
F. oxysporum1
F. oxysporum2
F. oxysporum3
F. oxysporum4
F. oxysporum5
F. oxysporum6
Toxins
measurement
Fumonisin, Zearalenone and T-2 toxin (ppb) excreted by 6 Isolates of
F. oxysporum isolated from Ghat area and effect of different
streptomyces strains on toxin concentration
Control
St.
plicatus
St.
rimosu
St.
rutgersensus
St.
griseus
Mixture
Fumonisin
815
253
290
333
441
239
Zearalenone
217
99
118
178
161
100
T-2
43
21
25
30
27
21
Total
1076
373
433
541
629
360
% of
reduction
0.0
65.4
59.7
49.7
41.5
66.6
Fumonisin
1141
286
301
394
349
277
Zearalenone
163
93
108
133
145
90
T-2
54
22
29
39
32
21
Total
1359
401
438
567
527
388
% of
reduction
0.0
70.5
67.8
58.3
61.2
71.4
Fumonisin
1280
374
419
472
473
348
Zearalenone
353
126
145
222
199
120
T-2
76
27
38
44
41
25
Total
1709
527
602
739
713
492
% of
reduction
0.0
69.1
64.8
56.8
58.3
71.2
Fumonisin
1033
313
333
350
349
308
Zearalenone
207
82
96
122
105
78
T-2
98
46
53
63
65
43
Total
1337
442
482
536
519
429
% of
reduction
0.0
67.0
64.0
59.9
61.2
67.9
Fumonisin
1413
451
495
539
570
443
Zearalenone
239
77
74
93
97
72
T-2
43
15
19
27
23
15
Total
1696
543
588
659
689
530
% of
reduction
0.0
68.0
65.3
61.2
59.4
68.7
Fumonisin
707
225
231
252
256
217
Zearalenone
272
97
96
139
129
93
T-2
54
20
23
32
29
15
Total
1033
342
349
423
414
326
% of
reduction
0.0
66.9
66.2
59.0
59.9
68.4
LSD at 5%: 2.25
638
Fig(1): HPLC histogram to determine the Alternaria toxins; AOH and AME
which produced by different six isolates of Alternaria alernata in SMKY media at
7 days incubation at 27±2°C.
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OP 31
Some Inorganic Salts for Production of Sterile Adults of the Red Palm
Weevil Rhynchophorus ferruginous (Coleoptera: Curculionidae)
Aziza Sharaby, F.M. EL-Hawari, S.A. Ibrahim
Pests and Plant Protection Dept. National Research Center, Cairo , Egypt
sharabyaziza@yahoo.com .
Abstract
Fifteen inorganic salts were evaluated as possible sterilants against the Red Palm
Weevil (RPW) Rhynchophprus ferrugineus, by applying the pupal dipping method in a
screening program. Results showed that all tested salts adversely affected the adult
emergence and reduced the egg laying capacity and egg hatchability. Among the tested
salts Ca(OH)2 , LiCI , CuCL2 , ZnSO4 ,, kBr and KI gave various 97 - 100% sterility at
the tested nontoxic concentration under different pupal dipping periods. Percentage of
sterility increased with the increase of the concentration and the dipping period. All
other salts induced degrees of sterility ranging from 63 to 96%.
Key words: Rhynchophorous ferruginous , chemosterilants , inorganic salts
Introduction
The red palm weevil Rhynchophorous ferruginous is the most important insect pest
for the date palm trees in the Middle East and Gulf states. The insect could be
distributed through most of the Arab world that cultivated the date palm trees by
transferring the infested shoots from place to another. The larvae of the insect feed
inside the tree trunk making dangerous tunnels and led to dumping off the tree . The
new trend of pest control is searching for safe and available alternative materials of
dangerous insecticides. Many attempts have been made to chemo sterilize the insects
in adult stages, less effort has been made on treatment of the pupal stages. [1] tested 18
alkylating compounds, 10 antimetabolites and 8 compounds belonging to
miscellaneous groups by pupal dipping methods, they found that the promising
chemosterilants for Sitotroga cerealella (Oliver) are to be found among the aziridines
especially Tepa, Metepa, Thiotepa and ENT50761 at low concentrations causing high
sterility. [2] recorded that sterility can be induced in adults of rice moth Corcyra
641
cephalonica (Stant) following pupal dipping methods in different amino acids :
Ornithin, ά-Glutamine, Valine, Serine and Alanine. [3]
found that twenty four
inorganic salts evaluated as sterilants for the lesser cotton leaf worm Spodoptera
exigua(Hubn.),by applying the pupal dipping techniques in a screening program. A
Compendium of inorganic substances used in European pest control before 1850 were
recorded by [4] list of 24 inorganic chemicals used in European agriculture up to 1850
for pest control is given ,together with description of their recorded. Attempts have
been made to assess their possible efficacy. In the present study some inorganic salts
were tested for the first time for their efficacy by pupal dipping techniques with a view
to evaluate the promising and effective chemosterilants against the RPW R .
ferruginous as a new approach in its control programs .
Material and methods
Tested insects were obtained from infested palm trees then maintained under
laboratory culture on bulb of date palm tree (main host) [5] at 28±2c º where its life
cycle not exceeding 55-60 day and the time for pupation from egg lying was 30days.
Pupae with its cocoon were collected for the experimental tests. Three different serial
concentrations 5,10and 15 % of fifteen inorganic salts were prepared by dissolving in
distilled water . Pupae , about 2-4days old were selected from the culture for dipping
treatment. The pupae were placed in a small wire basket (10×20cm.)and immersed
inside a beaker (500ml.)containing the tested salt solution. The experiments were
carried out by taking two different dipping periods 20and 30 min. Treated pupae were
washed with water and left to dry, then introduced into a glass jars ,left till adult
emergence. Both sexes of pupae were accordingly treated with the same treatment,
while the control was treated with distilled water and run side by side. Five pupae were
used for each test , all tests were repeated three times . Observations on the percentage
of adults emergence from pupae was recorded both from treated
and untreated
individuals . To evaluate the fecundity of adults and egg hatchability , pairs of one
male with one female of normal adults emerged out of treated pupae were confined in
a glass egg laying jars (1 Litre ) containing piece of date palm tree bulb as a site for
egg laying. Deposited eggs were collected every two days and counted. Data were
642
subjected to analysis of variance [6] for determining their significance. Sterilizing
percentages were calculated by the formula described by [7] as follows .
Corrected % effect = 100 ( Experiment % effect
Control % effect
% Sterilization is defined as 100 ( 1- f h), where f and h are the corrected decimal
fractions of percentages of fecundity and hatchability , respectively.
The tested inorganic salts were listed in Table 1
Hydroxides: NaOH, KOH, Ca(OH)2 .
Chlorides: LiCl, NaCl , MgCl2 , Cucl2 .
Nitrate: NaNO3
Acetate: CH2 COOLi
Sulphate: Na2 SO4 , Mg SO4 , ZnSO4
Bromide: KBr
Iodide: KI
Phosphate: Na2 HpO4 .
Results and discussion
Results for screening the 15 inorganic salts at various concentrations and
dipping
periods are given in Table (1) . It is apparent from the results that six salts gave the best
results where they gave 100% sterility at the different tested concentrations and the
dipping intervals 15 and 30 min. The six salts were as follows: Hydroxides: Ca(OH)2 and
koh ,Chlorides:
Licl and cucl2 , Sulphate: ZnSO4 , Bromide: KBr and Iodide: KI
However, all tested salts adversely affected the adult emergence and reduced the
egg laying capacity and egg hatchability comparing with the control test . The sterility
produced was of a high level for most of the tested salts, percentages of fecundity and
hatchability were significantly reduced by increasing the salt concentration and the
time interval of pupal dipping. [3] recorded that some inorganic salts had a promising
effect as chemosterilants against S.exigua by applying the pupal dipping method in a
screening program. Among the tested salts , Ca(OH)2 , NaOH2 ,LiCl2 ,CuCl2 ,NHCl2
,CH2COOLi , ZnSO4 ,KBr and KI gave 100%
sterility at the tested nontoxic
concentration under different pupal dipping periods , another salts induced various
degrees of sterility ranging from 31-97%.
643
Soluble inorganic salts are transported
across cell membranes [8] and [9] and it was suggested that the penetration of the salts
through the cell membrane is according to the order of ionic mobility which will
determine the order of effectiveness. Effect of the tested salts might be a result of
accumulation of salt ions in the tissues of the pupae after treatment and their
interference with the biological processes of the pupae, causing
retardation in
development of the reproductive system . It might be also of some adverse effect on
the viability of sperms and ovaries in either sex of R. ferruginous adults. [10]
recorded the toxic effect of three inorganic groups (Copper sulfate, Copper hydroxide
, Copper ox chloride) was great toxicity on some piercing sucking insects (Aphids,
Aphis gossypii. White fly ,Bemisia tabaci.,Gassyd, E. lybica.,and Mites,Tetranycus
urtica) that infested the small seedlings of cucumber plants . The tested materials have
a great toxicity as acaricidal and insecticidal agents for controlling the pests without
any harmful effect on the treated plant. [11] discussed the effect of ZnSO 4 on the
feeding and growth of Spodoptera littoralis and they found accumulation of Zink in
tissue of the larvae seems to be the factor inducing moths sterility .[12] conducted the
screening test with several compounds including aziridines, methanesulphnates,
nitrogen mustards, phosphor amides and urea derivatives by topical application or
feeding to adult Heliothis virescens. They showed that azirdines were the only
promising chemosterilants to the Lepidopteras insects. [13] indicated that only Tepa,
Metepa and Apholate caused complete sterility to male red bollworm Dispropsis
castanea , while no sterility was induced by using Hempa .
Conclusion
The present results cleared that the all solutions of the tested inorganic salts may be
able to be used as a chemosterilants for R. ferrugineus by injection in the infested tree
trunk or roots as alternatives to the harmful chemical insecticides , or as a new
approach to its control for mass production of this pest by sterile techniques . Thus the
present study highlights on the benefit of inorganic salts for pest control , further
investigations are need in this approach .
644
References
[1] G. Pandey, T.P.S. Teotia . Laboratory studies on chemosterilization of angoumios
grain moth Sitotroga cerealella ( Oliv. ) Screening of 33 compounds by pupal
treatments . Indian . J . Ent . 42 (1) (1980 ) 1-15
[2] A. Neerja , K.D. Upadhyay . Screening of certain amino acids as possible sterilants against
rice moth Corcyra cephalonica ( Staint . ) . Bull. Grain . Technol 20 (1) (1985) 37 – 42 .
[3] A . Sharaby . Screening of certain inorganic salts as possible sterilants against the
lesser cotton leaf worm Spodoptera exigua ( HBN. ) by pupal treatments . Bull .
Ent . Soc . Egypt . Econ . Ser . 16 (2 ) ( 1987 ) 269 – 282 .
[4] A . E . Smith , D . M . Secoy . Acompendium of inorganic substances used in European
pest control before 1850 . J . Agric . Food . Chem. 24 ( 6 ) ( 1976 ) 1180 – 1186 .
[5] M .AL – Dosary . Sensory receptors and behaviour of the red palm weevil
Rhynchophorus ferrugineus ( Oliv . ) ( Coleoptera : Curculionidae ) with reference
to attractants , repellents and their control . Ph . D Thesis in Science / Zoology
Arthropods and Parasites ( Entomology ) , Biology Department , Girls College of
Education Scientific Division in AL – Kharj , Riyadh , K . S . A . ( 2006 )P . 269 .
[6] D . B . Duncan . Multiple range and multiple F – test . Biometrics , 11 (1965 ) 1 – 41 .
[7] M . M . Crystal . Sexual sterilization of screw worm by N . N . Tetramethylene bis
( I- aziridure ) carboxamid : Influence of route of administration . J . Eon . Ent . 61
(1) (968 ) 143 – 139 .
[8] H . Frings . Gustatory thresholds for sucrose and electrolytes for the cockroach ,
Periplanata americana ( Linn. ) . J . Exp . Zool . 102 ( 1946 ) 23 – 50 .
[9] V . G . Dethier . The physiology and histology of the contact chemo receptors of
the blowfly . Quart . Rev . Biol . 30 ( 1955 ) 348 – 379 .
[10] M . M . Gehad , S . M . Saqabi ,A. G . EL- Sisi . Acaricidal and insecticidal efficacy of
some recommended cupreous fungicides against suking piercing pests. The Second
International Conference of Entomology ,8 -11 Dec . Cairo , Egypt . (2007) pp 96 .
[11] H . S . Salama , A . Sharaby . Effect of Zinc sulphate on the feeding and growth
of Spodoptera littoralis ( Boisd . ) Z . angev . Ent .72 (1963 ) 383 – 389
645
[12] H . M . Flint , W . Kalssen , J . F . Norland , E . L . Kressin . Chemo sterilization
of tobacco budworm , Survey of 16 compounds fed to adult moth . J . Econ .
Ent . 61 ( 6 ) (1968 ) 1726 – 1729 .
[13] D. G . Campion . Sterilization of Lepedopterous pest by radiation and
chemosterilization . J . Econ . Ent . 46 (4) (1967 ) 648 – 656 .
646
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648
OP 32
Observations on the biology and ecology of Oryctes agamemnon
arabicus Fairmaire, 1896 (Coleoptera: Scarabaeidae): pest of date
palm tree in Tunisia
Rasmi Soltani
Centre Régionale de Recherches Agronomiques, BP 357 Sidi Bouzid 9100, Tunisia
Correspondent author’s e-mail address: soltani_rasmi@yahoo.com
Abstract
Oryctes agamemnon was exogenous specie accidentally introduced in the oases of
Mrah Lahouar from Tozeur state, about 1980 and officially signalled in southwest
Tunisia in 1995. Few interested biographies on the biology and ecology of the specie
exist on the world. So, the aim of this work is to present some aspects of the biology
and ecology of this beetle under Tunisian conditions. This pest closely associated to
date palm was spread in some oases of southwest Tunisia, covering actually 3310 ha.
Preferred breeding sites are standing living palm tree. The attacks by this pest interest
different parts of the plant as respiratory roots, external components of the stem and
the oldest basal green palms of the crown. Breeding site can be the site of mating,
oviposition, larval feeding, pupation and adult emergence. In some cases, beetles are
attracted to heap manure, when exist inside oases, buried on the soil as compost. The
life cycle of immature stages, from egg to adult emergence, under natural conditions
still on average 319 days and is dominated by larval development which represent 89.6
% of the total cycle. Consequently, larvae constituted the harmful stages of the species.
Females prefer deposit their eggs in friable material inside hidden sites of the palm
tree. The mean registered fecundity of 43 females is 26.62 eggs/female. Eggs are laid
singly one by one many centimetres spaced inside several oviposition sites of many
palm trees. The threat of O.agamemnon consists on the attack of respiratory roots. In
fact, after many years of successive attacks of the respiratory roots by several
generation tunnels of larvae interpenetrate and evolve on a large hole which weaken
the basal support of the plant and lead to its sudden collapse by winds.
Keywords: date palm, life cycle, Oryctes agamemnon, southwest Tunisia
649
Introduction
The genus Oryctes Illiger (1798) is specific to the old world and includes about 40
species. It belongs to Rhinoceros beetles group which constitute a group of medium to
large-sized scarabs of the Dynastinae subfamily and whose males are distinguished by
a large cephalic horn [5]. Many species of this genus are associated with palm trees
and cause severe damages to coconut, oil and date palms [1, 3]. Three Oryctes species
develop specifically in date palms and are present in the Near- and Middle-East and in
North Africa: O. elegans Prell, O. agamemnon Burmeister and O. richteri Petrovitz.
The first two are common throughout the Near- and Middle-East. They are univoltine,
flying from spring to autumn [13]. Though not reported as major pests, O. elegans and
O. agamemnon inflict chronic damage in date palm and cause economic losses in Iran
and Iraq [10] contrary to O. agamemnon specie which has no direct effect on the
production in Tunisia [16, 17].Oryctes agamemnon is the representative species of the
genus in some oases of southwest Tunisia [16]. This pest was accidentally introduced
in 1980th from the oases of the United Arab Emirates using offshoots as part of varietal
exchange. It is signaled for the first time in 1995 in the oases of Mrah Lahouar from
Tozeur [11] and two years later in the oases of Redjim Maatoug from kebili [16].
During the first years after its introduction the specie is acclimated to the oases
conditions of Mrah Lahouar (380 ha); where it was spread by flight after the increase
of its populations to infest palm trees of the existent variety Deglet Nour and their
offshoots. Between 1980 and 1990, the lack of primary vegetal material, offshoots,
used in the extension of new plantations of Ibn Chabbatt 10 km apart, oriented farmers
to use offshoots originated from Mrah Lahouar. By this way and associated with the
ignorance of the existence of the specie favored its spread to this new extension
covering actually 800 ha. In this zone of Djerid the specie became well acclimated to
these oasis’ conditions, amplify considerably the number of its population to reach a
critical situation to date palm tree. Consequently, major palm trees and their offshoots
are invaded with this pest [16]. After 1987, the lack of offshoots to supply the new
project of Redjim Maatoug (1610 ha) from Kebili region oriented the responsible to
import offshoots from the oasis of Ibn Chabbatt to complete the plantation. So, we
attend to the same scenario as in Ibn Chabbatt. Other oasis covering about 520 ha are
650
infested by this pest in the region of Tozeur, these oasis are spread in the space.
Therefore, the pest is actually spread to 3310 ha which represent 10. 1 % of the total
area of date palm tree in Tunisia.
No study on the ecology of O. agamemnon is done in the oases of southwest
Tunisia. So, to understand the specie in its natural habitat, we undertook the study of
its ecology and biology. The work was based on a laboratory breeding trials and a field
monitoring using a light trap to catch adults and a meticulous survey of different stages
on palm tree.
This paper reports the main aspects on the ecological life history of the beetle under
the oasis conditions of southwest Tunisia. It describes some of the behavior, biology
and life cycle of the pest, oviposition activity, larval development, pupation and
adults’ activities. Such information can help in the establishment of a management
program when finding a cure and this by the knowledge of the specie and the
identification of dangerously and sensitive stages.
Research methodology
Study site
Sampling and monitoring were conducted during three years from Mai 2004 to June
2007 in Redjim Maatoug oases (1610 ha) situated in Saharan climate of Tunisia. This
zone localized on the southwest edge of Chott El Djerid was characterized by an
average rainfall lower than 100 mm/year, a mean temperature of 21°C with extremes
of 55°C in the shade in summer and 7°C in winter. Oases characterized by a sandy soil
type are planted from 1987 to 1995. They are continental and of modern type due to
the regular space of 9 m * 9 m between palm trees. The surrounding area inside oases
included mainly fodder cultures (Lucerne).
Life cycle and oviposition
All observations were made under natural conditions of temperature in the oases of
Redjim Maatoug in southwest Tunisia. Boxes were placed inside a cabin constructed
with dry palms in the oasis of the pilot station. Observations made on oviposition,
incubation, larval development, pupation, and adult feeding and mating are following
by a field survey of adult behavior (flight, mating and feeding).
651
Breeding methods- One male and one female were paired together inside opaque
plastic box of (20 cm × 15 cm × 10 cm). Boxes are filled with rotted sawdust collected
from natural breeding site of the specie, palm tree, used as oviposition middle. Several
holes are made on the cover to permit the ventilation of the box.
For duration of life history studies the boxes are daily inspected. They were emptied
and the middle checked for eggs which were carefully removed using a spoon and with
the surrounded sawdust to other small boxes of (10 cm × 5 cm × 5 cm) filled with the
same rotted sawdust and used as incubator. Eggs laid on the same day are placed by
group inside the same box which the date of oviposition is registered on the cover.
Eggs placed on the top of the substrate are three quarter of volume buried in to
facilitate their control and to minimize their manipulation because these boxes are also
daily examined. On hatching, new larvae rest for a day inside the same box after they
were removed by a group of three to six, hatching on the same day, inside big boxes of
(20 cm × 15 cm × 10 cm), with the same middle of breeding. Such a medium had
previously been advocated by [17] for rearing larvae of this pest. The larval food
middle was changed every two to three weeks for young larvae of first and second
stage and every one to two weeks for third larval stage. Boxes were checked daily for
mortality, signs of moulting and feeding behavior of larvae. When feeding activity is
relented larvae of third stage were removed to other boxes filled with the same middle
to observe the behavior of this stage.
Duration of the life cycle – The duration of the immature stages of O. agamemnon
under Redjim Maatoug conditions is studied.
Fecundity- A number of 43 couples of O. agamemnon were confined in plastic boxes
of 3 liters filled with rotting sawdust collected from infested palm trees. The boxes
were controlled every three days, the contents is sieved and examined for eggs which
were removed, counted and noted on the cover. At the end of experiment, when female
died the total numbers of eggs registered on the cover were summed. The calculated
number represents the fecundity (Fi) of the relative female. The mean of fecundity ()ܨ
is calculated using next formula:
ܨൌ
∑
సభ
Fi: Fecundity of each female, n: total number of female used in the
experience (n=43).
652
Adult feeding, dispersal and mating
The observations and the gathering of beetles from palm trees were usually made
along diurnal and nocturnal transects through plantations. These transects permitted
the collection of fundamentals information which contribute to understand a part of the
adults’ behavior in natural middle. The respiratory roots part of each chosen palm tree
was probed with a scythe. If a beetle or a couple was present, it was removed, and then
taken back to the breeding trials. The location of occupied burrow was described; also
the number of present individuals and eggs was noted. This procedure provided some
information on feeding, mating, and oviposition behaviors which were supplemented
by laboratory trials with beetle bred in the same middle.
Other beetles were caught in a light trap. This light trap, 1.8 m height, was
composed by: a 1.2 m basal parallelepipedic iron support, a funnel with
parallelepipedic top and four exits on the base linked to 4 stamps by four PVC tubes,
two crossed sheets of Plexiglas of 60 cm side fixed to a wooden support which was
inserted on the top of the funnel. As light source, neon was attached on the center of
the wooden support and linked to a switch always activated on the position On. The
electric system of the trap comprise: a solar sheet used to pick up the solar energy and
to convert it in electric energy which is stored up in the battery, a timer to control the
functioning either of the neon during night or the recharge of the battery during day
and the switch already cited. All these components are connected together in the same
order already cited using a system of cable.
At night, beetles attracted by light bump into the sheet of Plexiglas, fell in the funnel
and dispatched by the PVC tubes to the stamps. The trap was weekly controlled, the
stamps emptied, and the total of caught adults counted and separated by sex.
Results
Life cycle
The development of O. agamemnon is linked to date palm tree and occurred in moist
and dark parts. The duration of the immature stages is summarized in table 1. These
estimates are based on observations made under the oases conditions of Redjim
Maatoug, where the development of one generation from egg to adult emergence last at
mean 318.6±17.82 days. Although, O. agamemnon is univoltin species in this region;
653
there’s usually an overlap of generation in the field with all stages of the new generation
and the last stages (third larval stage and pupa) of the mother generation mainly
recorded between August and the first half of September at the respiratory roots part.
The larval development calculated from table 1 last on average 285.08 days and
represent 89.48% of the total duration of the cycle. From a comparison of these sets, it
emerge that the developmental time of third larval stage was the longest and dominate
as well the larval development as the total cycle of development. In fact, it represents
66.35% of the larval development and 59.37% of the total cycle. These results
demonstrate that larvae constitute the harmful stages of the O. agamemnon on
particularly the third stage.
Oviposition
After mating, almost any medium soft enough for female burrowing, yet firm enough
to provide compacted structure, well protected, and offering favorable conditions to the
development of all stages of the pest may be chosen as oviposition site. In the oases of
southwest Tunisia, favored breeding sites for O. agamemnon are standing living date
palm trees and heaps of compost when they exist. In fact, the female deposit eggs in
different parts of the palm tree, respiratory roots part and external components of the
stem (between matrixes of fibrilium), and in heaps of sheep manure buried in the soil to
prepare a compost which will be used in fertilization.
In palm trees receiving the attack for the first time, next steps are registered before
starting eggs’ oviposition: I) the female burrowed many centimeters (5 to 10 cm) below
the surface between intact hairy roots, ii) female chewed and transform a small part of
this vegetal material on small particles from which she extract the juice to feed and after
used them to construct a serpentine tunnel and, iii) laid eggs one by one many
centimeters spaced and compress the chewed material behind her using its posterior legs.
The same steps are registered on matrixes of fibrilium located between interior surface
of dry petiole and stem’s bark, but without feeding activity because of the dry material.
The respiratory root of the plant can receive multiple attacks by several generations
through the years. In fact, on respiratory roots of ancient infested trees, females prefer
deposit their eggs on decomposed vegetal material issued from previous attacks.
However, they start directly their oviposition activity without wasting time in the
654
preparation of a tunnel. So, this behavior permits to female to produce more eggs
which are scattered randomly many centimeters spaced inside the lodging. A female
don’t deposit all her eggs in the same lodging but disperse them on several palm trees.
Contrary to the observed behavior in respiratory roots part, female deposit eggs once a
time in the space between dry petiole and the bark of stem and no repeated attacks are
registered in high level of the external component of the stem. Hence, females show an
ascendant behavior of oviposition activity and this lead to conclude that attacks
throughout the stem are ascendant. NB the matrixes of fibrilium surrounding a level of
dry petioles is not completely invaded by the eggs of females but just some parts
situated under certain dry petioles which number varied from 0-2 eggs in basal level
situated on the top of respiratory roots part, 0-4 eggs in medium level and 0-6 eggs in
the upper level situated just under the crown.
The pollinator or male palm tree represent a special case; in fact, repeated
oviposition and young larval attacks are registered on the first level of dry petiole
situated just above the respiratory roots part. This phenomenon is mainly owing to the
height extension of the respiratory roots under the dry petiole of the first level.
Larval development
The eggs hatch in 9 to 19 days; the hatchlings consume the chorion to be liberated.
The first stage completely white with soft head capsule and mouth parts rest immobile
about 4–10 hours until the end of their hardening process. After, larvae start feeding
either on the chewed vegetal material composing the tunnel previously constructed by
female beetle or in particles of substrate of ancient attacks already decomposed. In soft
logs of respiratory roots, larvae can disperse freely from the oviposition site within the
even attacked volume.
Development of the two first larval stages coming from eggs laid under summer
conditions proceed rapidly and the third stage may be attained in 10-12 weeks, whereas
development of larvae hatching from eggs laid in September was firstly relented by the
unfavorable conditions of autumn and then blocked by winter conditions which lead to
the rest of all larval activities and the entry to hibernation. Larvae and adults constitute
the hibernation form of the specie. Analyze of hibernate larval population during winter
season show important dominance of third larval stage, with a percentage of 85% to
655
88.5%, issued from summer oviposition and a few number of the second larval stage,
10.3% to 14.1%, coming from the short duration of autumn oviposition. Larvae of first
stage are also present but only during December, 0.9% to 1.3%, and after that disappear
from the nature to reappear in the end of next spring.
At the end of each larval stage, a deceleration on the feeding and displacement
activities mark the preparation to moulting. This phase lasted less than two days with
first and second stage. However, larva of third stage marked at the end of its
development a continuous deceleration of the feeding and moving activities until their
total arrest. Thus, larva doesn’t molt but starts a non-feeding pre-nymphal period,
pseudo-stage, which last 12 to 22 days. Immobile and isolate inside the cell
constructed using the surrounding friable material or vegetal tissue at the extremity of
the larva’s feeding tunnel, movement of pre-nymph are limited to the pygidium
contraction-retraction. These repeated movements reduce the humidity inside the cell
and contribute to compress the friable material of its partitions making them more
cemented and compact. Larval development, which duration is 181-302 days, is
achieved by the nymphal moulting.
Field observations conducted for five years, within infested oases of Mrah Lahouar
and Ibn Chabbatt from Tozeur and Redjim Maatoug from Kebili, shown that cannibalism
phenomenon is unusual but it mainly occurred when larvae of third stage encounter the
prepupal and pupal stages. When developing in the same lodging, larvae of third stage
are spaced out and appear to ovoid the encounter of each other. Survey of this behavior
under laboratory trials show that inside the small space of boxes larvae live spaced out
and ovoid the encounter; cannibalism is usually observed when the breeding medium
becomes limited in supply and when larvae of third stage coexist with pre-pupa and pupa.
Pupation
The pupal stage (table 1) is spent inside the same cell of pre-pupa, and where pupa
took a dorsal position on the substrate with opened elytra and legs stick to the body. Its
movements are limited to the contraction-retraction of the abdominal part. The color
evolves progressively from creamy white at the moment of moulting to brownyellowish. Legs are progressively moved away from the body. At the approach of
adult moulting the head, thorax and legs becomes harden with dark color.
656
Adult feeding, dispersal, mating and sex-ratio
As other most of scarab beetles, O. agememnon needs the hot temperatures of
summer to accomplish its diverse vital activities (feeding, flight and reproduction). Molt
start by the liberation of legs followed by head, thoracic parts and abdomen. After the
end of moulting process, the body of new emerged adults is divided in two parts; both
head and thorax are harden and darken but, the rest of the body (abdomen) and elytra are
creamy white and soft. The newly emerged adult remains almost immobile inside its
pupation cell for 13 to 17 days. During this inactive period, called preoviposition period,
mainly initiated processes are the exoskeleton darkens and hardens and the
differentiation of internal organs as the digestive system and the genital organs.
When fully sclerotised and have undergone some maturation adults are ready to
start their activities (feeding, flight and reproduction). So, each beetle chews its way
out the pupal cell, usually using the larval tunnel, to be released in nature and flies to
other palm trees to feed or to search for a sexual partner.
The survey of adults’ feeding activity show that their main food was composed by
juice extracted from attacked hairy roots of the respiratory part. In fact, this activity is
registered when they chews their way out or enter to mate or to lie eggs (females), they
reduce fresh hairy roots gorged with water in small particles which are pressed
between mouth part to extract the contain juice. This operation permit the diminution
of relative humidity of particles which are used to build a tunnel where the female laid
its eggs. Dissection of 20 adults (12 females and 8 males) collected using the light trap
don’t show any solid material in their guts which are white in color. Also, digestive
system is not well developed and reduced in volume comparing to the genital organs.
Consequently, beetles do not ingest solid vegetal material thus they have a liquid
alimentary regime which is based on the extraction of juice that emerge from the
infested parts of the date palm tree.
Field works conducted in different oases of Tozeur (2003) and Redjim Maatoug
(2004-2007) shown that adults are never registered on the crown i.e., there’s no
recorded feeding and oviposition activities of adults in different levels of green palms
only damages of third larval stage on matrixes fibrilium, superficial injuries on the
petiole of green palms, and sometimes on stem bark.
657
In the laboratory, feeding activity is not registered with beetles reared inside boxes
yet proved by the absence of solid or liquid excrements. Adults mate normally and can
live for more than 50 days without feeding.
During the day, O. agamemnon hide in breeding places on the palm tree. The beetle
starts flying at twilight and is rarely observed out of the tree. Nightly survey,
performed in the previous cited oases shown next results, most of flight activity is
registered during the first half of night and appears to be limited to a few ten meters
because of the abundance of breeding and mating sites (palm trees) and the heavy
flight of adults imposed by their massive form; beetles are rarely even never observed
on the ground and walk as mean of dispersion is limited to the same palm tree when
the beetle passes or climbs from respiratory roots to the upper levels of dry petioles or
between different levels of dry petioles.
The period of flight of the beetle take place between June and October. The total
catch of three years included 672 males and 1203 females. This sex-ratio almost of 1:2
in favor of females let suppose that females beetles apparently makes more flight
during the dispersal period to search a partner for mating or to search new breeding
sites where to deposit their eggs. The fact that even females taken from the trap and
dissected, contained full-sized eggs.
Flight activity is limited inside the same plantation or between border plantations of the
same site because of the abundance of breeding sites. But, when adverse conditions will be
installed the beetle can develop a behavior of flight for long distance between near sites.
In the natural middle, the formation of couples and mating occurred in hidden
attacked parts of the palm tree and never on the ground or on exterior parts of the tree.
Night survey of beetles’ activities shown that one to three females groups are found in
the same log with one male. This result is usually observed in the oases let suppose that
female are aggregate together to facilitate the attraction of males using the additive
action of their pheromones. Couples are always located in the respiratory roots part and
never registered between the fibrilium’s matrixes between dry petiole and stem bark,
inside the basal part of dry petiole and on different levels of the crown. However, in
rearing trials, paired couples mates often inside the substrate, even if mating occurred
on the surface of substrate females burrows with the male on her back.
658
Basing on the feeding behavior, females choose and prepare the site of mating
between hairy roots then attract the male using their pheromone. One male can realize
several mating with several females in the same lodging. Once copulated, female pass
behind the site of mating by chewing hairy roots and many days later she start her
oviposition activity. In the same volume of respiratory roots, several females can
coexist together but they laid their eggs after the delimitation of their territory.
Fecundity and fertility
Egg production or the fecundity per female ranged from 21 to 37 eggs with a
calculated mean ( )ܨof 26.69±6.21 eggs per female for the whole experiment. The
mean duration of oviposition activity is 26.62±6.16 days. The female reproductive
success calculated for the all experiments was at mean 76.82%.
Mortality and growth rate- Interpretation of the table 1 reveals that eggs and pupal
stages represent the most sensitive stages of the specie. These higher percentages of
mortality are mainly caused by the decrease of temperatures at the end of the period of
oviposition activity and pupation in October. Between larval stages, the high rate of
mortality is registered with the first stage. This phenomenon is always observed at the
beginning of the development. Larvae of third stage are the most resistance and
mortality is frequently at the end of this stage with pre pupae which were killed by
other larvae of the same species. So, cannibalism phenomenon exists but it was rare.
Inside plantation, the registered cases of mortality are almost inexistent; but when
they exist they are caused by the decrease of temperatures (eggs and pupae in end
September and October) or cannibalism (pre pupa and pupa). No cases of mortality are
caused by natural enemies are registered because of the absence of predators and
parasitoids of this pest.
Based on data advanced in table 1, calculated rate of growth is 50.45 %.
Damage
The damage to date palms by O. agamemnon is only caused by larval stages. Inside
the invaded parts of palm tree, larvae of first and second stage start feeding either in
friable material issued from previous attacks of the specie (respiratory roots part) or on
the tunnel prepared by female before starting oviposition (respiratory roots part and
external components of stem). When larvae of these stages reach their full
659
development they can feed in hairy roots of small diameters (< 3 mm) in the
respiratory roots part and on the superficial layer of fibrilium’s matrixes. They dig
tunnels between invaded tissues. However, voracious larvae of third stage fed in all
encountered vegetal material within the lodging and attacked deeply the layers of
fibrilium, reach the stem bark and bore into more solid wood where they excavate a
cell. The real danger of the specie consists on the repetitive attacks of the respiratory
roots part. In fact, successive invasion by several generation through years lead to the
overlapping and interpenetration of larval tunnels, in which case we attend to the
formation of a large hole on the base of the plant weak its basal support and cause its
sudden collapse by winds. Consequently, respiratory roots part constitutes a zone of
multiplication to the specie and a threat to the survival of palm tree when invaded.
The attack of fibrilium’s matrixes and dry petioles is without danger to the plant
because these are dead tissues with no vital function to the plant. So, it can be
considered as just a zone of multiplication to the specie. There’s no crop loss by the
specie because fruit bunches are not interested by the attack of O. agamemnon.
Discussion
To assure survive and good development of the future descendants females prefer
deposit their eggs on living palm tree with a part of decaying material. This can partly
explain the choice of the respiratory roots part and the ascendance of the attack
throughout the stem. The same behavior towards the choice of oviposition site is
observed with other species belonging to the same genus is cited by [1]. For O.
rhinoceros, the most studied specie of the genus, it has been found that the breeding
sites of the beetle vary between countries; in fact female prefer laid their eggs in
rotting wood of dead standing coconut trees and cattle dung in Papua New Guinea, and
in decaying trunks on the ground in Malaysia [3, 6, 7, 8, 14].
Fecundity per female announced for the same specie by [15] ranged from 17 to 22
eggs and by [12] was at mean 30 eggs. However, [4] announced a fecundity of 100
eggs per female. All the differences with present results can be due to the biological
material (used under-specie), the development of immature stages (quality of food and
length of the cycle), and to the conditions of experimentations. Important differences
are registered when compared with results of fecundity relative to the specie O.
660
rhinoceros. Indeed, the fecundity per female cited by [18] ranged from 30 to 40 eggs,
[2] cited a range of 24-65 eggs with a mean of 51±16 eggs, [9] cited a mean of 49
eggs, and [8] ranged this fecundity between 70-140 eggs.
Results relative to the incubation period are similar to those cited by [12], 10-16
days and [15] 11-16 days. Compared to other species of Oryctes, results are
approximately the same with the O. rhinoceros specie which incubation of eggs last 11
days mentioned by [7] and 8-12 days cited by [2]. About the larval development of O.
rhinoceros in its Asian home land (from India to Indonesia) and on the South Pacific
Islands, [7] cited a period of 77-105 days well; [8] cited a period of 72-130 days.
These results are totally different from our finding (181-302 days) mainly prolonged
by the hibernation period which was absent for O. rhinoceros in its native home and
may be by the quality and availability of foods.
The prepupal period lasted at mean 17.14 days for O. agamemnon [17] under
laboratory conditions (23°C, RH: 55 %). It was described by [1] as having duration of 813 days for the Scarabaeidae: Melolonthinae. It was also mentioned by [7] and [3] for
O. rhinoceros and defined as a non-feeding period which starts after the construction of
the pupal chamber; it last approximately one week. [12] and [17] mentioned duration of
20-29 days for nymphs; these results are similar to the present work and also similar to
the results cited for O. rhinoceros specie, 21 days [7, 8] and 17-28 days [18].
In this context, it is indispensable to scrutinize the phrase “breeding site”. For O.
agamemnon, a log on the palm tree can be the site of mating, oviposition, larval feeding,
pupation and adult emergence. However, the site contributes to the perpetuation of the
population at one condition when the last stage, adult emergence, is achieved. These results
agreed with those cited by [7] for O. rhinoceros specie under the Samoan conditions.
Under natural conditions of Southwest Tunisia, the total life cycle of immature
stages, egg to adult emergence, last at mean 319 days. But, under favorable conditions
of the same zone and from end spring, May, until first half of autumn, September –
October, the same generation of Agamemnon can develop in 22 to 25 weeks. This
period of the year represent the best period of the development of all stages of the
specie due to favorable weather conditions, especially temperatures, and with no
period of arrest. So, feeding activity and growth rate are intense. For the O. rhinoceros
661
at Keravat, the total life cycle of immature stages lasts approximately 331 days [2], for
about 7 to 8 months [9], 4 to 5 months, with the third instar stage occupying most of
this period [2].The preoviposition period is cited by [7] and is called the teneral period;
it last 3 weeks during which the exoskeleton darkens and hardens. The duration
announced by [14] is 20-30 days after ecdysis for this distinct preoviposition period.
[2] advanced that the feeding activity of adults belonging to the species of Oryctes
genus is occasional, beetles in this case can live without feeding during their life.
Several authors were agreeing that the damage and crop loss by O. rhinoceros is only
caused by the adult beetles. They fly to the palm crown of living trees and fed by
burrowing galleries in the fresh tissues of palms’ petioles which may result in breaking
of the fronds during strong winds, digging into the stalks of fruit bunches or they cause
damage to growing points, in which case the beetle kills the palm. In the tropical
region, eggs and larvae of Oryctes species develop in the wood of dead trees: standing
palms, dead coconut trunks and stumps, and decaying cocoa pod humus. The two
feeding habitats are separate in space [2, 6, 13, 14, 18]. However, in Tunisia, larvae
and adults of Oryctes spp. live in the same living and standing trees. Larvae develop in
the respiratory roots part, at the periphery of the stem and on the basal part of the
crown, feeding at the interface of dead and living tissues [16].
Oryctes agamemnon arabicus has one generation per year, 319 days, under the
oasis conditions of southwest Tunisia. Its life history is mainly dominated by larval
development, 285.08 days, which itself dominated by third larval stage, 185.15 days.
Role of adults is limited to reproduction and oviposition which assure the perpetuation
of the population. Mating occurs inside dark places on the palm tree between June and
first half of October and when temperatures are hot. Fecundity per female is 26.69
eggs. Adults fed occasionally on the palm tree and their damage is without importance.
However, larvae constitute the harmful stages of the specie particularly the third stage.
Indifferently from the infested part, damage appears on the form of tunnels, but the
real danger interest the respiratory roots part. Repeated attacks from a year to another
year in this part lead to the interpenetration of tunnels and as a result generate the
formation of a large hole which engenders the sudden collapse of the tree.
662
References
[1] Balachowsky, A.S. 1962. Entomologie appliquée à l’agriculture. Tome I.
Coléoptères premier volume. Masson et Cie, Editeurs, 564 p.
[2] Bedford, G.O. 1976. Observations on the biology and ecology of Oryctes
rhinoceros and Scapanes australis (Coleoptera: Scarabaeidae: Dynastinae): pests.
J. Aust. Ent. Soc. 15: 241-251.
[3] Bedford, G.O. 1980. Biology, ecology and control of palm rhinoceros beetles. Ann.
Rev. Ent. 25: 309-339.
[4] Dhouibi, M.H. 2000. Les coléoptères Oryctes agamemnon. p. 122-123. In : Lutte
intégrée pour la protection du palmier dattier en Tunisie. Centre de publication
universitaire, Tunisie.
[5] Endrödi, S. 1985. The Dynastinae of the World. Dr. W. Junk Publisher, Dordrecht, 828 p.
[7] Hammes, C. and Monsarrat, P. 1974. Recherches sur Oryctes rhinoceros L. Source
Cahiers ORSTOM. Série Biologie 22 : 43-91.
[8] Hinckley, A.D. 1973. Ecology of the Coconut Rhinoceros Beetle, Oryctes
rhinoceros (L.) (Coleoptera: Dynastidae). Biotropica 5(2): 111-116.
[6] Howard, F.W., Moore, D., Giblin-Davis, R.M. and Abad, R. 2001. Insects on
Palms. CABI Publishing, 400 p.
[9] Hurpin, B. and Fresneau M., 1967. Contribution à la lutte contre les Oryctes nuisibles
aux palmiers. Elevage en laboratoire d’Oryctes. rhinoceros. Oléagineux 22 :1-6.
[10] Hussain, A.A. 1974. Date Palms and Dates with their Pests in Iraq. University of
Baghdad, Baghdad, 166 p.
[11] Khoualdia, O. and Rhouma, A. 1997. Premières observations sur Oryctes
agamemnon, ravageur du palmier dattier en Tunisie. Fruits, vol. 52: 111-115.
[12] Lepesme, P. 1947. Les insectes des palmiers. Editions Paul Lechevalier, Paris,
France, 904 p.
[13] Rochat, D., Mohammadpoor, K., Maloose, C., Avand-Faghih, A., Lettere, M.,
Beauhaire, J., Morin, J.P., Pezier, A., Renou, M. and Abdollahi, G.A. 2004. Male
aggregation pheromone of date palm fruit stalks borer Oryctes elegans. J. Chem.
Eco. 30: 378-407.
663
[14] Sivapragasam, A. 1988. The rhinoceros beetle, Oryctes rhinoceros (L.) : An
appraisal of its problem and control in young Coconut replantings in Malaysia.
This paper was presented at the Workshop on coconut replanting, UNDP/FAO
Projet RAS/80/032>> Improved coconut production in Asia and Pacific<<, 23-27
November, 1988, MARDI, Malaysia.
[15] Soltani, R. 2004. Oryctes agamemnon Burmeister (Coleoptera: Scarabaeidae):
Contribution à l’étude de sa biologie et des dégâts occasionnés dans les oasis du
Djérid au Sud Ouest Tunisien. Mémoire de DEA, Institut supérieur
AgronomiquesChott Meriam – Tunisie, 81 p.
[16] Soltani, R. 2009. Oryctes agamemnon arabicus Fairmaire, 1896 : Etude bioécologique et éthologique dans les oasis de Rjim Maâtoug au Sud Ouest
Tunisien. Mémoire de Thèse de Doctorat, Institut supérieur Agronomique, Chott
Meriam – Tunisie, 152 p.
[17] Soltani, R., Chaieb, I. and Ben Hamouda, M.H. 2008. The life cycle of the root
borer, Oryctes agamemnon, under laboratory conditions. Journal of Insect
Science 8:61, available online: insectscience.org/8.61.
[18] Waterhouse, D.F. and Norris, K.R. 1987. Biological control – Pacific Prospects.
Australian Centre for International Agriculture Research (ACIAR).
664
Table 1: Mean of development of immature stages of O.
agamemnon under Redjim Maatoug conditions
Stage
Number of specimens studied
Duration (mean days)
% of Mortality
Egg
220
12.95±2.18
23.18
L1
169
35.24±4.32
12.42
L2
148
60.69±38.69
8.11
L3
136
189.15±44.55
2.94
Pre-pupa
43
15.2±1.8
17.42
Pupa
132
20.81±2.6
Adult
109
-
665
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666
OP 33
A trap for auto dissemination of the Entomopathogenic fungus
beauveria bassiana by Red palm weevil adults in date palm plantations
R. El-Sufty1, S. Al Bgham2, S. Al-Awash2, A. Shahdad2 and A. Al Bathra2
Biological Control Project of Red Palm Weevil "3rd Phase"
Arab Organization for agricultural Development (AOAD)
1)
Prof. of Economic Entomology Plant Prot. Dept. Fac. Agric. Tanta Univ. Egypt
2)
Ministry of Environment and Water, United Arab Emirates.
Abstract
A trap was designed to allow red palm weevil adults to pass through it so that they
come out contaminated with a high density of the fungus conidia for spreading them
amongst the red palm weevil population in date palm plantations. A fungus inoculum
containing 10% conidia was prepared and used in the trap. An adult was contaminated
with 9.53x107 conidia per a tape visit with a lethal time of 8.25 days. Field trials were
carried out using 20 traps in 3 date palm plantations in the period from April 2006 to
May 2007 in the Northern Region of United Arab Emirates. Efficacy of the trap was
evaluated by assessing the monthly mortality caused with the fungus in the adult
population. In the last two months, mortality of adults caused by the fungus in the field
population ranged 41.2-51.3% compared by 4.8– 4.9% in the control. Results showed
that the trap is effective for spreading the fungus Beauveria bassiana among
Rhynchophorus ferrugineus population.
Key words: dissemination, fungus conidia, adult population, date palm weevil
Introduction
The red palm weevil (RPW), Rhynchophorus ferrugineus (Oliv.) (Coleoptera:
Curculionidae) is a serious insect pest infesting date palm, Phoenix dactylifera in
Arabian Gulf Region and other different countries including Egypt (Nirula, 1956; El
Ezaby et al. 1998; Saleh, 1992; Faleiro, 2006). In date palm plantations, adults occur
on the basal buried part of the trunk, in the soil and aggregate, feed, mate and lay their
eggs in the succulent tissues of the tree leaf axils. The larvae occur inside complicated
tunnels in the tree trunk, where they feed on the soft tissues, till the trunk is completely
667
hollowed out and the tree dies. The Biological Control Project (BCP) of Red Palm
Weevil (3rd phase) was conducted and directed by Arab Organization for Agricultural
Development (AOAD) in 2004 with the objective of developing integrated biological
control technology against the insect using local microbial control agents. Laboratory
and field investigations using a local strain of the entomopathogenic fungus, Beauveria
bassiana in United Arab Emirates indicated that the foliar application of the fungus
was efficient for the control of RPW (El-Sufty et al. 2007, 2009). However, more
advantage is essential to increase the control efficacy of the fungus in date palm
plantations to achieve long term management of this destructive insect. In the last few
years, the use of a proportion of the target insect population to facilitate the pathogen
dispersal in an insect population has received considerable attention (Vega et al., 2000;
Furlong and Pell, 2001; Vail et al., 2006). The present paper reports on a control
method that manipulates a proportion of the field adult population of the red palm
weevil to disperse the fungus in the wider insect population.
Materials and Methods
Fungus inoculum
The fungus used was a local strain of Beauveria bassiana (UAE-B2), which was
isolated for the first time by BCP and found to be potentially important for RPW
control. The fungus was identified by CABI Bioscience, UK and samples were also
dispatched to Brook University, Canada for finger printing. The fungus was
maintained on Sabouraud dextrose yeast agar (SDAY) as described by Goettel and
Inglis (1997) with regular passages through RPW-adults. For mass production of
conidia, the fungus was cultured on a medium composed of 40 g grinding rice grains,
8 g peptone, 10 g agar and 2g yeast extract per 1L water as described by El-Sufty et
al. (2007). Dried conidia were used to prepare a powder formulation composed of
55g sand, 35g Talkum and 10 g conidia. One g of the powder contained 5x109
conidia. The powder bio-preparation was fresh prepared and used as inoculums in the
auto dissemination trap.
The trap
The trap is designed as one way road. It allows passing of RPW adults (males and
females) inside it so that they come out with their bodies contaminated with a large
668
quantity of conidia to spread them in the field population by passive transfer from the
contaminated adults to the healthy ones and by indirect contamination by conidia
deposited in the habitat. The trap consists of two champers; an attractive chamber with
two entrances, which is baited with RPW aggregation pheromone bag and a bottle
containing 15 ml palm tree kairomone and a contamination chamber with one exit,
which furnished with a Petri dish cover (15 cm diameter) containing 15g of the fungus
inoculum. When the adult weevil is attracted to the trap and enters the attractive
chamber, it falls down to the contamination chamber. The trap was partially buried in
the ground up to the openings of its attractive chamber and the exit opening of the
contamination chamber is free so that the contaminated adults can leave the trap.
Bioassays
Adults of RPW used in all bioassays were collected from date palm plantations
using insecticide free pheromone-kairomone traps developed by BCP and maintained
under room conditions for a week in plastic containers provided with food (moistened
pieces of palm wood). Dead and injured insects were discarded and healthy ones used
in the experiments. A powder formulation composed of simple inert substances mixed
with dry B. bassiana conidia was prepared and used in dose-mortality assays against
RPW adults. Conidia were added at ratio of 5%, 10% and 20% to the carrier
substances and evaluated for their virulence as described by Lacey et al. (1994). Five
adult weevils were introduced into a plastic container closed with a cap. One g of the
preparation was added to the container. For each dosage, 4 replicates were used. The
containers were periodically rotated end-over-end for 2 min. Insects of a replicate were
removed and maintained inside a plastic container provided with palm wood pieces as
food. Control insects were treated with inert substances without conidia. Containers
were kept under room conditions. Treated and control insects were daily examined,
dead individuals removed and recorded. Mortality and lethal time were calculated.
To
determine the conidia density contaminating a RPW adult after a trap visit, 10
RPW adults were allowed to enter a trap supplied with 15 g fungus preparation of
10% conidia one after the other. Time need for an adult to leave the trap was recorded
and then each adult was introduced into a screw neck vial containing 10 ml water and
0.1% tween 80, vigorously agitated and number of conidia per cadaver was determined
669
by direct enumeration using haemocytometer (Goettel and Inglis, 1997).To determine
mortality and lethal time for the contaminated adults leaving the trap, 20 RPW adults
were allowed to enter a trap supplied with 15 g of the fungus inoculum (10% conidia)
one after the other. Each 5 adults were maintained inside a plastic container provided
with palm wood pieces as food. Control insects (20 adults) were treated with inert
substances without conidia. Containers were kept inside screen cages (50X50X50 cm)
placed in a date palm plantation. Insects were daily examined, dead individuals
recorded and mortality as well as lethal time was calculated.
Trap visiting rate and other insect visitors
To evaluate visiting activity of RPW adults to the trap in the field and identify the
other insect species visiting it, five traps free of fungus inoculum and without exit of
contamination chamber were installed at equal distances of 100 m apart in a date palm
plantation for 10 weeks starting on the 4th of March 2005. Readings were weekly
recorded, number of RPW adults and other insect species were identified.
Survival of the fungus inoculum in traps
To assess survival of the fungus inoculum in the trap, 4 traps (4 replicates) were
installed in a date palm plantation at equal distances of 100 m apart. Each trap was
furnished with 15 g of a fungus powder formulation containing 10% B. bassiana
conidia. Other four quantities of the fungus formulation inside Petri dishes were kept
in the laboratory and served as control. Samples, each of 0.5 g of the fungus
formulation were weekly taken from a trap as well as from a Petri dish and conidia
viability was determined by germinating propagules on a translucent SDAY medium
on slide (Goettel and Inglis (1997). Percent germination were assessed for 6 weeks.
Field experiments
Twenty auto dissemination traps were installed in 3 date palm plantations (DPP) at
Ras Al Khaima, United Arab Emirates; DPP-1, 8 ; DPP-2, 8 and DPP-3, 4 at equal
distances of 100 m apart. Traps were experimented from April 2006 to May 2007 and
each one was monthly supplied with a slow release bag containing 700 mg of the
aggregation pheromone (mrthyl-5 nonanol and 4-methyl-5-nonanone) which releases
an average of 10-20 mg /day ( Chemical International S.A., Costa Rica) and 25 ml
bottle containing 20 ml of a kairomone mixture extracted locally from date with 2 mm
670
hole in its cover allows the release of 0.3 ml kairomone/day (AOAD, Progress report,
2001). At 4 weeks intervals, each trap was supplied with 15 g of the fungus inoculum
in a Petri dish cover fixed in the center of its contamination chamber. A plantation
located about 5 km far from the experimental plantations ( DPP-4) served as control.
To evaluate the distribution of the fungus in the RPW adult populations of the 4
plantations, terrestrial food-baited aggregation traps were used and installed at equal
distances of 200 m apart ( 2 traps for DPPs 1, 2, 4 and 1 trap for DPP 3 ). The foodbaited traps were weekly examined and RPW adults were collected in the period
extended from March 2005 to May 2007. For each weakly trap sample, cadavers of
dead weevils were disinfected by immersing in 1% sodium hypochlorite for 30
seconds, washed in sterile water and exposed to mycosis test (Lacey and Brooks,
1997). The cadavers were individually placed on moistened filter papers inside Petri
dishes. The dishes were maintained at room temperature for 10 days and the fungus
growth was observed. The alive adults were individually maintained inside plastic
boxes provided with moistened pieces of palm wood for 7 days and the dead ones
were recorded and tested for mycosis as previously described. The cadavers showed
external growth of Beauveria bassiana were considered killed by the fungus.
Statistical Analysis
Mortalities were corrected according to Abbot’ formula (1925) and analysis of
variance was used to evaluate the impact in bioassays.
Results and Discussion
Fungus inoculum and time required for an adult in inoculation chamber
In order to obtain a suitable and effective fungus inoculum for the trap, three
powder formulations with conidia concentrations of 5, 10 and 20% were bio assayed
for their virulence against RPW adults. The results showed that the three formulations
caused 90, 100 and 100% mortality with lethal times of 10.3 ,7.6 and 6.8 days with
significant differences between the first and the other two concentrations. Therefore
the formulation of 10% conidia was used in the following bioassays and in the trap.
Assessment of conidia density contaminating a RPW adult after a visit of a trap
supplied with 15 g fungus inoculum containing 10% B. bassiana conidia indicated that
an adult was contaminated with 9.53X107 conidia. The adult visit duration to a trap
671
ranged 1-6 min. and averaged 2.3 min. Similar results were obtained by Furlong et al.
(1995) for an auto dissemination trap of the entomopathogenic fungus Zoophthora
radicans
by
adults of Plutella xylostella (Lepidoptera: Yponomeutidae), who
reported that the moth remained in the contamination chamber for 1.47 min. to be
contaminated with an amount of conidia enough to cause satisfied mortality.
Mortality response of adults
All the contaminated RPW adults visited the trap showed B. bassiana mycosis
symptoms (El-Sufty, 2009) and killed by the fungus after 5-11 days with a mean of
8.25 days (Fig. 1). It means that the contaminated adults could carry and transfer the
fungus conidia among its own population for a suitable period before their death.
RPW adult visiting rate and other insect visitors
Under date palm plantation conditions, the RPW adult visiting rate to a trap was
2.84 adults/week. The rate appears to be low and the trap may need more efforts to
facilitate its attraction power. On the other hand, the trap attracted other different
insects included species from order Orthoptera (grasshoppers and crickets),
Hymenopter (ants), Dictyoptera (roaches) and Coleoptera (soil beetles and chafers).
These insects have to increase the fungus effectiveness by transferring conidia from
site to another and spread them in RPW habitat during their activity. In a similar study,
Dowd and Vega (2003) reported that the sap weevil was able to transfer B. bassiana
conidia from an inoculative device to the overwinter sites of the insects.
Inoculum viability in inoculation chamber
Data illustrated in Fig. 2 indicate that the fungus conidia remained viable inside the
inoculation chamber of the trap for 4 weeks with germination percentages ranged 89.296.5%. Conidia viability
significantly decreased to 66.7% during the 5 th week.
Therefore, the fungus inoculum was replaced by a fresh one at 4 weeks interval.
Efficacy of the trap in date palm plantations
Figures 3, 4 and 5 demonstrated that the trap was efficient as autoinoculative device
to spread the entomopathogenic fungus B. bassiana amongst RPW adult population in
date palm plantations. In each of the three experimental plantations, the fungus killed a
proportion of the population during two successive seasons which extended from April
2006 to May 2007. In the three plantations, the fungus caused monthly mortality
672
ranged 3.6-16.7% in the first five months, then the mortality steadily increased to
reach 14.3-34.4% in the following 5 months with a fluctuation appeared to be due to
the natural population fluctuation of the insect. In the last four months, mortality
caused by the fungus sharply increased to reach its maximum values in the last two
months, being 45.7-51.3, 40.7-47.5 and 41.2-43.3% of the adult populations in DPP-1,
DPP-2 and DPP-3 , respectively compared by 4.8-4.9% in the control. In the control
plantation (DPP-4), which was about 5 km far from the treated one, RPW adults killed
by the fungus were detected about 7 months following starting of the experiment.
About in the same time (February – May 2007) mycosed RPW adults infected with B.
bassiana were collected in some other plantations, where the fungus was not applied.
These results indicate that the trap was designed to be effective enough to spread the
fungus by RPW adults in the wider population. It is clear that the adult weevils
themselves function as very efficient carriers of the fungus conidia. Because RPW
adults are susceptible to B. bassiana infection (El-Sufty, 2009), routes of infection of
the population individuals were direct contamination by passive transfer and indirect
transmission of conidia from the sporulating mycosed cadavers previously killed by
the fungus in the habitat. Furlong and Pell (2001) found that adults of diamondback
moth, could effectively transport the two entomopathogenic fungi, B. bassiana and
Zoophthora radicans from the source of inoculation to the field population. Huger
(2005) reported of a successful release the Oryctes virus in the population of the
coconut palm rhinoceros, Oryctes rhinoceros (Col.: Scarabaeidae) by the adult beetles
and considered this method as a classical biological control, which can bring the target
insect pest under control. These results represent the first report on the trap. It is clear
that, additional work is still needed to improve its effectiveness and to increase the
mortality rates.
Acknowledgment
The study was supported by Arab Organization for Agricultural Development
(Project " transfer of bio-control technologies as essential components of IPM to
compact red palm weevils in the Middle East.". We thank Ministry of Environment and
Water, United Arab Emirates for facilities we received during the course of the study.
673
References
[1] Abbot, W. 1925: A method of computing the effectiveness of an insecticide. J.
Econ. Entomol., 18: 265-267.
[2] Arab Organization for Agricultural Development (2001): Progress report on "The
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sap beetles (Coleoptera: Nitidulidae) to overwintering sites. Biocontrol Science
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[4] El Ezaby, F.A. ; Khalifa, O. and A. El Assal 1998: Integrated pest management
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S.A. Musa 2007: Biological control of red palm weevil, Rhynchophorus
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(Col. Curculionidae) by the entomopathogenic fungus Beauveria
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Pathogenicity of the fungus Beauveria bassiana (Bals.) Vuill. to the red palm
Weevil, .Rhynchophourus frrugineus (Oliv.) (Col.: Curculionidae) under
laboratory and field conditions. Egypt. J. Biol. Pest. Control, 19 (1),81-85.
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palm during the last one hundred years. International Journal of Tropical Insect
Science, Vol. 26, No. 3, pp. 135-154.
[8] Furlong, M.J. and Pell, J. K. 2001: Horizontal transmission of entomopathogenic
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[9] Furlong, M.J; Pell, J.K.; Ong, P.C. and A. Syed 1995: Field and laboratory
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entomopathogen, Zoophthora radicans (Entomophthorales) by the diamondback
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press,London.
[11] Huger, A.M. 2005: The Oryctes virus: its detection, identification and
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Oryctes rhinoceros (Coleoptera: Scarabaeidae). J. Inverteb. Pathol., 89, 78-84.
[12] Lacey, L.A. and Brooks, W.M. 1997: Initial handling and diagnosis of diseased
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Africa: collection No.22563. International Ins. Entom. 56Queen gate,
London, SW7 5JR.UK.
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and H.K. Kaya) pp. 153-177. Kluwer Academic, Dorddrecht.
675
100
% mortality
80
60
40
20
0
1
2
3
4
5
6
7
8
9
10
11
Days after treatment
Fig. 1: Mortality curve of Rhynchophorus ferrugineus adults attracted to the
trap and fell into the inoculation chamber contained 15g of a powder
formulation containing 10% Beuaveria bassiana conidia.
100
% germination
80
60
40
Control
treated
20
0
18th Apr. 25th Apr. 2nd May
9th May 16th May 23th May 30thMay
2005
weeks in test
Fig. 2: Survival of Beauveria bassiana conidia in the inoculation chamber of
the trap under field conditions compared with conidia maintained under
laboratory conditions.
676
60
Treated DPP-1
Control DPP- 4
50
% mortality
40
30
20
10
0
M.
2006
A.
M.
J.
J.
A.
S.
O.
N.
D.
J.
2007
F.
M.
A.
M.
Month
Fig. 3: Mortality of red palm weevil adults as a result of autodissemination of the
fungus Beauveria bassiana among its population in the date palm plantation -1
in Ras Al khiama, United Arab Emirates.
60
Treated DPP- 2
% mortality
50
Control DPP - 4
40
30
20
10
0
M.
2006
A.
M.
J.
J.
A.
S.
O.
N.
D.
J.
2007
F.
M.
A.
M.
Month
Fig. 4: Mortality of red palm weevil adults as a result of autodissemination of the
fungus Beauveria bassiana among its population in the date palm plantation-2 in
Ras Al khiama, United Arab Emirates.
677
60
% mortality
Treated DPP - 4
Treated DPP - 3
50
40
30
20
10
0
M.
2006
A.
M.
J.
J.
A.
S.
O.
N.
D.
J.
2007
F.
M.
A.
M.
Month
Fig. 5: Mortality of red palm weevil adults as a result of autodessimination of the
fungus Beauveria bassiana among its population in the date palm plantation-3in
Ras Al khaima, United Arab Emirates.
678
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680
OP 34
Electrophoretic mobilities of haemolymh protein in date palm pest
Rhynchophorus ferrugineus
Annie Thomas* and Nair,C.R.M.
*Department of Zoology,St.Joseph’s College for Women, Alleppey688001,Kerala,India, P.G and Research Department of Zoology, S. D. College, Alleppey
Abstract
The protein
pattern and the concentration of total protein in the haemolymph of
larval stages and pupa of Rhynchophorus ferrugineus have been analyzed. Native PAGE
analysis of haemolymph
shows that the number of protein bands in the older larval
stages is more than that of the younger larval stages in R. ferrugineus. In the pupa these
bands were much darker than others. A statistically significant difference was noted in
the haemolymph protein
concentration between the stages (p<0.001). The protein
metabolism of the insect can be studied by observing the haemolymph protein pattern.
Key Words: R. ferrugineus, date palm, haemolymph, protein, Relative mobility
Introduction
The red palm weevil, Rhynchophorus ferrugineus Olivier (Insecta: Coleoptera:
Curculionidae) also called the Indian palm weevil, is well known in the Middle East
where it causes severe damage on date palms. About 5 to 6 % of palms in the Middle
East region are infested with this pest. The occurrence of R. ferrugineus on various oil
palms in India was reported by many (Abraham et al.1989; Dhileepan, 1991; Rajan and
Nair1997).Integrated pest control techniques have been used to control the red palm
weevil but they are not efficient enough in the field in eliminating red palm weevil.
The present study has been undertaken to investigate the
protein
pattern as well
as the concentration of total proteins in the haemolymph of larval stages and pupa of
R. ferrugineus. The biochemical composition of haemolymph is highly variable
among the insect species at different developmental stages (Florkin and Jeuniaux,
1974). Since metabolic activities have been reported to be
different
at different
developmental stages in an insect ,the fluctuation in the metabolites could indicate
rhythmic changes that occur in the biochemistry of insect haemolymph. This could
681
be related to the rhythm of insecticide susceptibility
occurring in insects and
hence may be of importance in the study of the mode of action of insecticides on
the mechanism of resistance to development. Study of the biochemical composition
of the pest is useful in developing proper pesticides against this potent pest.
Insect haemolymph contained many different proteins with a variety of functions.
The total quantity of protein in the blood varied in the course of development. These
proteins were usually classified as storage proteins, lipid transport proteins,
vitellogenins, enzymes, proteinase inhibitors, chromo proteins, and a range of different
proteins that were
involved in various immune responses in insects. Most proteins,
though not all, in insect body have been reported to be synthesised in the fat body
(Chen et al., 1976; Xuwen Yue and Huang Fu Sheng,2003), released into the
haemolymph and at least some of them sequestered by the ovaries during
vitellogenesis. A correlation, therefore, should exist between the protein pattern of these
tissues not only in the number of protein fractions present in each tissue (haemolymph)
but also in the timings of their appearance and disappearance. Haemolymph proteins
served as carriers of neutral lipids , steroid hormone and juvenile hormone.
Quantitative changes of haemolymph proteins during development of lepidopterous
larvae have been reported by several authors .Temporal changes in the levels of
soluble proteins in various insect tissues during development have been reported
(Wyatt and Pan, 1978). In holometabolous insects, the developmental life of a pupa
was of paramount importance as radical transformation from larval to adult form took
place within the pupa. .Proteins having the ability to bind to Escherechia coli
were
isolated and purified by affinity precipitation from the larval haemolymph of the
silkworm Bombyx mori by Koizumi et al., (1997). Insects have been found to survive
microbial and parasitic infections by humoral and cellular defense mechanisms .In
response to invading microbes, insect defense is composed of this two-stage strategy,
haemocytic to be followed by humoral. The haemocytic defense was mainly by
phagocytosis, encapsulation, and nodulation of foreign matter by phagocytosis mainly
by plasmatocytes and granulocytes. The humoral response that follows the cellular one
takes time, in view of de novo synthesis of antibacterial proteins in the haemolymph.
A number of antibacterial proteins have been isolated and characterized from many
682
insects, which include lysosymes, cercopins, attacins, haemolins, diptericins,
coleoptericins etc (Hetru et al., 1998, El-Sadawy,H.A.,and S.AbdelShafy.2007).
Insects were known to lack lymphocytes and immunoglobulins and therefore have
developed a relatively simple sequence of inducible antimicrobial peptides. Axen et
al., (1997) isolated one inducible antibacterial insect protein called gloverin, from the
pupal haemolymph of the giant silk moth, Hyalophora gloveri. This protein was found
to inhibit the growth of Escherichia coli.
In the present study the protein pattern in the haemolymph of larval stages and pupa
of R. ferrugineus has been characterized. .The haemolymph native proteins have been
separated by Non- sodium dodecyl sulphate-Polyacrylamide gel electrophoresis. General
proteins in the haemolymph of the different stages were stained by specific staining
methods. The relative mobility and the total quantity of the proteins were also calculated.
Materials and methods
The native protein of the haemolymph of red palm weevil was analysed by 10%
polyacrylamide gel electrophoresis. The haemolymph protein pattern of different larval
stages and pupa
were compared. Determination of relative mobility was also done
electrophoretically. The polyacylamide gel electrophoresis (PAGE) method of Laemmli
(1970) was followed with necessary modifications. Estimation of
haemolymph
protein was carried out following the method of Lowry et al.,(1951).
Relative mobility (Rm) value.
The total length of the separating gel, the distance travelled by the marker dye in the
separating gel and the various distances migrated by the different protein fractions
were measured. The relative mobility /relative fraction values of each band was
calculated as follows:
Relative Mobility (Rm)= Distance travelled by the protein fraction
Distance travelled by the marker dye.
Results
Native PAGE analysis of the haemolymph of R. ferrugineus during the different
developmental stages were conducted to study the qualitative changes in the protein
pattern during the larval- adult transformation. Haemolymph of 20 day old larva of R.
ferrugineus displayed four protein bands, and the 40 day old larva had seven protein
683
bands while in the sixty day old larva, the total number of protein bands was
six.
Analysis of haemolymph in the pupa of R. ferrugineus permitted consistent resolution of
eight protein bands, although resolution between certain bands was poor (Plate-1).
The first band in all the three larval stages and pupa was very sharp, thick and dark
with an Rm value of 0.26.The forty day old larva, sixty day old larva and pupa had the
second protein band with an Rm value of 0.27, followed by the third and fourth protein
bands with Rm values of 0.31 and 0.34 respectively. Among these bands the third
protein band was much broader but diffuse. These three bands were absent in the
twenty-day-old larva. The fifth protein band which was present in all the three larval
stages and pupa had an Rm value of 0.47.This band was weak in the twenty day old
larva, but increased in intensity in the remaining larval stages and pupa. The sixth
protein band with an Rm value of 0.64 was present in all the three larval stages and
pupa with their intensity decreasing from twenty day old larva to sixty day old larva.
But in the pupa this band was much darker than others. The seventh protein band with
an Rm value of 0.86 was present only in the pupa, which was a lighter one. The eighth
protein band with an Rm value of 0.97 was present in the twenty day old larva, forty
day old larva and pupa; but was absent in the sixty day old larva. (Table-1). A critical
analysis of this shows that the number of protein bands in the older larval stages was
more than that of the younger ones.
The concentration of haemolymph protein of 20 day old larva was at a low
level (0.629±0.114 mg/ml) and increased steadily
through 40 day old larva
(0.796±0.069mg/ml) and 60 day old larva(0.883±0.108mg/ml).The concentration
of this metabolite was still higher in pupa(1.738±0.219mg/ ml ).(Table 2). There
was significant difference in the haemolymph protein concentration between these
four stages (p<0.001).
Discussion
In the present studies, native PAGE analysis of haemolymph
shows that the
number of protein bands in the older larval stages is more than that of the younger
larval stages in R. ferrugineus. Haemolymph of twenty old larvas displays only four
protein bands. During the transformation to forty day old larva, three more protein
bands appear. In the sixty-day-old larva, a total of six protein bands appear; while in
684
the pupal haemolymph, a total of eight protein bands are found. The findings in the
present studies regarding the increase in the number of protein bands in the course of
development appear to be consistent with that reported in Diacrisia obliqua by
Senthamizhselvan and Muthukrishnan (1983).The increase in the number of protein
bands in the course of each larval instar as well as during the entire larval stage in R.
ferrugineus may be attributed in part to the progressive increase in the feeding rate.
Pandian (1973) reports that to tide over the nonfeeding pupal and adult stages,
lepidopterous larvae feed and utilise the food at a faster rate. Change in the protein
pattern during moulting also indicates the possible relation between haemolymph
protein and cuticle deposition (Florkin and Jeuniaux, 1974).
Neilsen and Mills (1968) have postulated a hypothesis
for the appearance and
disappearance of protein bands. Accordingly, the midgut and fat body synthesise
some of the haemolymph proteins. Such proteins could be reabsorbed or hydrolysed to
form amino acids during the life cycle leading to the disappearance of protein bands in
the blood or they may be changed into one or more of the other protein components
of the blood, resulting in the appearance of others. However, any change in the protein
pattern during the development
can be considered as being directly determined by
the gene factor, which reflects the alteration in the metabolism of the developing organ
(Poster and Kastritsis, 1971 ,Blandin et al 2002, ).
SDS PAGE of haemolymph of Dysdercus koenigii during the last larval instars and
imaginal stages shows the presence of four protein bands, as major haemolymph
proteins in
both sexes during the developmental stages. (Venugopal and Dinesh,
1997). Haemolymph protein analysis in the last nymphal instar and adult of Dysdercus
cingulatus by SDS PAGE, shows the presence of eighteen protein bands in the four
day old nymph and twenty protein bands in the six day old nymph, whereas in the
adults, the haemolymph shows twenty two bands (Mohan and Muraleedharan, 2001).
The larval haemolymph of Eri silkworm, Samia cynthia ricini reveals the presence of
two different biliverdin- binding proteins of which the native biliverdin-binding
protein has a molecular weight of 48kDa (Saito, 1998). Analysis of haemolymph
proteins by SDS PAGE and densitometry showed that the quantities of haemolymph
proteins were reduced dramatically in the parasitized larvae of Pericallia ricini (Raja
685
et al., 2000); while in Spodoptera frugiperda parasitised by the braconid parasatoid
Cotesia marginiventris, several high molecular weight proteins were detected much
earlier in high concentration in the haemolymph of parasitised host larvae than in the
control ones (Ferkovich et al., 1983)Analysis of haemolymph proteins by SDS PAGE
in both sexes of silkworm, Bombyx mori during pupal development reveals several
polypeptides ranging from 200 to 14.5 kDa (Janarthanan et al., 1998). The protein
bands in various species are stage dependant and species specific (Elliot and Gillot,
1979;Kim and Seo 1980,1981; Scharf et al.,2005:)
As the haemolymph composition of insects reflects the nature and degree of
metabolism of the tissues bathed in this fluid, changes in protein of haemolymph may
show the
level of
modification
in the organism. Therefore by studying the
haemolymph protein pattern, it is possible to have a clear picture of the protein
metabolism of the insect. The low level of haemolymph protein concentration in the
twenty day old larva and forty day old larva seems to commensurate with the low
degree of growth and development in these instars. The sixty day old larva, on the
other hand shows a maximum growth and so a high level of haemolymph protein
concentration. The factors leading to this condition could be an accumulation of
storage proteins which are known to increase enormously, during the last larval instar
(Levenhook,1985).The voracious feeding habit of these instars may also tend to
increase the dietary source of proteins. During metamorphosis , it has been found that
the pupa requires more proteins for the construction of adult structures. This may be
the reason for the higher levels of protein concentration in the haemolymph of red
palm weevil as development proceeds. However, the variation of
protein fractions
during the different developmental stages of R. ferrugineus in the present investigations
apparently indicates both the synthesis and breakdown of specific proteins.
686
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688
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689
Table-1 R m values of haemolymph proteins of R. ferrugineus in the
differentlarval stages and pupa.
20 day old larva
40 day old larva
60 day old larva
pupa
0.26
0.26
0.26
0.26
0.27
0.27
0.27
0.31
0.31
0.31
0.34
0.34
0.34
0.47
0.47
0.47
0.47
0.64
0.64
0.64
0.64
0.86
0.97
0.97
0.97
Table- 2.Concentration of total proteins in the haemolymph of R. ferrugineus. (mg/ ml).
Stage
Total protein
Larva-20 day old
0.629±0.114
Larva-40 day old
0.796±0.069
Larva-60 day old
0.883±0.108
Pupa
1.738±0.219
separation of haemolymph proteinsof Rhynchophorus
ferrugineus during the different larval and pupal stages.
Lane1-20 day old larval haemolymph. Lane2-40 day old larval haemolymph.
Lane3-60 day old larval haemolymph. Lane4-Pupal haemolymph.
PLATE-1 Electrophoretic
690
ا ر ل ا و ه ا
ا ا اء
Rhynchophorus ferrugineus
س* و س.ر.م ،
ا
!" # $وآ او ا ا اء ا ي ا
&,و+درة ''& ا !%ا"اء .ferrugineus Rhynchophorusأو!" ./ PAGEأن :د ا,9%ت او 8 &%ا !7ا,ت ا &- $ه أآ 4ذ 2
ا !7ا,ت ا" .&4-أ &7 8ادرة > 8ه=< ا,9%ت او &%اآ&, 4
ا .-+Fو E7وDد 8و,ت &-%Cإ 8 ً@A7آ او ا ي G
ا !7ا"ة ) ،(٠J٠٠١>Pو -درا'& ا'M$ب او 8ا"ة M+ل
ا &,او ا ي.
691
692
OP 35
Activity of insect fauna during night in date palm orchards of Central Iraq
1. Mohammed Zaidan Khalaf; Ali Khaywon Shbar; Falah Hansh Naher
;Bassim Hasson Hassan; Noel Franso Jabo and Rajaa Aboud Sami
IPM Center ,Ministry of Science & Technology ,P.O.Box:765 ,Baghdad, IRAQ ,
mzkhalaf2007@yahoo.com :
Abstract
The study was carried out at three palm orchards, each of 2.5 hectare in Al-Madain
palm orchards(30 km. South Baghdad) contain the palm varieties ( Prem, Osta omran,
Zahdi , Berhee, Khadrawi , Khastawi ,Oueedi and Tebarzel ) in 2010 Season aiming to
determination of population density of Palm Borers and to know other insect
occurring in Date Palm orchards. Three light traps with solar energy ( Magna Traps
with lamps of 320 – 420 nm wavelengths are produced by Russell IPM) were used as
one trap in each orchard. The number of Fruit Stalk Borer, Oryctes elegans and Frond
Borer, Phonapate frontalis adults were calculated per week in each trap aiming to
determine the population density of adults in the three orchards under this study.
Results of using light Traps ( Magna Traps ) indicated that the number of O.
elegans adults catch per trap during the period from April to December 2010 were
254, 217 and 240 adult per trap in orchards No. 1, 2 and 3 respectively , the higher
population densities were 87 ,79 and 81 adult /trap per month, respectively reported in
July. Also, the study indicated that the population density of Frond Borer, Phonapate
frontalis catch per Trap during the period from April to December 2010 were 34, 58
and 54 adult /trap for orchards No. 1, 2 and 3 respectively, the higher population
densities were 10, 21 and 15 adult/trap per month, respectively reported in June. Many
insect group were catch in light traps, the Insect fauna composed of nineteen insect
group ,12 are Coleoptera 1 Hemiptera, 1 Hymenoptera, 1 Homoptera, 2 Dermaptera
and 2 Orthoptera, and the numbers of this insect were recorded per month a year round
in Palm orchards.
Key words : Insect, Population density, Palm ,Borers, Light trap.
693
Introduction
The Palm-tree, Phoenix dactylifera is the tree most adapted to climatic conditions of
mad and south of Iraq, these trees and their fruits subjected to attack by many serious
pests causing the considerable loss ( Hussein,A.A.1974 ,Al-Baker,A.I.1972 ,AlBahely,A.Z.2004 , Zaid,A.2002 , Martin,H.1986 , Dhiab,I.M.1979 and BaAngood,S.A.2009 ). The Fruit( Bunches ) Stalk Borer Oryctes elegans, Palm Frond
Borer Phonapate frontales and Trunk (Stem ) Borer Jebusaea hammerschmidti are
from important pests infesting Date Palm trees in many places of the world and a
damaging levels could be found in Iraq and surrounding countries ( Hussein,A.A.1974,
Abass,A.H. 2000, Al-Khunji,A.S.2000, Al-Jboory,I.J.1999 , Al-Jboory 2000.,
Dhiad,I.M. etc.1979 , Kassuma,M.S.2004, Bedford,O.1973 and Atia,M.Z. etc.2009 ).
Oryctes elegans caused many damage to the bases of fronds and bunches making a
long tunnels in the tissue, which are acting as a weakening and breaking factors for
these parts. The opened tunnels may attack by other insect pests and plant pathogens
leading to the complication of the problem ( Abass,A.H.2000 , Al-Beker,A.J.1972 ,
Al-Khunji,A.S. and E.H. Turaihi 2000 , Moustafa,A.T. etc.2004 , Al-Khawaja,H.K.
1999 and Al-Jboory,I.J.2000 ) the larvae and adults are feed on heart of palm causing
weakness and small size of the palm fruits ( Khalaf,M.Z.2010 , Bedrord,O.1973 and
Kaaka,W.A.2009 ) have been found differences between date palm varieties in degree
of sensitivity to infest with O. elegans.
Palm Frond Borer Phonapate frontales is one of the important pest infesting date
palms in Libya ( Ba-Angood,S.A .etc.2009 , Atia,Z.M. etc. 2009 ). Three important
insect infest affected Date Palm trees in Qatar, these insect, according to their
economic importance were Red Pam Weevil Rhynchphorus ferruginens ,Fruit Stalk
Borer Oryctes elegans and long horned Stem Borer Pseudophilus testaceus ( AlKhunji,A.S. and E.H. Al-Turaihi 2000 ). The most important pests affecting Date Palm
in Kingdom Saudi Arabia and United Arab Emirates have been identified as the Red
Palm Weevil
Jebusaea
Rhynchphorus ferruginens , Trunk-Borer O. elegans , Stem Borer
hammerschmidti
and
Rhinoceros
Beetles
Oryctes
Agamemnon
(
Moustafa,A.T.2004 , Kassuma,M.S.2004 ). Biological control has its importance as a
solution responding the economic, sanitary and environmental requirements, The IPM
694
can conserve the biodiversity by natural balance and minimizing and rationalizing to
pesticide use ( Alrouechdi,K.2000 ) .
Al-Bahi et.al.,.2000 , Steibaner,M.J.2003 , Muirhead,R.C.1991 and El-Sebay, Y.
etc.2000 have pointed to possibility of using Light Traps in management strategy of
insect and Date Palm Pests, This study aiming to determine the insect fauna and
Biodiversity conservation in Date Palm orchards in Central Iraq .
Materials and methods
The study was carried out at three palm orchards, each of 2.5 hectare in Al-Madain(
30 Km.South Baghdad ) palm orchards contain the palm varieties ( Prem,Osta
omran,Zahdi ,Berhee, Khadrawi , Khastawi ,Oueedi and Tebarzel ) . Three light traps
with solar energy ( Magna Traps with lamps of 320 – 420 nm wavelength are produced
by Russell IPM) (Fig.1) were used as one trap in each orchard . The number of
economic important pests Fruit Stalk Borer Oryctes elegans , Frond Borer Phonapate
frontales and others were calculated per week in each trap a year round aiming to
determine the population density of this insect in the three orchards under this study .
Result and discussion
1 : Population density of Fruit Stalk Borer Oryctes elegans :
The results presented in table 1 indicated that the number of O. elegans adults catch
per trap during the period from April to December 2010 were 254 , 217 and 240 adult per
trap, the higher population densities were 87 ,79 and 81 adult /trap per month in orchard
No. 1, 2 and 3 respectively reported in July. The data showed that the flight activity of
the adults started from the beginning of April until October , While the Harib,A.1970 ,
Al-Khawaja,H.K.1999 , Swayer,I. A. etc.1979 pointed that adults showed in March till
November. Notably a large body of this insect may help on transfer many pathogens
among Palm trees. The studies in Iraq indicated to isolate many pathogens from Oryctes
elegans larvae ( Al-Jboory,I.J. etc.2006 ). This pest was controlled by using chemical
pesticide ( Al-Jbooyi,I.J. etc.2001 , Al-JbooryI.J. and S.J.Salh 2001 ), pathogenic
nematode (Al-JbooryI.J. and S.J.Salh 2001 ) and Virus ( Bedford,O.1973 ). The highest
period of adults activity during period of May , June and July, therefore, possibility of
application control programs to adults in this period . High efficient of light traps in catch
of adults to possibility will be used in IPM programs to this pest .
695
Population density of Frond Borer ,Phonapate frontalis Fahr.:
The numbers of frond Borer , Phonapate frontalis Fahr. Were calculated in weekly
and monthly bases during the season 2010 . The highest population densities were 10 ,
21 and 15 adult per trap /month for the orchards No. 1, 2 and 3 respectively with an
average of 15 adult per trap /month during in June 2010 Table 2. The data showed that
the highest flight activity of the P. frontales in period of May, June and July. Light
traps have proven highly efficient in catch of P. frontales, therefore, it can be used in
IPM programs to this pest for purposes of monitoring and control.
3. Palm Stem Borer ,Jebusaea hammerschmidti Reiche:
The Palm Stem Borer , Jebusaea hammerschmidti Reiche was not recorded in south
Baghdad Palm orchards with a middle –age trees, but was present in old trees in Palm
orchards in North Baghdad and Diwaniyah Region trees .
4. Identification of some Insect types caught by Light Traps
( Magna Traps ) in date Palm orchards :
The insect caught by Light Traps were screened and identified by the Iraqi Natural
History Museum /University of Baghdad specialist, Dr. Mohammed Saleh Abdul
Rassoul . The Insect were recorded weekly and monthly (Table. 3 ), the insect fauna
composed of Nineteen insect group, 12 are Coleoptera, 1 Hemiptera, 1 Hymenoptera,
2 Dermaptera Fig. 2 and others, some of these insect were pests , non pests, natural
enemies unknown, this subject needs more studies and contacts with specialized
persons or centers . Data showed that have been found differences between insect
groups in the activity period of these insects during the year ( Table 3 ). The
Coleopterous insect are important in various-agro-ecosystem some play in active role
as predators or saprophagous insect and others serve as a biological indicators in the
place where exist ( Ramadhane,M.A, 2000 ).
Acknowledgments
The Authors are grateful to the International Center for Agricultural Research in the
Dry Areas (ICARDA) & The International Fund for Agriculture Development (IFAD)
This work was supported by ICARDA & IFAD , it is part from project" Improved
livelihoods of small farmers in Iraq through integrated pest management and organic
fertilization" { IRAQ – ICARDA – IFAD Project(IFAD GRANT NO. 1001 – 1Q)}
696
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Table 1.The number of Oryctes elegans adults caught in Light Traps ( Magna
Traps) in Date Palm orchards of south Baghdad during season 2010 .
Number of Oryctes elegans adults
Month
Orchard No.1
Orchards No.2
Orchard No.3
January
0
0
0
February
0
0
0
March
0
0
0
April
27
21
23
May
45
39
38
June
56
41
56
July
87
79
81
August
24
27
31
September
14
10
11
October
1
0
0
November
0
0
0
December
0
0
0
Total
254
217
240
Table 2. Number of Phonapate frontalis adults caught in Light Traps
( Magna traps) in Date Palm orchards of south Baghdad during season 2010 .
Number of Phonapate frontalis adults
Month
Orchard No.1
Orchards No.2
Orchard No.3
January
0
0
0
February
0
0
0
March
0
0
0
April
3
4
7
May
6
5
8
June
10
21
15
July
9
19
13
August
4
7
2
September
2
2
0
October
0
0
0
November
0
0
0
December
0
0
0
Total
34
58
45
700
Table 3. Some Insect fauna of activity night in Date Palm orchards of south
Baghdad during season 2010.
Number of insect caught in Light Traps ( Magna Traps) per month
Insect
May.
0
Apr.
27
45
Jun.
56
Jul.
87
Aug.
24
Sep.
14
Oct.
1
Nov.
0
Dec.
0
0
0
105
247
509
137
147
69
73
11
0
0
0
0
19
42
30
6
32
10
6
0
0
0
0
0
1706
3737
4605
2554
1296
224
24
0
0
0
0
0
304
607
123
23
39
24
0
0
0
0
0
0
3
36
16
5
4
2
0
0
0
0
0
0
1
1
131
170
0
0
0
0
0
0
0
0
1
3
195
170
0
0
0
0
0
0
0
0
0
601
1179
2953
291
35
0
0
0
0
0
0
0
0
81
256
139
5
0
0
0
0
0
0
16
108
276
176
59
17
0
0
0
0
0
0
0
0
0
0
7840
9633
3722
1104
0
0
0
0
9
56
87
27
3
7
2
5
0
0
0
0
0
0
9
4
0
0
0
0
0
0
0
0
84
3988
603
706
46
81
21
44
12
Dermaptera,Forficulidae
0
0
0
16
58
28
8
4
1
0
0
0
Orthoptera,Acrididae,
Lacustidea
Orthoptera,Gryllidae,
Blattidae
Coleoptera,Cerambycidae
Jebusaea hammerschmidti
0
0
0
12
77
77
105
13
2
3
1
0
0
0
0
23
75
233
265
285
83
57
13
0
Coleoptera,Scarabaeidae,
Oryctes elegans
Coleoptera,Dynastidae
Pentodon bispinosas
Coleoptera, Copridae
Onitis alexis
Coleoptera, Copridae
Onthothagus sp.
Coleoptera,Tenbrionidae
Opatroides punctatus
Coleoptera,Bostrychidae
Phonopate frontalis
Coleoptera,Rutelidae
Anisoplia leacaspis
Coleoptera,Melolonthidae
Cyphonoxia praestabilis
Coleoptera,Cicindelidae
Cicindela melancholica
Coleoptera,Carabidae
Distichus planus
Coleoptera,Carabidae
Chlaenius spoliatus
Hemiptera,Cydnidae
Geotomus sp.
Hymenoptera,Formicidae
Dorylus fulvus
Homoptera,Cicadidae
Cicadatra alhageos
Lepidoptera
Jan.
0
Feb.
Mar.
0
0
Found in north Baghdad and Diwaniyah site orchards but did not calculated (old Palm
trees)
701
Fig.1 : Solar insect light traps ( Magna Trap, Russell IPM ) used to
determine the population density of insect in Date palm orchards
Coleoptera
Hemiptera
Hymenoptera
Homoptera
Dermaptera
Orthoptera
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702
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703
704
OP 36
Effect of Fusariosis on the metabolism of phenolic compounds of
date palm leaflets, resistant and sensitive to the disease.
Saida OUAFI, Nicole BOUNAGA.
Research Laboratory on Arid zones (L.R.Z.A) Faculty of Biology (USTHB). BPn° 32
El-Alia, Bab Ezzouar, 16111, Algiers, Algéria. Saida_ouafi@yahoo.fr
Abstract
The date palm, Phoenix dactylifera L. is the only species to be adapted to the most
arid regions of the planet. At the base of the diet of traditional agricultural societies of
the Sahara, the date palm is probably for agriculture in desert regions the best source in
the future. Because of socio-economic importance of date palm in North Africa, the
scientific community has always followed the progression of parasitic disease caused
by the fungus Fusarium oxysporum f.sp.albedinis identified by Malençon in 1934. The
definition of flavone content of the species seemed to us interesting to acquire, since
the’ ‘small molecules’ ‘are recognized both as genetic markers and as molecules of
relationship. The study was performed on population of thirty palm trees belonging to
three cultivars of the oases of south-west Algeria TOUAT Gourara . The analytical
protocol consists of a hot hydrochloric acid hydrolysis of 1 g of leaf material dried
and ground. Structural analysis of compounds present in extracts was conducted by
chromatographic and spectrophotometric techniques. Through the analysis of all
HPLC profiles of phenolic acids,we have established three chemotypes: resistant with
the highest heights.Sensitive, with the lowest height, finally low sensitivity or tolerant
with intermediate heights. The increased synthesis of C-glycoflavones, within diseased
palms, can be seen as the first sign of plant response to fungal attack, or even as a
defence mechanism so these flavonoids correspond to''pre-inhibitines.''
It has not been found qualitative differences between different cultivars studied .The
merits of our work show a negative correlation between flavonoid content and the
level of lignification in the date palm ,suggests that the process of lignification is more
rapid in resistant cultivars. We can say that there is a constitutive resistance.
Key-words: date palm tree, Fusariose, chemotype, resistant, phenolic compounds
705
Introduction
The date palm, Phoenix dactylifera L. is the only species to be adapted to the most
arid regions of the planet. At the base of the diet of traditional agricultural societies of
the Sahara, the date palm is probably for agriculture in desert regions the best source
in the future.
In Algeria, the park covers about 1 million hectares, almost all agricultural land
areas lying below the isohyet 100 mm / year. The number of date palm tree is
estimated at 17 million for one-third crop farming, mainly on the cultivar of date
Deglet Nour, the other two thirds, producing 120 000 tons of dates of various cultivars,
feed producers and the local market.
Because of socio-economic importance of date palm in North Africa, the scientific
community has always followed the progression of parasitic disease caused by the
fungus Fusarium oxysporum f.sp.albedinis identified by Malençon [1 ].
Reported for the first time there are over 120 years, fusariosis date palm tree, or
bayoud attacks best cultivars and destroyed 10 million trees in Morocco and 3 million
in Algeria. Present exclusively in these two countries, this disease constitutes a serious
threat to date palm of the other countries of North Africa and the Middle East.
According to the work of Rahmania
[ 2]., Matheron and Benbadis [3 ]., the
penetration of Fusarium oxysporum f sp albedinis is through the roots and then
invades the vascular elements of the palms causing the decay and the bleaching and
death of affected tree from which the name of the disease "Bayoud."
The definition of flavone content of the species seemed to us interesting to
acquire, since the ''small molecules'' are recognized both as genetic markers and as
molecules of relationship. Initially, a flavone type cultivars was proposed, essentially
based on the profile flavones / flavonols and open the way to the taxonomy and
genetics infra specific
[4 ]. [5 ]. [ 6]. [7 ]. We present results on more physiological evolution of
polyphenols (phenolic acids and flavone C-glycosides) response to Fusarium
infestation, seeking a possible causal relationship between the metabolism of the
parasite and the date palm tree.
706
Materials and methods
Plant material
The study was conducted on population of thirty palm trees belonging to three
cultivars of the oases of south-west Algeria TOUAT Gourara (rainfall below 30 mm /
year). Harvesting of foliage (palm leaflets) was performed in November 2003
Methods
The analytical protocol was developed by Lebreton [8 ]. It consists of a hot
hydrochloric acid hydrolysis of 1 g of leaf material dried and ground, causing release of
phenolic acids. Exhaustion of the aqueous phase with diethyl ether hydrolytic allows
extraction and assay. From the remaining aqueous phase were extracted the C-glycosides
by nbutanol. Spectrophotometry (at 340nm) allows the measurement of C-glycosylflavonoides.Results are expressed (in mg.gl MS), as vitexin for C-glycosyl flavonoids.
Structural analysis of phenolic acids:
Structural analysis of compounds present in extracts was performed by
chromatographic and spectrophotometric techniques. The use of a solvent gradient is
more efficient. Regarding the composition of the solvent: acetic acid was chosen,
acetonitrile eluent with properties superior to those of methanol has been preferred.
The following Solvent A: 0.5% acetic acid in distilled water. Solvent B: 0.5% Acetic
acid in acetonitrile. The linear gradient from 0% to 20% B in A in 45 minutes,
followed by a return to initial conditions for 15 min with a constant flow of 1.5 ml /
min. Detection is at 260 nm.
Results and discussions.
Results of structural analysis.
The study of the HPLC profiles of the leaflets of palm trees belonging
to the three cultivars studied showed a total of 10 peaks (Fig. 1). Each of these peaks is
characterized by its retention time and its absolute content (peak area).
Through the analysis of all HPLC profiles of phenolic acids. This is a descriptive
work that aims to research and the differentiation between the profiles of the most
original and most common profiles. Original profile is represented by a homogeneous
group of plants that combine the same way all the studied phenolic acids metabolism,
such a profile will be designated by the term "chemotype". Highlighting the different
707
patterns will finally approve the status of each cultivar has its status in relation towards
Fusariosis. We reported on table-1 the absolute content mean for each phenol acid
present on profile of the individuals of the three cultivars studied (GH: Ghar TK:
Takerboucht, DN: Deglet Nour).
of each cultivar.
From this table we have established the histogram
These histograms correspond to chemotypes: resistant with the
highest heights(TK:takerboucht).Sensitive(DN:deglet nour), with the lowest height,
finally low sensitivity or tolerant(GH:ghars) with intermediate heights.
(Fig.2)
Result of quantitative analysis of C-glycosyl-flavonoids
The situation is almost symmetrical to the last, except that changes in these
compounds are accompanying the infestation early as before, there are increasing (32%)
significantly (t = 8.30, p <0.001)(Table-2)
The increased synthesis of C-glycoflavones,
within diseased palms, can be seen as the first sign of plant response to fungal attack, or
even as a defence mechanism so these flavonoids correspond to''pre-inhibitines.''
It should be noted however that this does not necessarily imply a direct causal
relationship, as underlined precisely [9 ]. in such a context,’ the increased synthesis of
these substances may not be directly related to disease resistance, but may simply be a
symptom of the disease’’.
Modelling results (see diagram )
Chemical processes of defence can also be coupled with physical mechanisms,
including alteration of the vessels, previously reported, the deposits of lignin and / or
tannins. This effect opposes to the propagation of Fusarium, but also a double-edged
sword in that the diffusion of nutrients and metabolites is also affected. The parasite
attack is generally recognized at least in part, the phenomenon may be related to
increased activity of PAL (phenylalanine ammonia-lyase), which controls the flow of
the amino acid to cinnamic acids, common precursors of phenolic acids, flavonoids
and lignin. Perhaps the difference in sensitivity to the parasite expressed by the various
cultivars of date palm results in the rate of balance achieved between the two channels
respectively, positive (1) and negative (2) for the plant (Fig.3).
Conclusion
In conclusion, it has not been found qualitative differences between
different cultivars studied. These were distinguished by their content of
708
different phenolic constituents. The quantitative aspect could therefore be used in the
behaviour of date palm cultivar in relation to the bayoud. Thus, resistant genotypes
accumulate levels of phenolic acids significantly higher level of leaflets . This criteria
could serve as a biochemical marker of date palm resistance to Fusarium oxysporum
fsp albedinis. These phenolic compounds are often used in relation to plant pathology
in several plant species. Especially some are precursors of lignin. The merits of our
work show a negative correlation between flavonoid content and the level of
lignification in the date palm suggests that the process of lignification is more rapid in
resistant cultivars; we can say that there is a constitutive resistance.
709
References
[1] MALENÇON 1934 – Les palmeraies du Draa et Bayoud. Bull. soc. Hist. Nat. Afr.
N., 25 :112 – 117.
[2] RAHMANIA F., 1982 – Contribution à la connaissance du palmier dattier,
(Phoenix dactylifera L), et de l’agent du bayoud, fusarium oxysporum fsp.
albedinis (Killian et Maire).Gordon. Aspects ultrastructuraux des relations Hôte –
parasite. Thèse de 3eme cycle.U.S.T.H.B. Alger
[3] MATHERON B., et A. BENBADIS 1985 – Etude comparée des premières phrases
del’infection du palmier dattier (Phoenix dactylifera L) par deux formes spéciales
de Fusarium oxysporum : La fsp albedinis (agent du Bayoud) et la fsp melonis.
Bull. Soc . Fr. 132, lettresbot., 203 – 212 p.
[4] OUAFI S., 1987– Etude chimiotaxinomique par les flavonoïdes des cultivars de
palmiers dattier de la station de l’INRA de ADRAR. Thèse de magister,
U.S.T.H.B, 125 p.
[5] OUAFI S., GACEB – TERRAK R., BOUNAGA N et LEBRETON PH, 1988 –
Les flavonoïdes marqueurs infraspécifiques chez le palmier dattier Phoenix
dactylifera L. C.RAcad. Sci. Paris, t.306, série III, p- 399 – 404.
[6] OUAFI.S, BOUNAGA.N. 2008-Les glycosides flavoniques marqueurs de
cultivars algériens du palmier dattier Phoenix dactylifera L.Acta Bot.Gallica,
2008,155(2) ,307-315.
[7] OUAFI .S, 2011- Les composés phénoliques des folioles du Phoenix
dactylifera .Editions universitaires européennes (May 9, 2011) ISBN-10:
6131573484 ISBN-13: 978-6131573484
[8] LEBRETON PH., JAY M., VOIRIN B. et BOUCHEZ M.P. 1967 – Sur
l’analysequalitative et quantitative des flavonoïdes. Chimie analytique, 49
(7), 375 – 383.
[9] HARBORNE J.B. 1977 – Flavonoïds and the evolution of the Angiosperms.
Biochem, Syst.Ecol, 5(1), 7 – 22.
710
Table-1. The absolute content mean for each phenol acid present on profile of the
individuals of the three cultivars studied (GH: Ghar TK: Takerboucht, DN: Deglet Nour).
A
B
C
D
E
F
G
H
I
J
GH 14 487,50
1 761,30
15 860,30
72,14
5 124,25
1 860,66
3 892,15
1 980,80
90,3
36 001,25
TK 14 978,21
18 325,30
1 478,04
603,04
4 914,10
1 623,20
192,9
2 724,80
861,02
45 375,74
DN 14 978,21
3 741,80
1 577,16
612,22
5 243,00
1 636,30
113,16
1 443,33
723,53
30 210,34
Table-2. Mean levels of C-glycosyl flavanoids in three cultivars
Mean levels of C-glycosyl flavonoids
( mg.g-l M.S.)
Takerboucht(n=10)
3.46 +/- 0.19
Deglet nour healthy (n=10)
2.49 +/-0.22
Deglet nour diseased (n=10)
2.98 +/-0.25
Ghars(n=10)
3.32 +/- 0.21
A:Catechol, B:protocatechuic acid, C:gentisic acid, D:parahydroxybenzoic acid, E:vanillic acid, F:caffeic
acid, G:syringic acid, H:p.coumaric acid, I:ferulic acid, J: sinapic acid.
711
Figure 1: Profile H.P.L.C of phenolic acids of leaflets of the Phoenix dactylifera L.
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Fig.2. Histograms correspond to chemotypes: resistant with the highest
heights(TK:takerboucht).Sensitive(DN:deglet nour), with the lowest height,
finally low sensitivity or tolerant(GH:ghars) with intermediate heights.
712
Fusariosisا ا آت ا
" ) رات ا '& ،وا $ا #و" وا ا ا ض
Saida OUAFI, Nicole BOUNAGA
ا ا وSaida_ouafi@yahoo.fr:
ا
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أ# Iت أه ا) FG" ،ا/وEط ا دا C" 6ر ا#/اض ا Cا( AAB
Fusarium oxysporum albedinis.#Cا( ده Malenconم A .١٩٣٤و أن
" S #ا (2ى P O Qأ Oاع اN +5ت ا# 73ة ا #ة Qه( م اآ(Bب ا T " 5و%
ا (#ف GآQت ورا E Xاء وآ "#ف N +5Gت ا.Q
أ? F #ارا XQX P 5 Eن أ<5ر ا) ا( ( #ن إ XQXأYف ا ? دة
وات ? ب #-ب ا+5ا " Gourara #6ات#G .و" آ ل ا( G 2" +!"# 2اCE
] ا روآ ر \ ا P[Bل #- ١ام Pاوراق ا . 5و أ?#ي ا( 2ا Aي #آAت
ا ? دة ا ( Cت Q[ Pل " ت ا!#و" #-ا HPLCوا#(!ABو " (.#
Q[ Pل " 2آ ا(_! Qت Pا /ض ا ،أ`_Oت : chemotypes XQXو
و /ر Pا #آAت b EB،ار" ع أد ، Oوا EB2ا ) 9أ[ #ا أو ا ( b
ار" ت ( CEوا 2eا #? (Bام .و ! Pرؤ ز دة glycoflavones -Cدا[ ا)
ا ،G3وأول Qا G5(Eا 5 (Aم ا ، #Cأو ( hا ع (" g 2Gا هf%
ا ( .inhibitines pre O Qا ر ا[( Qت P G OاY/ف ا )( ا>G5(E
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lignificationا) ،
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713
714
OP 37
Population dynamics of the Red Palm Weevil Rhynchophorus ferrugineus
(Oliv.) on date palm plantations in 6th October Governorate.
El-Lakwah, F. A. M. *; EL-Banna, A. A.**; El-Hosary, Rasha A* and
El-Shafei, W.K.M** .
*Plant Protection Department, Fac. Of Agricultural (Moshtohor) , Benha Univ., Egypt.
**Date Palm Research Lab. Agricultural Research Centre(ARC), Giza, Egypt
Abstract
The present work aims to study the Population dynamics of RPW Rhynchophorus.
Ferrugineus (Oliv.) adults
in relation to certain climatic factors using attracting
pheromone traps on date palm plantation . Experiments were conducted during two
successive years (2009 and 2010) in Wardan and Abu-Ghalep villages, 6th October
Governorate. The diurnal flying activity of RPW were also considered .The obtained
results showed that adults emerging continually throughout the year. The lowest adults
population was recorded during December and January. The population showed four
peaks each year. There were four peaks of emergence during 2009 at 2nd week of
April, 1st week of June, 1st week of August and 2nd week of November; in 2010, four
peaks were also recorded during 4th week of March , 3rd week of June, 3rd week of July
and 2nd week of November.
Data indicated that there was significant positive
correlation between average temperature and adults population abundance of the RPW
during the studied two year. While relative humidity had negative effect. The diurnal
flying activity of RPW adults was studied during the tested period. The obtained data
revealed that the maximum flying activity of RPW adults during day hours was at time
6.00 am followed by the time at 16.00 pm.
Introduction
The
red
palm
weevil,
Rhynchophorus
ferrugineus
(Oliv.)(Coleoptera:
Curculionidae), is an economically important, tissue-boring pest of date palm in many
parts of the world. The insect was first described in India as a serious pest of coconut
palm (Lefroy, 1906) and later on date palm (Lal, 1917; Buxton, 1918). The weevil was
recorded later in Seri Lanka, Indonesia, Burma, Punjab, and Pakistan (Laskshmanan,
1972).The insect is a major pest of date palm in some of the Arabian Gulf States
715
including Saudi Arabia, United Arab Emirates, Sultanate of Oman, and Egypt (Cox,
1993; Abraham et al. 1998). The agroclimatic conditions prevalent in this region and
the unique morphology of the crop, coupled with intensive modern date palm farming,
have offered the pest an ideal ecological habitat (Abraham et al., 1998).
The recent discovery of the male-produced aggregation pheromone [ferrugineol, 4methyl-5-nonanol] for R. ferrugineus (Hallett et al. 1993) made the implementation of
pheromone-based monitoring and trapping of this weevil possible for the management
of this pest. Rhynchophorus palmarum is managed in Central and South America
without insecticide spray by pheromone trapping and sanitation practices in oil,
coconut and palmito palm. It is a strong flyer traps which are normally placed at
densities of 3-7 hectare (Chinchilla et al., 1993). El-Ezaby et al. (1998) reported
maximum catches in March and April. Aggregation pheromones have been reported as
effective tools for monitoring and trapping RPW in the field (Gunnawardena and
Badarage, 1995). The objectives of this study were to study the population dynamics
of RPWand Investigate the diurnal flying activity of RPW.
Material and methods
These experiments were carried out during 2009/2010 in certain date palm
plantations, which infested by the Red Palm weevil (RPW).The chosen area are
Wardan and Abu Ghaleb Villages, situated in the 6th October Governorate , Egypt.
1. Population dynamics of Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Oliv.).
To study the population dynamics of Red palm Weevil (RPW) R. ferrugineus
(Oliv.), three infested locations at Wardan Village, the 6th October Governorate were
taken .The experiment was conducted during 2009 – 2010.Three pheromone traps
were distributed in the investigated area. Each trap contained the following materials:
1- 250g of dates, 2- dispenser of the R. ferrugineus male aggregation pheromone
(Pheromone lure was manufactured by Chim Tica international S.A. Company, Costa
Rica. Trade Name PO28 Ferroluree+,700mg lure ,consists of a mixture of 4-methyl 5
– nonanol and 4- methyl 5-nonanone (9:1part purity of both components
95% release
rate 3-10 mg/day), 3 – dispenser of the kairomone containing 45 mL of the active
ingredient ethyl acetate in gel, at 95% minimum purity, release rate 200-400 mg/ day
colorant added (Kairomone manufactured by ChimTica international S.A. Company,
716
costa Rica. Trade Name PO80A Weevil magnet 45 mL lure), 4 – 5 L of water. Traps
were fabricated using a 10 L polypropylene bucket .The outer surface of the bucket
was rough with plastic net (1-2 mm).Traps were without cover and buried in the
ground down to the level of 15 cm to facilitate entrance of R. ferrugineus adults. Partburying of the trap also prevented it from being over-turned by wind or animals or
water of flooded irrigation. Each trap was 4 meter away from date palm trees (to avoid
that any adult could miss the trap and lay eggs on the palm tree) in the shade to avoid
evaporation. The water was always replenished every two weeks to keep sufficient
moisture in each trap for avoid escaping of the adult and it help to kill the insects.
Food bait (dates) was renewed every two weeks. Based on the dispensing rates, the
pheromone and the kairomone were replaced every 2 months. Trapped weevils were
collected, removed, counted, sexed and recorded every two weeks. Correlation
coefficient ,and regression values were calculated
2-Diurnal flying activity of RPW .
The diurnal activity of RPW in date palm plantation was studied at infested date
palm plantations at Abu-Ghalep Village, the 6th October Governorate by recording
data of the weevil captures in 5 food baited pheromone traps, during May of two
successive seasons, of 2009 & 2010. Two hourly Observations of the number of the
trapped weevils were recorded on a daily basis at 6 am, 8 am,10 am,12 am,14 pm,16
pm,18pm and 20 pm. Trapped weevils were removed from the traps and recorded
every day.
Statistical analysis
The obtained data were subjected to regular analysis of variance of randomized
complete block design (RCBD) , outlined by Gomes and Gomez (1984) .
Results and discussion
1. Population dynamics of RPW Rhynchophorus ferrugineus (Oliv.):
1.1.Population dynamics of RPW R. ferrugineus (Oliv.)on date palm plantation
during 2009.
Data presented in Table (1) and illustrated in Fig. (1.a&b) showed the adult
population abundance of RPW is existed all over the year and there are four peaks all
over the year 2009, the mean of total captured adults start with low number in the
717
beginning of January with 5.33 adults / trap and it fluctuated then increased gradually
to reach the first peak at the second week of April with 56 adults / trap. Then the mean
of total captured adults declined to 21.33 adults / trap at end of April and it stayed
stable for while then from the fourth week of May it started to increase drastically to
reach the second peak at the second week of June with mean of total captured adults
68.33 adults / trap (highest value) ,then it decreased rapidly until the third week of
June with 15 adults / trap and it stay stable for while after it increased gradually to
reach the third peak at the first week of August with mean of total captured adults
28.67 adults / trap Then it declined gradually to 2.33 adults / trap at the third week of
August then it stayed stable for a while until the second week of October it start to
increase slowly to reach the fourth peak at the second week of November with a mean
of total captured adults 19 adults / trap then it declined gradually to reach 6.67 adults /
trap at the first week of December and it stayed with the same rate until the end of
December. The results also indicated that significantly more females were caught
by 1.43 times than males during the whole year of 2009.The population fluctuation
of the male and female adults of RPW showed the same trend as indicated by the total
adults population during 2009 .The average mean value of the total adult population,
male and female adults populations was17.79 , 7.31 , 10.49 respectively .
1.2. Population dynamics of RPW R. ferrugineus (Oliv.)on date palm plantation
during 2010.
Data presented in Table (2) and illustrated in Fig. (2.a&b) showed that the adult
population abundance of RPW is existed all over the year with four peaks. The mean
of total captured adults started with low number in the beginning of January with
4.67(low value) adults / trap then it increased gradually to 17 adults / trap at the second
week of March then it increased sharply to reach the first peak at last week of March
with 81.67total adults /trap (highest value) then the average captured RPW adults
declined rapidly to 10 adults / trap at the second week of April. Then the average
captured RPW adults increased gradually to reach the second peak at the third week
of June with 66.33 adults / trap then it declined to 40.33 at the first week of July and it
increased again to reach the third peak at the third week of July with 75.67 adults /
trap. Then the average captured RPW adults decreased sharply to 21.67 at the first
718
week of August then it fluctuated to reach 5.67 adults / trap at the fourth week of
October then it showed the fourth peak at the second week of November with 30.67
adults / trap then it declined gradually to reach the lowest average captured RPW
adults 3 adults / trap(lowest value)
at the first week of January . The results also
indicated that significantly more females were caught by 1.76 times than males
during the whole year of 2010.The population fluctuation of the male and female
adults of RPW showed the same trend as indicated by the total adults population
during 2010 .The average mean value of the total, male and female adults population
was 22.81, 8.24,14.56 respectively . The mean of total adults population during 2010
was slightly higher than mean of total adults population during 2009,which could be
due to slight variation in the average of daily temperature during 2010.
The obtained results are in agreement with the findings of Abdallah and Al-Khatri,
(2003), who observed that RPW adults emerging continually throughout the year. The
minimum number of insects was recorded during December and January. In 1996,
there were four peaks of emergence during March, May, July and October; whereas in
1997 the peaks were recorded in April, May and September, In 1998, four peaks were
recorded during April, May, August and October . These data insure the previous data
obtained by and the same observation was obtained by Qin et al (2004) who found
that, the population monitoring of red palm weevil occurred in four peaks a year in the
area of Wenchang, Hainan Province. On the other hand El-Sebaey (2003) in Egypt
indicated that R. ferrugineus had two main active seasons annually. The first adult
brood was observed in April and the second one was in November. Female density
was higher than male density and it constituted 52.8-57.35% of the total population in
the field.In addition, Faleiro (2005) also found that the weevil captures were female
dominated and for every male weevil trapped two female Weevils were captured.
Furthermore, Al-Saoud,(2007) showed that the adult RPW were present throughout the
year, and the number of females was higher than the number of males.
1.3. The effect of weather factors on the population abundance of RPW during
2009 and 2010:
Data in Table (3) showed the effect of weather factors (mean daily temperature
&RH) on the population abundance of RPW during 2009 and 2010. The simple
719
correlation coefficient ( r ) value indicated significant positive correlation between
average temperature and adults population abundance of the RPW during the tested
two years of 2009 and 2010,while the means of daily relative humidity had significant
negative effect on the population abundance of the RPW adults during the two years.
The analysis of variance revealed that the two factors (Temp.+RH.) are responsible for
about 22.9% in the variability of the RPW populations during the studied years of
2009 and 2010.
This results is in harmony with the findings of Faleiro (2005) who found that
Maximum temperature and rainfall had a significant impact on the weevil activity in
India while, the maximum temperature was positively correlated (r = 0.51) with weevil
captures, rainfall was negatively correlated (r=-0.61) with the weevil catch .In this
respect. Also, Huang et al.,(2008) in China indicated that the climatic conditions had
an obvious influence on the trapping effect of pheromone for RPW. The trapping
population was significantly reduced in the conditions of rain and low temperature. In
addition, Abdallah and Al-Khatri, (2003), reported that there is an effect of the
climatic conditions of maximum and minimum temperature and the Relative Humidity
on the population fluctuation of RPW.
Contrarily El-Sebaey (2003) found that there was no relationship between seasonal
population fluctuations of RPW and weather factors.
2. Diurnal flying activity of RPW.
The diurnal flying activity of RPW adults was studied during the tested period of
(May 2009 and May 2010).The obtained data given in Table (4) and illustrated in
Fig.(4&5) revealed that the maximum flying activity of RPW adults during the day
hours was at 6.00 am with an average captured RPW adults of( 7.20 ,6.80 per trap) in
the two seasons of 2009 and 2010, respectively, followed by the time at 16.00 pm with
an average of (5 , 4.40 per trap) for the two seasons respectively. Otherwise, the flying
activity of RPW adults were lower showing fluctuation in the average captured values
and could be arranged in descending order as follows : (3.20,3.20), (3.00.2.60),
(2.00,1.60),(2.00,1.60),(1.60,1.20)at 14.00,12.00,10.00,18.00,20.00 hours during the
two seasons respectively .The lowest flying activity of the insect during the day was at
8.00 am with an average of 0.2 adults /trap and zero adults /trap during 2009 and 2010,
720
respectively . Data showed also that there were no significant difference between the
flying activity of the insect during the day hours at 12.00 and 1400 hours. The data
also showed that there were no significant differences between the RPW flying activity
at day hours at 10.00, 18.00, 20.00 hours depending on the average captured adults.
In this respect, Faleiro (2005) in India studied the diurnal activity of RPW in
Coconut by recording during May2002. Six hourly Observations on the number of
weevil trapped were recorded on a daily basis at 6 am, 12 pm, 6 pm and 12 am. The
maximum number of weevils trapped was at 6 am, while no weevils were observed at
12 am. This indicates that RPW was most active between 12 am (mid night) to 6 am
(morning). The highest mean cumulative catch per trap was between 12 am and 6 am
while, the least cumulative catch per trap was between 6 pm to 12 am .The findings of
this study therefore indicate that RPW adults prefer to fly in the coconut plantations of
Goa between
12 at mid night and 6 in the morning .Also, Gunawardena and
Bandarage(1995) in Seri Lanka reported that the daily activity of RPW adults were
between 6:00-8:00am and 6:00-8:00pm.In Sultanate of Oman, Abdallah and Al-Khatri,
(2003) found that the daily activity of RPW in date palm plantation (was maximum
Number of weevils trapped) were between 6.00 -9.00Am which the population
percentage was 39.4% followed by the time between 6.00 -9.00pm which the
population percentage was 33.3%
while no weevils were observed in the time
between 9.00am to 3.00 pm and 12 am -3 00am ,and the population percentage at the
time 3:00-6:00 am , 3:00-6:00 pm were 1.9% and 15.2% respectively.
721
References
[1] Abdallah F.F. and Al-Khatri S.A.(2003). Seasonal fluctuation of Rhynchoporus
ferrugineus (Oliv.) Coleoptera –Curculionidae) in the Sultanate of Oman . International
conference on date palm (2003) kingdom of Saudi Arabia king saud University
[2] Abraham, V. A., M. A. Al-Shuaib, J. R. Falleiro, R. A. Abozuhairah, and P. S. P.
V. Vidyasagar. (1998). An integrated management approach for the red palm
weevil Rhynchophorus ferrugineus Oliv. A key pest of date palm in the Middle
East. 3: 77-83.
[3] Al-Saoud, A.H. 2007. Importance of date fruit in Red Palm Weevil
Rhynchophorous ferrugineus (Oliv.) (Coleoptera: Curculionidae) Aggregation
Pheromone Traps. Acta Hort. (ISHS) 736:405-413
[4] Buxton, P. A. 1918. Report on the failure of date crops in Mesopotamia in 1918.
Agric. Directorate, M. E. F. Bassarah Bull. No. 6.
[5] Chinchilla, C.M.; Oehlschalger, A.C. and Gonzalez, L.M. (1993). Management of
Red Ring Disease in Oil Palm Through Pheromone-based Trapping of
Rhynchophorus palmarum (L.). Palm Oil Research Institute of Malaysia
International Palm Oil Congress, Kuala Lumpur, Malaysia, September.
[6] Cox, M. L. 1993. Red palm weevil, Rhynchophorus ferrugineus, in Egypt. FAOPlant-Protection-Bulletin 41: 1, 30-31.
[7] EL Ezaby, F.A.; Khalifa, O. and EL Assal, A. (1998). Integrated pest management
for the control of red palm weevil Rhynchophorus ferrugineus Oliv. In The United
Arab Emirates, Eastern Region, Al Ain. In RAHMAN-AL AFIFI, M. A. AND ALSHERIF AL-BADAWY, A. (Eds) Proceedings of the First International
Conference on Date Palms. Al-Ain, UAE, 8-10 March 1998. Faculty of
Agricultural Sciences, UAE University, pp. 269–281.
[8] El-Sebaey, Y. (2003). Ecological studies on the red palm weevil, Rhynchophorus
ferrugineus Oliv. (Coleoptera:Curculionidae) in Egypt. Egypt. J. Agric. Res., 81
(2): 523-529.
[9] Faleiro J.R. (2005). Pheromone technology for the management of red palm weevil
Rhynchophorus ferrugineus (Olivier) (Coleoptera: Rhynchophoridae)–A key pest
of coconut, Technical Bulletin No.4,ICAR Research Complex for Goa.40pp
722
[10] Gomes, K.A. and Gomez, A.A. (1984).Statistical procedures for Agricultural
research .2nded,john wilew and sons, New York, USA.
[11] Gunawardena, N.E. and Bandarage, U.K. (1995). 4 methyl 5- nononal
(ferrugineol) as an Aggregation pheromone of the coconut pest Rhynchophprus
ferrugineus, F. (Coleoptera: Curculionodae) synthesis and use in a preliminary
flied assay. Journal of the National science council of Srilanka, 23 (2): 71-79.
[12] Hallet, R.H. ; Gries, G.; Gries, J.H.; Borden, J.R.; Angerilli, N.P.D. and Rauf, A.
(1993). Aggregation pheromone of two Asian palm, weevil, Rhynchophorus
Ferrugineus and R. vulneratus Naturwissenschaften 80:228-331.
[13] Huang Shan-chun,MA Zi-long,QIN Wei-quan,LI Chao-xu,YU Feng-yu and
HAN Chao-wen (2008). The Trapping Effect of Aggregation Pheromone for the
Red Palm Weevil (Rhynchorus ferrugineus (Olivier)) and Its Traps
Development. China Forestry Science and Technology, 2008-03.
[14] Lal, Madan Mohan. (1917). Rept. Asst. Prof. Entomol; Rept. Dept. Sagr. Punjab,
for the year ended 30th June, 1917.
[15] Laskshmanan, P. L., P. B. Subba Rao, T. R. Subramanian. (1972). A note on the
control of the coconut red palm weevil Rhynchophorus ferrugineus with certain
new chemicals. Madras Agric. Journal 59: 638-639.
[16] Lefroy, H. M. (1906). The more important insects injurious to Indian
Agriculture. Govt. Press, Calcutta, India.
[17] Qin Weiquan Ma Zilong Wu Duoyang Cai Xizhou Wang Yongzhuang Zhao and
Hui Han Chaowen (2004). Trapping of Red Palm Weevil with Several
Attractants and Monitoring of Its Population in the Field. Chinese Journal of
Tropical Crops. ;2004-02.
723
Table (1):Population dynamics of red palm weevil Rhynchophorus
ferrugineus (Oliv.) on date palm plantation during 2009.
Average of RPW captured
adults/trap
Mean
Inspection Date
Total
Male
Female
Temperature
C˚
Humidity
(RH)
19-Jan-2009
5.33
2.00
3.33
15.00
55.57
2-Feb-2009
10.00
4.00
6.00
16.93
49.71
16-Feb-2009
20.33
7.00
13.33
17.50
46.21
2-Mar-2009
19.00
4.00
15.00
15.86
45.93
16-Mar-2009
33.00
24.00
9.00
16.73
58.60
30-Mar-2009
34.33
16.00
18.33
17.71
50.64
13-Apr-2009
56.00
24.33
31.67
20.57
51.93
27-Apr-2009
21.33
8.00
13.33
20.86
53.50
11-May-2009
20.67
8.33
12.33
24.29
48.29
25-May-2009
23.67
8.67
15.00
25.07
50.29
8-Jun-2009
68.33
28.33
40.00
26.50
51.29
22-Jun-2009
15.00
5.33
9.67
27.29
52.86
6-Jul-2009
17.33
5.33
12.00
30.14
51.21
20-Jul-2009
14.33
4.33
10.00
28.64
59.14
3-Aug-2009
28.67
10.67
18.00
30.93
56.93
17-Aug-2009
2.33
2.33
0.00
28.07
60.36
31-Aug-2009
3.33
0.67
2.67
29.57
60.00
14-Sep-2009
3.00
1.33
1.67
27.79
61.07
28-Sep-2009
3.33
1.33
2.00
25.79
61.00
12-Oct-2009
2.67
1.33
1.33
25.71
62.00
26-Oct-2009
10.33
2.33
8.00
25.21
62.93
9-Nov-2009
19.00
7.67
11.33
24.43
57.00
23-Nov-2009
14.00
5.67
8.33
19.21
59.00
7-Dec-2009
6.67
3.00
3.67
17.36
59.36
21-Dec-2009
5.67
2.00
3.67
15.64
55.93
4-Jan-2010
5.00
2.00
3.00
14.71
63.21
Total
462.67
190.00
272.67
Mean ± S.D.
17.79
± 3.8
7.31
±2.2
10.49
± 2.3
22.60
55.54
L.S.D. at 5%
6.23
724
Table (2):Population dynamics of red palm weevil Rhynchophorus
ferrugineus (Oliv.) on date palm plantation during 2010.
Average of RPW captured
adults/trap
Mean
Inspection Date
Total
Male
Female
Temperature
C˚
Humidity
(RH)
18-Jan-2010
4.67
2.00
2.67
11.93
65.50
1-Feb-2010
8.67
3.00
5.67
11.86
64.57
15-Feb-2010
11.33
4.67
6.67
14.00
62.93
1-Mar-2010
14.33
5.33
9.00
13.80
64.20
15-Mar-2010
17.00
7.33
9.67
18.64
55.86
29-Mar-2010
81.67
31.00
50.67
22.86
42.93
12-Apr-2010
10.00
3.67
6.33
19.36
50.14
26-Apr-2010
12.33
4.00
8.33
25.07
46.86
10-May-2010
17.33
5.33
12.00
22.29
49.00
24-May-2010
22.67
5.33
17.33
25.43
45.64
7-Jun-2010
51.00
16.33
34.67
27.14
48.93
21-Jun-2010
66.33
21.33
45.00
28.14
48.71
5-Jul-2010
40.33
14.00
26.33
29.64
51.57
19-Jul-2010
75.67
32.00
43.67
29.43
59.07
2-Aug-2010
21.67
6.00
15.67
28.93
58.00
16-Aug-2010
22.33
8.67
13.67
29.50
59.43
30-Aug-2010
11.33
4.33
7.00
30.50
58.29
13-Sep-2010
13.00
6.33
6.67
28.57
61.79
27-Sep-2010
10.33
4.00
6.33
29.36
51.86
11-Oct-2010
10.67
3.67
7.00
26.29
55.86
25-Oct-2010
5.67
1.33
4.33
23.43
61.38
8-Nov-2010
30.67
12.33
18.33
21.64
63.50
22-Nov-2010
12.00
4.67
7.33
20.36
62.79
6-Dec-2010
11.00
3.33
7.67
20.14
51.07
20-Dec-2010
8.00
3.33
4.67
18.00
56.36
3-Jan-2011
3.00
1.00
2.00
14.50
63.64
Total
593.00
214.33
378.67
Mean ± S.D.
22.81
±4.8
8.24
±2.6
14.56 ±
4.3
22.72
56.15
L.S.D. at 5%
7.77
725
Table (3): The effect of weather factors on the population fluctuations of RPW.
Factors
r
B
Mean of daily
Temperature
0.252
0.872
R2
S .E.
y^ = a+b x
6.3%
0.270
x= Temperature degree
0.001
0.002
y^=Number of RPW
adults at this degree
- 0.444
- 1.451
y^ = a+(-)b x
P
Mean of daily RH
P
Prediction equation
19.2%
0.000
Temp. & RH
0.236
0.000
- 0.168
x= RH
^
y =Number of RPW
adults at this degree
y^ = a+b1 x1+b2x2
22.9%
r = simple correlation coefficient value
b = simple regression coefficient value
R2 = relative contribution
S.E= standard error
P = probability
Table (4) : Diurnal flying activity of RPW during 2009 and 2010.
Average captured RPW adults during day hours per trap
Day hours
Years
6
am
8
am
10
am
12
am
14
pm
16
pm
18
pm
20
pm
LSD at
0.05
2009
7.20 a
0.20
e
2.00 d
3.00
c
3.20
c
5.00 b
2.00 d
1.60 d
0.948
2010
6.80 a
0.00
e
1.60 d
2.60
c
3.20
c
4.40 b
1.60 d
1.20 d
0.795
726
Daily mean of temperatue & RH
70
60
50
40
30
20
10
0
19-Jan-09
2-Feb-09
16-Feb-09
2-Mar-09
16-Mar-09
30-Mar-09
13-Apr-09
27-Apr-09
Temprature
11-May-09
25-May-09
22-Jun-09
6-Jul-09
Humidity (RH)
/ŶƐƉĞ Đ ƚŝŽŶĂ ƚĞ
8-Jun-09
20-Jul-09
3-Aug-09
17-Aug-09
31-Aug-09
14-Sep-09
28-Sep-09
12-Oct-09
26-Oct-09
9-Nov-09
23-Nov-09
7-Dec-09
21-Dec-09
4-Jan-10
Fig. (1.a): Mean daily temperature and relative humidity during 2009.
727
Av era g e ca ptured RPW a dult
80
60
40
20
0
19-J an-09
2-Feb-09
16-Feb-09
2-M ar-09
16-M ar-09
30-M ar-09
Total
13-A pr-09
27-A pr-09
11-M ay -09
25-M ay -09
Male
22-J un-09
6-J ul-09
20-J ul-09
Female
/ŶƐƉĞĐ ƚŝŽŶĂ ƚĞ
8-J un-09
3-A ug-09
17-A ug-09
31-A ug-09
14-S ep-09
28-S ep-09
12-O c t-09
26-O c t-09
9-Nov -09
23-Nov -09
7-Dec -09
21-Dec -09
4-J an-10
Fig.(1.b) : Population dynamics of RPW Rhynchophorus
ferrugineus(Oliv.) on date palm plantation during 2009.
728
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730
OP 38
The study of fungal infections of dates during storage
Oustani Mabrouka
Assistant professor at the University of Kassdi Merbah Ouargla (Algeria)
E-mail belsam.oustani@yahoo.fr
Abstract
The presence of fungal flora in the dates for food of man can lead to serious
consequences for his health. The quality control during the storage can reduce the loss
of product caused by these fungi. It is a means of prevention and risk management of
infections by fungi, some of which may be highly toxigenic. The study of fungal
contamination of samples stored dates was performed on 12 samples from six varieties
of dates (Chars, Deglat Nour, Deglat Beida Tantbouche, Kentichi, Tenissine) collected
from two local conservation in two regions located in southern Algeria (Ouargla Oued
and righ). The results showed a wide range of fungal flora contamination. In fact, ten
species belonging to 8 genera of fungi were isolated (on PDA medium incubated at 25
°C) of 6 varieties of dates in each region. The genera Aspergillus is the principal
representative of the flora of contamination dates studied. This type was represented
by two species: Aspergillus niger and Aspergillus flavus, supported directly by the
genera Penicillium in turn represented by two species: Penicillium expensium and
Penicillium isolandicum. Finally, Fusarium solani, Paecilomyces variotii, Candida
albicans, Cladosporium sp, Mucor sp are relatively less frequent in the samples
analyzed dates. In addition, our study showed that the importance of fungal spoilage
and is directly related to the water content and acidity of dates. This study shows the
need for quality control in post-harvest dates, during storage and until
commercialisation thereby limit the loss of product caused by fungi.
Keywords: Dates, Storage, Fungi, pH, Moisture, Aspergillus, Penicillium, Yeasts.
Introduction
The date palm Phoenix dactylifera L, is the most important tree in the world.
Studies have indicated that Arab countries are the main sources of dates [1]. In
Algeria, the production of date palm plays an important role in the field of agriculture;
it is the mainstay of national agricultural economy [2]. Heritage phoenicicole Algeria
731
is estimated at about 10,475,150 feet of many related in an area occupied by palm trees
de154 372 ha with a production of dates of 4,921,880 quintals of yield and 47.0 kg /
tree Despite the importance of this crop to the economy of our pay, the date palm is
subject to various problems that hinder its development and extension. The problem
dates alterations during storage and storage ranks first among these problems because
of their depressive effects on fruit quality.
In addition, dates are fruits that have high nutritional value and energy. However the
poor conditions of conservation of these fruits can lead to serious drawbacks that are
causing the loss of nutritional quality of dates, which negatively affects their market value.
Among the microbiological changes that may occur during storage, it cites the
fungal damage caused by several fungi. In fact this type of contamination is one of the
main causes of deterioration of the fruits of date palm [6]. The presence of this flora in
dates is favoured by several parameters. Mention is mainly:
• The initial load of fungal micro flora,
• The presence dates injured
• The humidity and temperature during storage.
Fungal contamination is a direct relationship with both the physical initial dates and
environmental conditions of the premises including the storage temperature and
humidity can alter the organoleptic parameters of dates, and consequently decrease the
market value. The sensitivity of dates to fungal spoilage is related to its poor
conservation of places of production and storage is a big problem for national
operators. This alteration is particularly important as the storage conditions and storage
are inadequate [5, 7].
It is in this context that we do. We have chosen to study the fungal spoilage during
storage of dates and to do this, we set the goal of our work involves isolation and
identification of molds that can contaminate the dates during storage and that can
cause and serious consequences for human health. A particular interest is deemed
toxigenic molds [8], since they begin to grow beyond 15% humidity.
732
Materials and methods
1. Choice of varieties
We chose six varieties of dates. Some of them are considered among the most
abundant varieties in the domestic market and the most appreciated by the consumer,
these varieties Deglet Nour, Ghars. Others have other economic interests, these
varieties Deglat Beida Tinissine, Kentichi, Tantboucht. The analysis focused on 12
samples taken at random dates of the crop of 2009 at two local conservation, one in the
region of Ouargla and the other in the region of Oued Righ.
2. Méthodes studies
2.1. Physicochemical analyses
Determination of moisture
Measures moisture samples of dates were performed on a sample of 10 g weighed
and placed in an oven at 105C ° for 24 hours. After drying, the sample is cooled in a
desiccator and reweighed [9].The moisture content is calculated by taking the weight
test sample before and after drying to an oven using the following formula:
Pre-dry weight - weight after drying
Water (%) =
Initial weight of sample
PH measurement
It was measured using a pH meter, by making a suspension of 5 g of fresh dates (cut
into pieces for each sample separately) with 50 ml of distilled water, subsequently
determines the value of pH.
2.2. Microbiological analyses
Fungal isolates
Isolation of fungi was carried out by depositing fragments of dates from 1 cm 3 cut
from the fruits of six varieties of date palm random sample collected from each of the two
study areas. The surface of these fragments were sterilized with chloral (sodium
hypochlorite 10%) for 5 minutes, then rinsed several times with sterile distilled water and
placed in petri dishes containing sterile filter paper, moistened with sterile distilled water.
The boxes are prepared and incubated at 25 ° C ± 1 ° C for 7 days. After this period, the
733
fragments of dates with a mold growth are planted in separate Petri dishes containing
culture medium to which sterile PDA was added acetic acid (40%) late to avoid bacterial
contamination [10]. For each sample analysis was conducted in three replicates.
Identification
After 5-7 days of incubation at 25 °C, visible colonies were identified according to
[11, 12, 13, and 14]. So identification for fungal species was done according the
morpholological i.e. sporangiophores, sporangia, conidia…etc.
Results and discussion
1. Results of physicochemical analysis of dates
1.1. Water content Dates of Ouargla
Examination of histograms related to the moisture content of the dates of Ouargla
(Figure 1) shows that the water content of the varieties studied is from the margin of
7.10% to 18.10%. The highest humidity were recorded by the varieties marrow
supported by those half-marrow. With values of about 18.1% and 16.90 % for the
variety and Ghars Deglatnour.
Varieties and Tinissine Tantbouche recorded intermediate water contents in the
range
of
16.10,
15.20%
respectively
for
the
two
varieties.
In contrast, the lowest humidity were recorded by the dry varieties with water contents
of 13.10 and 7.10% respectively and Kentichi Deglat Beida.
As for the dates stored Oued Righ, humidity levels recorded are relatively high
compared to those recorded in the region of Ouargla. The resulting rates vary between
13.1 and 18.10%. Figure (2) shows that the largest humidity levels were recorded by
the variety Ghars seconded by Deglat nour variety, with rates of around 18.1 and
17.50% respectively for the two varieties.
So what the lowest levels were recorded as it is the case of dates of Ouargla, by
varieties and Kinitchi Degla beida with water contents in the range of 13.10 and
13.50% respectively for the two varieties. Intermediate position rank varieties and
Tinissine Tantbouche with water contents in the range of 17.40 and 15.50%
respectively for the two varieties.
734
.2. pH measurement
Dates of Ouargla
Analysis of the results illustrated in Figure (3), show that the pH values studied
varieties of dates vary between a minimum value of 5.11 recorded by the variety
Deglat Beida, and a maximum value of 5.91 recorded by the variety Kentichi. The
classification of six varieties of viewpoint acid obeys the following order: Deglat
beida> Tantbouche> Tinissine>DeglatNour>Ghars>Kenitchi.
Dates of Oued Righ
Dates of Oued Righ represent a relatively high acidity compared to that recorded at
dates in the region of Ouargla. Indeed, a comparison of six varieties at a time (Figure
6) revealed the rankings following order: Deglat beida> Tantbouche> Deglat nour>
Tinissine> Kenitchi> Ghars
2. Microbiological test results
Ten species of fungi belonging to 8 genera were isolated from 12 samples taken
from two local dates for the conservation of date palm fruits in two different regions:
6 samples taken from six varieties of the region of Ouargla.
6 samples taken 6variètes the region of Oued Righ.
Dates of Ouargla
The results shown in fig (4) relative to the percentages of contamination by various
fungi isolated from six varieties show that:
The genera Aspergillus is the major contaminant in all varieties of dates of Ouargla,
it is represented with two species: Aspergillus niger and Aspergillus flavus.The rate of
infection by Aspergillus niger recorded a maximum of about 64.70% for the variety
Deglat nour, and a minimum value of about 22% for the variety Degalt Beida.The rate
of infection by Aspergillus flavus register their towers to a maximum of about 63.63%
for the variety Deglat nour, and a minimum value of about 11.11% for the variety
Deglat Beida.The Genera Penicillium dominance scored lower compared to
Aspergillus. For this kind of two fungal species were identified: Penicillium
islandicum which was recorded in five varieties, with the percentages of contamination
include: 27.77, 2, 22, 18.75, 11.76, 11.11% for the varieties Deglat Beida; Tinissine;
Kentichi ; Deglat Nour and Ghars.
735
It should be noted that the species Penicillium islandicum scored a total lack in the
variety Tantbouche. Regarding the Penicillium expansium, this species is less frequent
compared to the previous one, with a total lack in the variety Deglat Beida. However,
we have record low percentages of contamination in other varieties: 33.33% for
Tinissine and Ghars, 6.25% for the variety Kentichi; 5.88% for the variety Deglat
Nour, 5.55% for the variety Tantbouche.
The presence of Paecilomyces variotii was recorded at five varieties of dates with
35.29% for the variety Deglat Nour, 33.33% for the variety Ghars, 22.22% for the variety
Tantbouche; 22.22% for the variety Tantbouche, 11, 11% for the variety and Tinissine
Deglat Beida. The species Mucor sp was recorded only in Deglat Beida (11.11%).
The yeasts represented by the species Candida albicans, were also present in the flora
of contamination dates of Ouargla, especially among varieties and a half spinal marrow:
the percentages of contamination in the range of 16.66 and 33.33 % for both varieties.
The actinomycetes were very common represented by percentages of infection
varied between a minimum value of about 11.11% (Kentichi) and a maximum value of
about 81.25% (Tantboche).
Dates of Oued Righ
The results shown in fig (5) relative to the percentages of contamination by various
fungi isolated from six varieties show that: The Aspergillus is the most dominant genre
in the fauna of contamination of samples studied. This type is represented by two
different species: Aspargillus niger and Aspergillus flavus. However, Aspergillus niger
is the answer especially in two varieties Ghars and Nour Deglat with infection rates of
about 90 and 88.88% respectively for the two varieties. However, other varieties have
shown infection rates relatively less important in the order of 50, 44.44, 41.44 and
26.66% for the varieties Tenissine, Tantbouche, and Kentichi Deglat Beida.
The results show that the percentage of recovery of Aspergillus flavus is less
important than that of Aspergillus niger with infection rates of around 38.88, 27.77,
25,16.66, and 6.66% for the varieties Ghars and Deglat Nour,
The genera Penicillium is not as dominant as Aspergillus, it is represented by two
different species: Penicillium expansium, Penicillium islandicum. Penicillium
expansium is well presented in the variety Kentichi (16.66%), while it has a low rate of
736
contamination in the variety Deglat Beida (6.66%). However, this species represents a
complete lack in varieties and Tinissine Tantbouche.
Although the results show a total lack of Penicillium islandicum in the variety of
Tinissine, this species showed high levels of contamination in the variety Ghars (50%).
The other varieties showed low frequencies of appearance and the order of 5.55, 6.66,
16.66, and 22.22% for the varieties Tantbouche, Deglat Beida, and Kenitchi Degalt
Nour. With regard to the actinomycetes, these organisms show a frequency of
abundance similar to that of Aspergillus. Thus, their presence is more pronounced
among varieties and nour Deglat Ghars with percentages of contamination in the range
of 77.77 and 83.33% respectively for the two varieties. Rates lower contamination
were recorded for varieties Kentichi 41.66% and 53.33% Deglat Beida.
For Paecilomyces variotii has appeared in five varieties of dates, with relatively low
infection rate of about: 27.77% in the two varieties and Ghars Tantbouche; Tinissine
22.22 for the variety, 20% variety Deglat Beida and 16.16% forDeglatNour.
The results show the involvement of Candida yeasts in the flora of contamination
dates studied, it is the species Candida albicans, whose presence was recorded only
four varieties of dates. With a marked absence of this
species in other varieties.
Infection rates recorded are: 41.66% for the variety Kentichi; 33.33% for both varieties
and Tinissine Deglat Beida and 27.77% for the variety Tantbouche.
The genera Fusarium represented by a single species Fusarium solani was recorded
only in two varieties Ghars Deglat Nour and a similar percentage of contamination in
both varieties and did not exceed a percentage of 8.83%.
For the kind Mucor also represented by a single species Mucor sp was detected in
the varieties and dry Deglat Beida Kentichi respectively rates of about 46.66and8.33%.
Finally the kind presented by Cladosporium Cladosporium sp was recorded in the
variety of Deglat nour with a rate of 9.99%, with a marked absence in the other varieties.
Discussion
The water contents obtained justify the classification of the date and Ghars Deglet-Nour
respectively in the category of dates and half spinal marrow, making them susceptible to
different types of alterations. These results are consistent with those of [15]. [16],
development of microbial flora is in the same direction as the water content. Thus, dates
737
and half-marrow marrow are the most susceptible to fungal spoilage in relation to dried
dates. Storing dates with such water content, can be dangerous by causing a proliferation
of microorganisms, development of a musty smell and an acceleration of the progress of
other reactions alterations (enzymatic, biological, biochemical ) as against the dried dates
(Kenitchi, Deglat beida) are less sensitive. Their low water content does not provide a
good atmosphere for the fungal flora of contamination.
Regarding acidity dates, the results allow us to say that this factor is of the same order
of magnitude as the moisture factor, it is considered in turn as a powerful agent of
microbial contamination such as fungus. This has been justified by differences in
infection rates observed among the varieties studied, according to their degree of acidity.
On the other hand, [13] have shown that fungi are species which are acidophilic or
acidotolérants good conditions for development in an acidic medium. This is in
agreement with our results, through what we have shown that dates Daglat beida were
a substrate conducive to mold growth contaminants due to their relatively high acidity.
After microscopic analysis of the fungal flora of contamination, eight genera have
been isolated and identified from the dates analyzed: the genus Aspergillus is
represented by two different species: Aspergillus niger and Aspergillus flavus,
Penicillium is represented in turn by two different species: Penicillium islandicum and
Penicillium expensium, the genera Fusarium by a single species: Fusarium solani,
Paecilomyces The gerera a single species, Paecilomyces variotii; The genera Candida
is represented by a single species: Candida albicans; The genera Cladosporium is
represented also by a single species: Cladosporium sp; The genera Mucor by a single
species: Mucor sp, a type of unidentified actinomycetes.
Moreover, the results of mycological analysis showed a clear dominance of the two
genera Aspergillus and Penicillium in the flora of contamination dates studied
compared to other genres. However, a clear superiority in the frequencies of
abundance was noted for the genus Aspergillus that be justified by the different
behavior of gender vis-à-vis the humidity.According [17], high humidity growth of
Penicillium disadvantage, while it strongly stimulates that of Aspergillus. This latter
consistent
with our results showing that the rate of contamination
738
in Penicillium
dates
marrow
is less than Aspergillus.
The dominance of the genus Aspergillus in
the flora of contamination of dates has been delayed by several works [17, 18].
Thus we can consider the species of the genera Aspergillus as storage molds.
The abundance of Candida yeasts presented by the species C. albicans at dates that
are high in sugar (as is the case of varieties and Ghars Deglat Nour), can be explained
by the fact that these varieties have to against yeast, an important energy substrate
required to perform the fermentation. Moreover, this study showed that fungal
contamination is strongly influenced by the health status of dates. In fact, the presence
of injured dates can only support the development of sources of contamination and
therefore can only encourage the development of source of contamination and
therefore it can only be to the disadvantage of storing dates in long-term.
In fact, while making use of climate data for the two regions for the crop year [19],
we see a significant rainfall was recorded in the region of Oued Righ specifically
during the harvest period (September and October) and almost zero in the region of
Ouargla, why we noted that the dates of Oued Righ were much more susceptible to
fungal spoilage, the latter which is favored by injuries caused by the strong rainfall
that has characterized this region.
Finally, the diversity of flora contamination occurred at the dates studied both for
1
the varieties of Oued Righ or those of cOuargla, explains the need for research on the
quality of dates. This covers all operations from harvesting to marketing are designed
to preserve the nutritional value of this fruit [20].
739
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741
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Ϭ
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Fig.2: Water content of dates stored of Oued Righ
742
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dĂŶƚďŽƵĐŚĞ ĞŐůĂƚEŽƵƌ ĞŐůĂƚĞŝĚĂ dŝŶŝƐƐŝŶĞ
sĂƌŝĞƚŝĞƐŽĨĚĂƚĞƐ
Fig.3. The pH values dates preserved of Ouargla.
Ɖ,
ϳ
ϲ
ϱ
ϰ
ϯ
Ϯ
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Ϭ
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'ŚĂƌƐ
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dĂŶƚďŽƵĐŚĞ ĞŐůĂƚEŽƵƌ ĞŐůĂƚĞŝĚĂ
sĂƌŝĞƚŝĞƐŽĨĚĂƚĞƐ
ϱ͘ϲϳ
dŝŶŝƐƐŝŶĞ
Fig.4: The pH values dates preserved of of Oued Righ.
743
Fig. 5. Percentage of contamination of preserved dates of Ouargla
744
Fig. 6 : Percentage of contamination of preserved dates of Oued Righ.
745
The board below shows some pictures of microscopic fungi, yeasts and
actinomycetes isolated from different varieties of dates studied.
746
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747
748
PP 11
Acaricidal , Ovicidal and Repellent Activities Of Some Plant
Extracts On The Date Palm Dust Mite, Oligonychus afrasiaticus
Meg. (Acari: Tetranychidae)
Badr El-Sabah A. Fetoh 1 (2) and Kholoud A. Al-Shammery2
1- Plant Protection Research Institute, Dokki, Giza, Egypt
2-Department of Biology, College of Science, Hail University,1441 Hail, Saudi Arabia
Corresponding Author: badrelsabah@yahoo.com
Abstract
The ethanolic extracts of Demsisa, Duranta and Cumin plants were tested on the
adult females of the date palm dust mite, Oligonychus afrasiaticus Meg. (Acari:
Tetranychidae). The recorded results showed that all the tested plant extracts were
effective on O. afrasiaticus , however Demsisa extract was the most virulent one. The
concentration 1x101 p.p.m. gave the lowest mortality percent of O. afrasiaticus
39.00%, 33.33% and 12.00%, while the concentration 1x105 p.p.m. resulted in the
highest mortality percent 93.33% , 69.00% and 64.67% for Demsisa, Duranta and
Cumin extracts, respectively. The LC50 values were 47.16, 1102 and 8433.2 p.p.m.
The LC90 values were 3.5x105, 2.47x108 and 1.30x109 p.p.m., respectively. Also,
Demsisa plant extract showed the highest toxicity index being 100%. The slope values
of the toxicity lines were 0.445, 0.240 and 0.402 for Demsisa, Duranta and Cumin
extracts, respectively. Furthermore, all the tested plant extracts exhibited ovicidal and
repellent activities and showed significant effects on the reproduction and feeding
behaviours of O. afrasiaticus.
Key words: biological control , plant extracts , acaricides , ovicides , repellents , date
palm , mites ,Oligonychus afrasiaticus
Introduction
The date palm dust mite, Oligonychus afrasiaticus Meg., is a serious pest in North
Africa and the Near East and is known to occur in most date palm growing areas in the
world including Egypt, KSA, Iraq, Algeria, Kuwait, UAE, Morocco, Bahrain, Sudan,
Sultanate of Oman, Yemen, Mauritania, Iran, Chad, Mali, Niger and USA (Abdul
Hussain, 1985 ; Bass’haih, 1999). In Saudi Arabia was reported to infest date fruits in
749
orchards in Dirab, Unayzah and Al Sulayell districts and spreads in Hail, Qassium,
Rayaid, Al-Kharg and Dammam regions (Talhouk, 1991 and Al-Shammery, 2008). It
attacks the dates from their early stages of development, spinning its webs around the
date bunches and multiplies in large numbers. Dust collected in the webs plus the
exuviae of different developmental stages of the date dust mite, affect the date bunches
giving it a dusty appearance. (Saleh and Hosny, 1979). Furthermore, it attacks
cucumber, citrus, eggplants, bean and strawberry (Al-Shammery, 2008). It feeds on the
juices, sucked from the different infested plants and dates, rendering them unfit for
human consumption (Mohamed, 2005). O. afrasiaticus has 6 generations/year, the
peak number of the mite was recorded around the middle of July/in Iraq (Hussein,
1969). The infestation of this mite species usually starts around mid of May to June
when it builds dusty, creamy silky webs on date bunches around date fruit stands,
where adults and immature stages live. Sever infestation with mite results in economic
reduction in the quality and quantity of crop production (Aldosari , 2009).
Over the last several decades, various attempts to control insect pests have taken an
effort toward effective eradication or prevention through the development of
synthetic insecticides and acaricides. Synthetic chemicals have given many profits
and convenience to mankind, but a lot of them have revealed serious environmental
problems and threatening to human life (Kim et al., 2005). Natural products and
plant extracts seem to resolve environmental problems caused by the synthetic
pesticides, and many researchers are trying to find out effective natural products to
replace synthetic chemicals. Plants may provide potential alternatives in the place of
currently used insect pest control agents because they constitute a rich source of
bioactive chemicals (Wink, 1993). They are also selective to pests, have no or
little harmful effects on non-target organisms and the environment, and act in many
ways on various types of pest complex (Arnason et al., 1989; Hedin et al., 1997).
Many of them are applied to crops in the same way as other agricultural chemicals.
All of the plant-derived compounds that have been characterized as having pesticidal
activity are plant secondary metabolites (Schmutterer, 1995). Plant secondary
metabolites may have to affect insect growth and behaviour, acting as anti feedents,
toxins and insect growth regulators (Campagne et al., 1992). Since the world's
750
tendency today is to reduce chemical pesticides and use botanical extracts and natural
enemies as safe controlling methods; the aim of the present study is to evaluate the
acaricidal, ovicial and repellent activities of some plant extracts on the date palm dust
mite, Oligonychus afrasiaticus.
Materials and methods
Three plants were used to obtain their ethanolic extracts, these plants were:
Demsisa, Ambrosia maritimal ( Fam. Compositae), Duranta, Duranta plumeria (Fam.
Verbenaceae) and Cumin, Cuminum cyminum (Fam. Labiaceae). The whole plant parts
of Demsisa , leaves of Duranta and seeds of Cumin were used in this experiment
(Table 1). The plant materials were dried under room temperature in dark condition
and ground; 150 gm of the powder were extracted in 95% ethanol according to
Freedman et al., (1979). Five different concentrations of the ethanolic extracts were
prepared as 10, 100, 1000, 10000, 100000 p.p.m. A culture of the date palm dust mite,
Oligonychus afrasiaticus was reared at 25±1 ◦C and 65±5% R.H. on bean plants ,
Phaseoulus vulgaris in the laboratory. 150 adult females of the date palm dust mite,
O. afrasiaticus were collected from the stock culture and divided into three replicates
for each treatment. The aciricidal effects for the tested plant extracts was evaluated by
the leaf dipping technique according to Siegler (1947). Bean leaves were dipped in
each concentration of the tested three plant extracts for 10 minutes, then left to dry.
Each treatment was repeated three times. For each replicate used the bean leaves used
were similar in shape and size. In untreated control the bean leaves were dipped in
ethanol only.
The mortality percentages of the treated date dust mites after 24 hour were
calculated, corrected by using Abbott’s formula (1925) and statistically computed
according to Finney (1971). Computed percent mortality was plotted with
corresponding concentrations on logarithmic probability paper to obtain the
corresponding Log-Concentration Probit (LCP Lines). The lethal concentrations of
50% and 90% (LC50 and LC90) for O. afrasiaticus were determined for the established
regression lines. Also, toxicity index was calculated according to the equation of Sun
(1950). After calculation of lethal concentrations of LC
751
50
of the different plant
extracts, their effects on the amount of laid eggs , ovicidal and repellent responses on
O. afrasiaticus were evaluated.
For the determination of the effect of the tested plant extracts on the amount of laid
eggs by the females of O. afrasiaticus , 30 gravid females were transferred to bean
leaf discs. These leaf discs were sprayed with the calculated LC 50 of each plant extract
for the treated test and 95% ethanol for untreated control. The number of laid eggs was
counted for 24, 48 and 72 hours. This test was replicated three times for treated and
control tests. Those eggs that did not hatch after this period were regarded as nonviable (Sarmah et al., 1999). Percent reduction in hatchability of the eggs was
calculated by using the following formula:
Egg mortality (%) =
100 – (No. unhatched eggs /treatment ÷ Total No. of eggs /treatment) x 100
The repellent effect of the tested plant extracts was carried out by leaf discs choice
test. Leaf discs (2x2cm) were put on cotton pads in petri dishes, then sprayed with the
calculated LC50 of each plant extract, then the treated and untreated discs were placed
in the petri dish in three replicates. Also, 30 adult females of O. afrasiaticus were
transferred in the middle of the treated and untreated leaf discs. After, 24, 48 and 72
hours the number of mites on treated and untreated leaf discs counted. Repellence
index was calculated according to Kim et al., (2005) as following:
R= (C-T/ C+T) / 100, where: C= Number of mite on untreated (control) leaf disc,
T= Number of mite on treated leaf disc. Treatments and control means were compared
by Duncan's multiple range test at p≤0.05 (SAS, 1998).
Results
Toxicological effects of three plant extracts on the date palm dust mite, O. afrasiaticus :
Results in Table (2) showed that, all tested concentrations of
the ethanolic
Demsisa, Duranta and Cumin extracts exhibited high toxicity effects on O. afrasiaticus
and the high toxicity rate was concentration dependent. Demsisa extract was more
potent than Duranta and Cumin extracts. The lowest concentration (10p.p.m.) elicited
a toxicity response being 39.00±1.00% , 33.33±1.53% and 12.00±2.00% for Demsisa,
Duranta and Cumin extracts, respectively. The mortality percentages of the date dust
mite increased in ascending manner with increased the concentration reaching the
752
maximum of 93.33±1.52% , 69.00±0.71% and 64.67±1.53% for Demsisa, Duranta
and Cumin extracts at the highest concentration (100000p.p.m.), respectively.
Results tabulated in Table (3) included the relative toxicity of Demsisa, Duranta and
Cumin extracts against O. afrasiaticus . The LC50 values were 47.16, 1102 and 8433.2
p.p.m. The LC90 values were 3.5x105, 2.47x108 and 1.30x109 p.p.m., respectively. The
toxic effect of Demsisa plant extract was more virulent than Duranta by 23.37 and then
Cumin by 178.82 . Also, Demsisa plant extract showed the highest toxicity index (100%).
Figure (1) shows the probit lines of predicted percentage of death of O. afrasiaticus
treated with Demsisa, Duranta and Cumin extracts, respectively. The slope values
were 0.445, 0.240 and 0.402 for Demsisa, Duranta and Cumin extracts, respectively.
Ovicidal efficacy of some plant extracts against O. afrasiaticus:
The result in Table (4) showed that all tested plant extracts caused lowering in the
amount of laid eggs by the females of O. afrasiaticus. Damsisa extract was the most
effective one , the quantity of laid eggs was decreased from 1.30 eggs after 24 hour to
0.30 eggs after 72 hour. In the contrary Cumin extract registered 3.30 eggs after 24
hour and increased to 7.75 eggs after 72 hour. All tested plant extracts caused a
significant effect on egg mortality percent at LC50 being 87.33 and 70.67 % with
Demsisa and Duranta extracts, respectively, whereas in Cumin the lowest egg
mortality recorded was 30.67 %.
Effect of ethanolic plant extracts on the feeding behaviour of O. afrasiaticus:
The plant extracts of Demsisa, Duranta and Cumin exhibited high repellent
activities against the date palm dust mite, O. afrasiaticus as showed in Table (5). The
repellent rate of three tested plant was clearly potent and no significant difference
appeared after 24 hours. The repellent index was 97.80, 95.50 and 95.33 % for
Demsisa, Duranta and Cumin extracts, respectively. This repellent effect extended for
72 hour, after which , Demsisa extract showed the highest repellent rate (93.33%),
followed by Duranta extract , which exhibited 84.40%, while Cumin extract gave the
lowest repellent effect (53.33%).
Discussion
The obtained data from our study showed that the ethanolic extracts of Demsisa,
Duranta and Cumin plants were potent and exhibited acaricidal, ovicial and repellent
753
activities against the date palm dust mite, O. afrasiaticus. No reports are available on
using of the Demsisa and Cumin plants in controlling mites; whereas Duranta plant has
been used rarely against insects (Nikkon et al ., (2008a, 2008b, 2009) , so we recommend
utility the plants extracts as effective eco-friendly agents for the pests control.
The use of plant extracts for pest control was reported earlier by Slama (1974), who
found that the incomplete blastokinesis and abnormal breakage of extra embryonic
membranes in the embryo or unequal penetration of plant extracts through the egg
chorion to different parts of egg at different times of the sensitive period could also be
associated with observations on variability of morphological effects. These findings also
corroborate with present work on ovicidal activity of the chosen plant extracts against O.
afrasiaticus. Petroleum ether and acetone extracts of P. hydropiper exhibited more than
80% egg mortality in the red spider mite (El-Naggar and Mosallam 1987; Sarmah et al.,
1999); also Raja et al., (2003) screened 9 plants with various solvent extracts against the
armyworm Spodoptera litura in relation to ovicidal and ovipositional deterrent activity
and varied responses were noticed irrespective of the concentrations and the solvents
used for extraction. Kim et al., (2005) tested methanol extracts from 28 samples of 22
plant species in 17 different plant families at concentration 10,000 p.p.m. for detecting
their acaricial , insecticidal, ovicial and repellent effects on Tetranychus urticae Koch,
Aphis gossypii Glover, Myzus persicae Sulzer, Trialeurdes vaporarioum (Westwood) and
Panonchus citri (McGregor). Shi et al., (2006) used petroleum ether, chloroform, and
methanol. extracts of an annual herbaceous plant, Kochia scoparia to determine their
acaricidal effects against Tetranychus urticae Koch, Tetranychus cinnabarinus
(Boisduval), and Tetranychus viennensis Zacher (Acari: Tetranychidae) in the laboratory.
The toxicity of Duranta as in the present study was previously reported by Nikkon et al .,
(2008a, 2008b, 2009) who found that the crude extracts (both stem and fruits), their
fractions and fresh fruit juice of Duranta repens were highly effective larvicidal
agents against I, II, III and IV instar larvae of the Mosquito Culex quinquefasciatus. The
increase in mortality with increase in exposure period could be due to several factors,
which may be act either separately or jointly. For example, the uptake of the active
moiety of the compound could be time dependent, leading to a progressive increase in
the titer of the plant-derived compounds tested and its effect on the larval body. The
754
active moiety of the compound could be converted into more toxic metabolites in the
larval integument and alimentary canal, resulting in time- dependent effects insecticidal
properties against the larvae of Culex pipiens and Spodoptera littoralis, and the adults of
Musca domestica and C. pipiens. They have also reported antibacterial, antifungal, brine
shrimp lethality, acute toxicity and insecticidal activity on Tribolium Castaneum
(Herbst) associated with the stem and fruits of Duranta repen Linn. Their findings
suggested that the stem and fruits of Duranta repens could be explored as potent natural
larvicidal agent. However they revealed that fruits compared to stem are more convenient
for larvacidal activity as both fresh juice and crude extract of t h e fruit have shown
their larvacidal activity. Similarly Hatem et al., (2009) used the hexane, petroleum ether
and ethyl alcohol extracts of three species of p l a n t s : a n n u a l s o w t h i s t l e ,
Sonchus olearcues (L.) (Solanaceae), black mustard, Brassica niger (Koch.), and red
radish, Raphanus sativa var. surtus (L.) and both of them from family Cruciferae for
testing insecticidal toxicity and antifeedant activity in fourth instar larvae of Egyptian
cottonworm, Spodoptera littoralis. All extracts showed a certain degree of larval toxicity.
The hexan extracts of Sonchus olearcues L., the petroleum ether extracts of Brassica
niger Koch., and the ethyl alcohol extract of Raphanus sativa var. surtus L., were highly
toxic (LC50s = 218.36, 96.11 and 5574.66 ppm, respectively). The antifeeding activity
of crude extracts of selected weed plants, petroleum ether and hexane extracts of B. niger
were chosen based on their high insecticidal activity to be tested. The antifeeding activity
of hexane crude extracts was more effective than petroleum ether extracts of B. niger.
Sarmah et al., (2009)
evaluated four aqueous plant extracts of Acorus calamus,
Xanthium strumarium, Polygonum hydropiper and Clerodendron infortunatum under
both laboratory and field conditions at 2.5, 5.0 and 10.0% (w/v) concentrations against
tea red spider mite, Oligonychus coffeae (Nietner). Chemical substances present in the
chosen plants may block the micropyle region of the egg thereby preventing the gaseous
exchanges that will ultimately kill the embryo in the egg itself. The results in our study
revealed that the Demsisa, Duranta and Cumin plants have the potential to be used in
mite control in the form of ethanolic extracts. The discovery of acaricidal properties in
native plant species can aid in future production of safer crops by small farmers, based on
application of natural acaricides as a control method against phytophagous mites.
755
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Table (1): The plants tested for their acaricidal activities against Oligonychus afrasiaticus.
Common name
Scientific name
Family
Parts used
Source
Demsisa
Ambrosia maritimal
Compositae
Whole plant
parts
Giza, Egypt
Duranta
Duranta plumeria
Verbenaceae
Leaves
Giza, Egypt
Cumin
Cuminum cyminum
Labiaceae
Seeds
Local markets,
Egypt
Table (2): Acaricial activities of three ethanolic plant extracts on the
date palm dust mite, Oligonychus afrasiaticus.
Concentration
p.p.m.
Demsisa
Duranta
Cumin
10
39.00±1.00
(38-40)
33.33±1.53
(32-35)
12.00±2.00
(10-14)
100
53.33±2.08
(51-55)
40.33±0.71
(39-42)
22.67±2.52
(20-25)
1000
75.00±2.00
(73-77)
43.67±2.12
(42-45)
31.67±1.53
(30-33)
10000
83.67±1.53
(82-85)
61.67±0.70
(60-63)
56.33±1.53
(55-58)
100000
93.33±1.52
(92-95)
69.00±0.71
(68-70)
64.67±1.53
(63-66)
The numbers between the brackets refer to the range.
Table (3): LC50 , LC90, resistance ratio , index and slope of three ethanolic
plant extracts on the date palm dust mite, Oligonychus afrasiaticus.
Toxicity parameters
Demsisa
Duranta
Cumin
LC50
47.16
1102
8433.2
5
8
1.30x109
LC90
3.5x10
Resistance ratio
1
23.37
178.82
Index
100
4.28
0.56
Slope
0.445
0.240
0.402
2.47x10
Index and resistance ratio compared with Demsisa.
759
Table (4): Efficacy of LC50 of some ethanolic plant extracts on the quantity of
laid eggs by O. afrasiaticus and their ovicial activity.
Mean number of laid eggs ± S.D.
Plant extract
Egg Mortality
%
After 24 h
After 48 h
After 72 h
Total laid eggs
Demsisa
1.30±0.50
0.50±0.30
0.30±0.10
2.10d
87.33 a
Duranta
2.20±1.9
1.65±0.94
6.95±4.43
10.80c
70.67 b
Cumin
3.30±1.45
3.70±2.77
7.75±5.80
14.75b
30.67 c
Control
6.10±0.35
6.35±0.37
11.66± 1.21
24.12a
-
The same letter in the same column is non-significant.
Table (5): Repellency % of LC50 for ethanolic plant extracts on O. afrasiaticus:
Repellent index %
Plant extract
After 24 h
After 48 h
After 72 h
Demsisa
97.80a
97.80a
93.33a
Duranta
95.50a
88.95b
84.40b
Cumin
95.33a
66.67c
53.33c
The same letter in the same column is non-significant.
Fig. (1): Log- Probity curve of three ethanolic plant extracts
on the date palm dust mite, Oligonychus afrasiaticus.
760
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762
PP 12
Occurrence of some diseases in date palm trees in Upper Egypt and its control
Eman S. H. Farrag1 and Sabry Y. M. Mahmoud2
1)
Agricultural Botany Department (Plant Pathology), Faculty of Agriculture,
South Valley University, Qena, Egypt.
2)
Agricutural Microbiology Department; Faculty of Agriculture, Sohag
University, 82524 Sohag, Egypt.
E-mail: emfarrag@gmail.com
Abstract
In 2007, date palm trees surveyed for different symptoms on leaves and fruits in
Upper Egypt. The samples were collected from symptomatic rachis, fronds and fruits
in four localities (El-Minia, Assiut, Sohag and Qena Governorates). The major disease
noticed was leaf spots, off-shoot decline, black scorch, leaf basal rot and fruit spots.
Seven different fungal species were isolated from date palm exhibited different
symptoms. The results of pathogenicity test proved the ability of Thielaviopsis
paradoxa, Alternaria alternate and Negrospora oryza to infect date palm rachis, fronts
and fruits, respectively. Helminthosporium sp. and Epicocum nigrum were involved in
the leaf spots. E. nigrum was associated with off-shoot decline, black scorch and leaf
basal rot, while Fusarium proliferatum and Aspergilus niger were associated with fruit
spots. The pathogens were identified based on morphological approach. T. paradoxa
as a major pathogen found produce microconidia and chlamydospores. Antagonistic
effect of E. nigrum against A. alternate and T. paradoxa was examined by dual culture
technique, which leads to clear inhibition zone. Calcium chloride (CaCl 2) and E.
nigrum filtrates effectively controlled the leaf spot disease when applied at suitable
time (when disease severity was low).
Key words: Date palm, Black scorch, Thielaviopsis paradoxa, Alternaria alternate,
Negrospora oryza and control.
Introduction
Date palm (Phoenix dactylifera L.) is considered one of the most important fruit
crops in Egypt (14 million trees, FAO, 2010). It is cultivated all over the country
especially in New Valley, Aswan, Sinai, Sharkiya, Beheira, Kafr El-Sheikh and Giza
763
Governorates. Date palm infected with several fungi resulting in decline of the growth
and production. There are various economically important diseases infecting date palm
trees in all date palm growing countries [1-3]. Among these diseases, bayoud caused
by Fusarium oxysporum f. sp. albedinis [4] leaf spot caused by Alternaria spp. and
Mycosphaerell tassiana [5, 6]; inflorescence rot caused by Mauginiella scaetae [7] or
T. paradoxa [8], black scorch caused by T. paradoxa [9-11], leaf blight caused by
Glomerella cingulata [12] and off-shoot decline caused by Chalara paradoxa [13].
According to available literatures, no previous data was published on date palm fungal
diseases in Upper Egypt. So that, the study was carried out to evaluate the distribution
and incidence of various fungal diseases on date palm major growing areas of Upper
Egypt. The intend of pathogenic capability to provide update information on the most
important diseases which affecting date palm in Upper Egypt regions. On the other
hand, the study was conducted to evaluate the E. nigrum filtrates and CaCl2 in control.
Materials and Methods
Survey of diseases incidence
Surveys were conducted on date palm trees of all ages in El-Minia, Assiut, Sohag
and Qena Governorates in 2007 season. Each site was inspected once a month from
March to October. Percentage of infection with leaf spot, black scorch, leaf basal rot
and off-shoot decline was recorded. Percentage of infection was calculated according
to the following formula: Infection percentage (%) = (Number of infected date palm
off-shoots / Total number of date palm off-shoots) x 100.
Isolation and identification of causal fungi
Random samples of diseased rachis, fronds and fruits were collected from date palm
off-shoots in surveyed area then kept in plastic bags in a refrigerator until used for
isolation. Collected samples were washed with tap water then tissues from the margins of
lesions on rachis and frond were surface sterilized with 2% sodium hypochlorite solution
for 3 min, rinsed in sterile distilled water, allowed to dry and plated on Potato Dextrose
Agar (PDA) at 30 ºC for 8 days. The developed fungal colonies were purified using
single spore isolation technique according to Baudion [14]. The purified fungi were
identified according to fungal morphological characteristics as described by Barnett and
Hunter [15] and Moubasher [16], and then confirmed in plant Pathology Department,
764
National Research Centre (NRC), Dokki, Egypt. Frequencies of the isolated fungi from
date palm parts were calculated according to the following formula: Frequency (%) =
(Number of isolates of each fungus / Total number of all isolates) x 100.
Pathogenicity test
Inoculation was carried out by techniques used by Hooker [17] on date palm (Saidy
var.). A 8 mm in diameter and 5 mm deep wound (hole) was made at the center of offshoot rachis by using sharp flamed needle, and inoculated with a PDA disc (8 mm in
diameter), which was cut from a 10 days old culture plate of Helminthosporium sp., T.
paradoxa, E. nigrum, F. proliferatum, N. oryza and A. niger. Agar discs which were
containing conidia, chlamydospores and mycelial fragments, were inserted into the whole
cuts, covered with small pieces of wet, sterile cotton and covered with polyethylene bags
to maintain high relative humidity necessary for infection according to Molan and ElHussieni [18]. Fruits and fronds were pin prick and sprayed with the fungal suspensions.
Rachis, fronds and fruits free from fungi were used for control test and ten replicates
were used in each test. One month after inoculation, the disease severity index (damage
necrotic or dark-brown area around the point of infection) was calculated using a scale
(0-5), where 0= No apparent infection, 1= 1-10% damage necrotic or dark – brown area
around the point of infection, 2= 11-25%, 3=26-50%, 4=51-75%, and 5= 76-100%
according to Baudion, [14]. Re-isolation was carried out from infected tissues and the
isolated fungi were compared with the original cultures used.
Control of leaf spot caused pathogen and disease
1- Antagonistic effect of E. nigrum in vitro
The antagonistic effect of E. nigrum isolate against A. alternata and T. paradoxa (as
main pathogens) was tested by dual culture method according to Hazarika and Das
[19]. E. nigrum was inoculated 48 hrs. earlier before the fungi tested. After 5 days of
inoculation at 28±2ºC on PDA medium, the radial growth of the fungi tested and
inhibition zones were measured.
2- Effect of CaCl2 and E. nigrum exudates in controlling leaf spot disease under
field conditions
The experiment was conducted in date palm trees (5 to 8 years old) off-shoot (Saidy
var.) growing on privet farms located at Qena Governorate. All palm trees used in this
765
study showed high disease severity (as a nature infection) at the start of the
experiment. The 3rd whorl leaves from the bottom of tested trees were selected and
labeled. Three trees were used as a replicates for each treatment. E. nigrum culture
filtrate (diluted to 50%) and CaCl2 (0.5 gm. l-1) were used. Treatments were sprayed
once a month starting from March to October 2009. The disease severity ranged from
3.1 to 6.7 at the initiation of spray. At starting November 2009, another set of leaves,
the 6th whorl from the bottom was selected and labeled for observation. Less than 2.6
disease severity was observed at the start of spray, which continued until October
2010. Five leaflets from each labeled leaf were selected at equidistant and disease
severity was calculated using the following formula of James, [20]: Disease severity =
(Mean area of plant tissue affected / Mean total area of the leaflet) x 100.
Results
Survey
The main symptoms noticed during survey were leaf spots, black scorch, leaf basal
rot, off-shoot decline and fruit spots. Leaf and fruit spots had the highest disease
incidence, compared with the other disease symptoms in most regions (Figure, 1).
Black scorch was found in Assiut and Sohag Governorates (ranged from 1.8 to 2.3%).
Fruit spots found to be the highest symptoms in three over four surveyed Governorates
(ranged from 3.2 to 6.4%). El-Minia regions showed lowest symptom incidence,
whereas, leaf spots only was noticed (2.2%).
Field symptoms
Leaf spots: Symptoms appeared as small scattered, irregular, brown to black (about
3-7 mm). The spots appeared on the upper and lower surface of rachis and fronds
(Figure, 2A). Off-shoot decline: The symptoms appeared firstly on one or more of the
inner leaves then appeared on the outside ones. It started on fronds as light brown
blights with dark brown edges (Figure, 2B1 and B2) then extended to the rachis midrib
on both surface as a black color (Figure, 2B3) causing death of midrib followed by
their drooping down. Black scorch: Symptoms appeared firstly as grayish
discoloration at leaves which turned into black discoloration followed by dwarfing and
malformation (Figure, 2C). Leaf basal rot: The symptoms appeared as brown to dark
brown rot at the base of leaves (figure, 2D1). The central leaf cluster might die before
766
the older leaves and can easily detached (Figure, 2D2). Fruit spots: Symptoms
appeared as circular, black and stone spots in all parts of the fruits (Figure, 2E).
Isolated fungi and pathogenicity
Seven fungi associated with date palm disease symptoms were identified to species.
The majority of isolates were obtained from leaves exhibited leaf spots, black scorch,
off-shoot decline and fruit spots. The isolated fungi belong to fungal species: A.
alernata, Helminthosporium sp. and E. nigrum. The percentages of frequency show
the predominance of A. alternate from samples exhibited leaf spot and basal rot (43.1
and 33.3%, respectively. T. paradox (Table, 1 and Figure, 3) was the most frequently
isolated fungi from samples exhibited black scorch and off-shoot decline symptoms
(52.7 and 37.4%, respectively). N. oryza was the most dominant fungi isolated from
fruit spots (27.5%). Among seven fungal isolates obtained from date palm parts
exhibited various disease symptoms, A. alternate, T. paradoxa and N. oryzae were
pathogenic Table, 1 and (Figure, 3). The rest isolates, Helminthosporium sp., E.
nigrum, F. proliferatum and A. niger were non-pathogenic. Helminthosporium sp. and
E. nigrum were associated with leaf basal rot and leaf spots, E. nigrum with black
scorch and off-shoot decline and F. proliferatum and A. niger with fruit spots.
1- Dual culture antagonism
The antagonistic effect of E. nigrum against the most frequent fungi (A. alternate
and T. paradoxa) was studied. The obtained results demonstrated that the radial
growth of the two fungal species was significantly inhibited by E. nigrum. The mean
inhibition zone was 0.82 and 0.66 for A. alternate and T. paradoxa, respectively.
2- Application of CaCl2 and E. nigrum exudates against date palm leaf spot disease
When the spray was initiated in March 2009, the disease severity on the 3 rd whorl
leaves ranged from 3.1 to 6.7. Although the E. nigrum exudates and CaCl2 were
sprayed every month until October 2009, the disease didn’t decrease, but increased
gradually (Figure, 4). The spray was continued and new disease that developed on the
6th whorl leaves was recorded from November 2009 to October 2010. The disease
severity on the 6th leaves ranged from 1.2 to 2.6% at time of the first spray. In two
treatments, the disease severity decreased gradually. The disease severity didn’t vary
767
significantly between two treatments, although the differences between the treatments
and control were highly significant (Figures 5 and 6).
Discussion
Fungi are known as the most pathogens on date palm trees [3]. The present study
showed limited spread of fungal diseases on date palm trees in the surveyed area
because of the dry conditions and high temperature in surveyed regions. The results
indicated that leaf and fruit spots are widespread. High incidence of two diseases may
be due to the absence of control measures in Upper Egypt. Diseases are common on
similar conditions in Saudi Arabia [11], Kuwait [9], Sultanate of Oman [1] and Iraq
[21, 22]. Despite the widespread of leaf spot disease, the author noticed absence of
Graphiola leaf spot in Qena Governorate; this may be due to the dry climate. Black
scorch and leaf basal rot are low spread. A. alternate, T. paradoxa and N. oryza were
the most predominant fungi isolated from date palm trees showed visible symptoms. It
was pathogenic 13-19 days after inoculation, confirming Koch's postulates. Similar
results were reported by El-Meleigi et al., Sarhan, Al-Sharidy and Molan and
Abdullah et al. [23, 22, 11 and 24]. There has been evidence in recent years of the role
calcium in plant nutrition relative to lessening the impact of Pythium, Sclerotium,
Botrytis and Fusarium in many crops. Calcium has important roles in the integrity of
cell walls, which infers a resistance mechanism to reduce penetration of fungal
pathogens. Among the fungi that we isolated, E. nigrum selected because of ability to
inhibit growth T. paradoxa and A. alternate on PDA through production of diffusible
metabolites. The inhibition of T. paradoxa and A. alternate by E. nigrum is similar to
the inhibition growth of Phytophthora erythroseptica and T. paradoxa by Trichoderma
virens and Tr. longibrachiatum, respectively [25, 26]. Although the E. nigrum
exudates and CaCl2 were sprayed every month until October 2009, the disease didn’t
decrease, but increased gradually. This implies that treatments used were not effective
in controlling the disease when the disease severity was high (above 3.1) at the initial
time of application. From the results obtained, it can be concluded that spraying E.
nigrum exudates or CaCl2 before the initiation of the symptoms can reduce the
incidence of leaf spot disease.
768
References
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[2] Saadi, M. (1992). FAO report on date palm protection and training activities.
Ministry of Agriculture and Fisheries, Oman. 121pp.
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Salinas, J.; Lopez-Lorca, L. V.; Jansson, H. B., and Gauarro, J. (2005). Occurrence
in Elx, SE Spain of inflorescence rots disease of date palm caused by Mauginiella
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[9] Mubarak, H. F.; Riaz, M.; As-Saeed, I. and Hameed, J. A. (1994). Physiological
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Chalara radicicola on date palms. Plant Dis., 85: 80-83.
769
[11] Al-Sharidy, A. and Molan, Y. (2008). Survey of fungi associated with black scorch
and leaf spots of date palm in Riyadh area. Saudi J. Biol. Sci., 15: 113-118.
[12] Al-Rokibah, A. A. (1991). Leaf blight of date palm caused by Glomerella
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[13] Molan, Y. Y.; Al-Obeed, R. S.; Harahash, M. M. and El-Husseini, S. (2004).
Decline of date palm offshoots infected with Chalara paradoxa in Riyadh
region. J. King Saud Univ., 16: 79-86.
[14] Baudion, A. B. (1988). Laboratory Exercises in Plant Pathology: An Instruction
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[17] Hooker,A. L. (1957). Factors affecting the spread of Diplodia zea in inoculated
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[18] Molan, Y. Y. and El-Hussieni, S. (1999). Pestalotiopsis Leaf Spot of Date-palm
(Phoenix Dactylifera L.) in Saudi Araba. Alex. Sci. Exch., 20: 189-200.
[19] Hozarika, D. K. and Das, K. K. (1998). Biological management of root-rot of
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Res., 13(2): 101-105.
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their preparation and usages. Canadian Plant Dis. Survey, 15: 39-65.
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in middle of Iraq. Proceedings of the 2nd International Conference of Date Palm,
March 25-27, Al Ain, UAE, Pp: 424-430.
[23] El-Meleigi, M. A.; Al-Rokibah, A. A. and Ibrahim, G. H. (1986). Study of fungal
leaf spots of date palms in Al-Qassim region, Saudi Arabia. Proceeding of the 2nd
Symposium on the date palm, March 3-6, Saudi Arabia, Pp: 401-410.
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[24] Abdullah, S. K.; Asensio, L.; Monfort, E.; Gomez-Vidal, S.; Salinas, J.; LopezLorca, L. V. and Jansson, H. B. (2009). Incidence of the two date palm
pathogens, Thielaviopsis paradoxa and T. punctulata in soil from date palm
plantations in Elx, South East Spain. J. Plant Protection Res., 49: 276-279.
[25] Etebarian, H. R.; Scott, E. S.; Wicks, T. J. (2000). Trichoderma harzianum T39
and T. virens DAR 74290 as potential biological control agents for Phytophthora
erythroseptica. Eur. J. Plant Pathol., 106: 329-337.
[26] Sánchez, V.; Rebolledo, O.; Picaso, R. M; Cárdenas, E.; Córdova, J.; González,
O. and Samuels, G. J. (2007). In vitro antagonism of Thielaviopsis paradoxa by
Trichoderma longibrachiatum. Mycopathologia, 163: 49-58.
771
Table (1): Frequency and pathogenicty of fungal pathogens isolated from date palm.
Symptoms
Isolated fungi
Frequenct (%)
Disease severity
Helminthosporium sp.
A.alternata
E. nigrum
4.9
43.1
14.8
2.1
36.7
2.5
Black scorch
T. paradoxa
E. nigrum
52.7
14.6
65.8
1.2
Off-shoot decline
T. paradoxa
E. nigrum
37.4
16.2
57.3
1.4
Leaf basal rot
A. alternata
E.nigrum
33.3
9.1
44.5
2.8
F. proliferatum
A. niger
N. oryza
8.5
3.7
27.5
2.1
2.0
36.4
Leaf spots
Fruit spots
7
6.4
Infection (%)
6
5.6
4.9
5
4.2
4
3.2
3
2.6
2.3
2.2
2
2.2
1.9
1.8
1
0
Leaf
spots
El-Minia
Leaf
spots
Black
scorch
Fruit
spots
Leaf
spots
Black
scorch
Assiut
Fruit
spots
Offshoot
decline
Sohag
Leaf
spots
Fruit
spots
Leaf
basal
rot
Qena
Localities and Symptoms
Figure (1): Survey for symptoms and incidence of some diseases
in date palm growing Governorates on Upper Egypt.
772
Figure (2): Different symptoms occurred in date palm trees as nature infection. Leaf spots in
rachis (A), Off-shoot decline in off-shoot (B1); fronds (B2) and rachis (B3), Black scorch in leaf
(C), Leaf basal rot at the base of leaves (D1) then central leaf cluster die (D2) and fruit spots (E).
Figure (3): Microscopic photograph of T. paradoxa isolate showing microconidia: which were hyaline
and cylindrical form, also, chlamydospores: have dark brown with thick walls (A). Pathogenicity test
for T. paradoxa on rachis (B) and A. alternate on fronds (C).Control of leaf spot disease
773
E. nigrum (50% dilution)
CaCl2
Control
70
65.7
60
58.2
57.1
Disease severity (%)
52.3
50
47.6
43.2
42.7
40
43.8
43
40.9
36.8
34.1
32.7
30
35.2
26.525.7
23.2
18.7
20
11.3
10
8.2
6.7
4.2
3.1
4.8
0
Mar.
Apr.
May
June
July
Aug.
Sep.
Oct.
Time of spray
Figure (4): Disease severity of leaf spot disease on the 3rd
whorl leaves of date palm from March to October 2009.
5
4.7
E. nigrum (50% dilution)
CaCl2
Control
4.5
Disease severity (%)
4
3.8
3.5
3
2.6
2.5
2.4
2.5
1.9
2
1.5
1.7
1.5
1.2
1
0.81
0.57
0.5
0.22
0
Nov.
Dec.
Jan.
Feb.
Time of spray
Figure (5): Disease severity of leaf spot disease on the 6th whorl
leaves of date palm from November 2009 to February 2010.
774
70
E. nigrum (50% dilution)
CaCl2
Control
63.8
Disease severity (%)
60.2
59.1
60
51.3
50
44.5
39.5
40
30
20
12.7
10
5.8
0
0.141.05
0 0.73
0 0.55
0.050.31
0 0.18
0 0.12
0.120.08
0 0.07
Mar.
Apr.
May
June
July
Aug.
Sep.
Oct.
Time of spray
Figure (6): Disease severity of leaf spot disease on the 6th
whorl leaves of date palm from March 2010 to October 2010.
775
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776
Post Harvest and Storage
777
778
OP 39
High-Rise Fully Automated Refrigerated Warehouses, New Trend
for Date Storage in KSA
Atef M.Elansari* & Sultan Al-Thagafi**
*Professor of Food Engineering Faculty of Agricultural Engineering
Alexandria University, Egypt.
Email: aansari1962@yahoo.com Mob: +20168891464
** Vice president of Samara Refrigeration Company
Sultan@samara.com.sa , Mob: +966544474141
Riyadh, Saudi Arabia.
Abstract
Saudi Arabia has the highest population in GCC with more than 26 million people
and a growth rate of 1.5% annually. This imposes greater pressure on improving the
status of post-harvest technologies including its cold chain. However, the
concentration on agricultural operations in order to increase production remains the
focal point, while the ways to reduce post-harvest loss through the application of
appropriate technologies do not meet the same importance.
This paper examines potential advantages offered by constructing High-rise fully
automated refrigerated warehouses in KSA for Date storage. Based on a recent study,
unfulfilled refrigerated space demanded in Saudi has reached more than one million
tons. Which makes it very feasible to apply the high-rise automated warehouses
concept. In this paper, we aim for discovering key advantages of shifting from
conventional refrigerated warehouses to fully automated high-rise technology and how
it can enhance the quality of the cold chain of date handling. The paper will also be
describing challenges facing cold store chains in Saudi Arabia as one of the leading
economies in GCC & Middle East.
Key words: High-rise, fully-automated, dates, cold storage
Introduction
The public cold store is characterized by its flexible quantities from freight cars to
cases and also with its flexible lengths- short, medium, or long term. In addition, it has
flexible access where the pricing is on volume bases.
779
The success of a public cold storage facility hinges upon the local demand for the
services provided by the facility, as well as the demand in the larger marketplace for
the products supplied by local users. A public cold storage facility depends on the
local demand as well as the likely success of local users. The local demand can be
generated by processors, harvesters, or other members of the public. The market for
the public cold store is related to the potential requirements as well as the supply of
resources that might be held in the facility.
Public cold storage of dates in Saudi Arabia has been facing a real crisis since the
year 2009, according to warnings of agricultural experts. That is because the season of
Ramadan is being preceded by entry of harvest dates, which requires storing dates for
a period of 11 months to enable consumption during the holy month. Moreover, The
current cooling capacity in Saudi Arabia does not serve the purpose of storing entire
production of dates.
For dates and according to estimates, the current capacity of the warehouse cooling is
around one hundred thousand tons, while the production of Saudi dates is more than one
million tons per year; in which over half of the production is consumed during the holy
month of Ramadan alone. Based on a recent report by the Saudi Agriculture Bank,
Qassim region alone is expected to build cooling warehouses with a total cost of 200
million SR (table 1), since the area is one of the main date production zones in the region.
The literature dose not reveal any numbers of the existing cold storage capacity in Saudi
Arabia either for dates or any other commodities. Such figures is needed for any further
planning and funding to fill the gap between what is there and what should be available.
In order to maximize the outcomes of the investment directed towards the
refrigerated warehouse in Saudi Arabia, a new and recent trends should explored and
evaluated on order to avoid to avoid any negatives impacted that might be exist. One
of the most recent concepts in the area of cold storage is the high-rise fully automated
refrigerated warehouse. The net consequence of this technology is not only to save a
very large amount of labor, but also to make the building much smaller and more
thermally efficient. The much smaller footprint also reduces the size of the required
site, resulting in significantly lower construction and land costs for date’s cold storage.
For date’s industry in Saudi Arabia, high-rise automated buildings represent the future,
780
and refrigeration design engineers should understand the implications for the design of
a refrigeration system.
Therefore and based on the above facts, the first objective of the current study was
to determine the projections of required cold storage capacity in Saudi Arabia within
the coming ten years. The second objective was to present the most recent trends in
refrigerated warehouse, which is the high-rise fully-automated cold store to the Saudi
agricultural and cold store community.
International trends in refrigerated warehouse
Public refrigerated warehouse (PRW) storage capacity is increasing around the
world, according to the International Association of Refrigerated Warehouses (IARW)
Global Cold Storage Capacity Report. IARW has collected data for the 2008 report
from over 40 countries, more than ever before. In addition, IARW has compiled
profiles of national public refrigerated warehouse markets in 18 countries, including
several emerging and developing markets. This is also the first year that the report has
shown a full decade of industry growth.
IARW monitors PRW capacity in 45 nations and regions. In those places where
data was collected in 2006, capacity increased in 17 places while it remained flat in six
nations. Data in other areas was insufficient to draw any conclusions. Areas showing
the greatest increases were France, Germany, the Netherlands, Spain and Brazil. Total
global capacity for these countries for 2008 is 179.82 million cubic meters, which
represents a 15 percent increase from 2006.
The total refrigerated warehouses storage capacity in the United States is about 94
billion cubic meters (USDA 2008).This growth suggests a worldwide trend toward
increasing cold storage capacity driven by a greater reliance on the cold chain to meet
growing trade and consumption rates of perishable products. Overall global capacity in
2008, including the 25 original countries surveyed, is approximately 247.77 million
cubic meters.
In both China and India, there has been significant increase in capacity since 1998.
China shows a 20 percent increase and India‘s capacity has more than doubled since 1998.
Projection of Cold storage size needed for Saudi Arabia
According to this statistics that is shown in Table (1), the refrigerated warehouse
781
capacity in 2008 were 3.5 Million cubic meter for the Middle East. Using the
recommendation of the International Institute of Refrigeration that each 200 kg of
products occupies 1m3, its means that 3.5 Million cubic meter equals to 700,000 Tons
refrigerated storage.
Tables 2 to 5 represent a list of the most important cold storage projects in the
Kingdom. A previous study funded by the Riyadh Chamber of Commerce and
Industry in 2006, showed that 25% of the total refrigerated warehouse capacity in the
Kingdom is located in Riyadh. Based on this fact and according to table (2) that list
major cold stores in Riyadh, it indicates that the capacity is about 110,000 tons. By
other words the total refrigerated capacity in Saudi Arabia is about 440,000 tons or 2.2
Million cubic meter. This means that 62% of the total refrigerated cold stores in the
Middle East is in Saudi Arabia.
Comparing by India, it is estimated that cold-storage capacity of 19.5 million ton
which is less than 15 per cent of the annual horticulture production and is dominated
by potatoes (80 per cent of capacity). For Saudi Arabia and for the same crop, potato
production, the capacity available in the potato production areas is less by about 90%.
For Dates the same problem applies.
To compare by the EU country and the USA in general, the estimated size of the cold
chain industry is around Euro 1.4 - 1.7 billion and is expected to grow at 20 - 25 per cent
annually. By comparing the available storage capacity per capita for different countries,
we find that for Japan, it is 0.21 m3/capita; for France, it is 0.141 m3/capita, for USA, it
is 0.313 m3/capita; while for Saudi Arabia it is 0.096 m3/capita. By other words, there a
gap between what is exist and what should be there by more than one million tons based
on the international trends. This figure implies that the current cold storage capacity is
not adequate to the harsh weather conditions and the vast areas of the kingdom. This
capacity should to be tripled within the next few years in order to meet different changes
of the increasing population and growing consumption rates.
Total projections of the amount of cold storage tonnage that will be built in Saudi
Arabia is difficult to judge, but all indications are that they are likely to be high. Larger
growers and other Community and Agricultural Development associations already
send tens of funding requites to the agricultural bank for their projects as shown in
782
tables (1). These suppliers and growers are planning to expand and improve their
marketable yields each year, so eventually we believe that available produced volumes
will be even higher. The development of refrigerated warehouse is likely to be a
catalyst for further increased production and demand.
Dates cold storage economics in Saudi Arabia
Refrigerated storage of dates and vegetables for investments point of view is highly
economical feasible, as shown by comparing of the costs of cold storage and the price
of wholesale in and out of season for dates. The indicators shown in table (6) that for
the most important dates varieties grown in Qassim region as an example ranges of 3.0
to 4.0 SR/kg and this represents a five to six times the storage costs.
Therefore, the profit is ranging from 25% for Sukkary to 55% to Alrziz where the
average profit for the various varieties is 40%. This is a large profit margin and
encouraging investment in cold storage for dates, even with low off-season prices.
High-rise fully automated cold store
In high-rise automated warehouses we usually have two cranes working in the same
aisle, putting and picking pallets so quickly in around a minute. Some automated cranes
can hold two pallets at a time. Conveyers in the middle of the building normally feed
cranes so that the cranes need not travel the entire length of the aisle. Buildings with 30,000
pallets often have only two aisles and two cranes. High-rise automated warehouses change
the role of the refrigeration design engineer considerably (Dohlen, 2011) :
1. Refrigeration loads are reduced significantly. The system size is reduced
commensurately. The buildings are very tall and gravity helps distribute refrigeration
in the building.
2. The internally generated loads during operations are much lower, allowing offpeak operations, saving the cost difference between on- peak rates and off-peak rates.
The system must be designed to capitalize on the off-peak opportunities in warehouses
particularly those that operate in de-regulated states as well as those in which the states
have legislated premiums for on-peak operations.
3. The design process of an automated building is significantly different from the
conventional building. The crane manufacturer/system integrator, the rack company,
and the operating company design the material handling system, and the building is
783
simply the enclosure of the material handling system.
4. Rack-supported buildings distribute loads relatively evenly on the floor plate,
changing the design of the floor plate particularly when pilings are used. The engineer
designing the floor structures needs to coordinate with the manufacturer of the racksupported building. Again, this gives an advantage to the rack manufacturer for
coordinating the entire project.
5. Automated cranes and the pallet license plate readers are very moisturesensitive; neither tolerates any frost or ice coating. Dehumidification, particularly of
dock areas, is essential. Pallet readers will not read; the conveyors and the cranes can
become jammed by ice. The system is very intolerant of moisture.
6. Product might be stored in bulk on docks for long periods of time, in order to
buffer the cranes from the trucks, so the customer sometimes wants –18°C docks. One
design engineering firm in Europe designed a continuous vestibule across the truck
doors, with the air between the truck doors and the inside wall dehumidified with a
desiccant system. The dock itself was kept around –23°C for ice cream. The entire
dock, door-design and dehumidification system requirements and design are affected
by these material-handling decisions.
Accordingly, it is necessary to adopt a comprehensive strategy that aims towards
increasing the capacity of the cold storage available for dates by transferring the
technology of the High-rise fully automated cold store due to the vast advantages
that offer. The statistics shows a big gap between the existing vacancy of the cold
store and the needed one.
Characteristics of Cold Chain Challenges of dates in Saudi Arabia:
The term “cold chain” refers to steps from harvest to consumption that extends the
natural shelf life of a product.
Typical components of a cold chain may include
post-harvest handling, Precooling, refrigerated transport, refrigerated storage,
controlled atmosphere storage, chilled or frozen processing, cold storage holding and/or
distribution, retail refrigeration, institutional refrigeration, and home refrigeration.
There has been significant shortage in research that address postharvest losses in
Saudi Arabia in particular and in the GCC countries in general. There are diverse
problems and challenges related to the establishment of an appropriate cold chain
784
program that minimize losses in Saudi Arabia and it can be summarized as follow:
1) High ambient temperature and harsh conditions.
2) Absence of any information concerning postharvest losses.
3) Lack of technical labor and modern pack house facilities.
Compliance issues (Quality standards, Sanitary & Phyto-sanitary issues, Food
safety, Pesticide residues, Traceability).
4) Absence of any standard R&D facility for postharvest and cold chain.
5) Lack of integration between research, academia and extension.
For most of the farms there are no precooling facilities even for the major
agricultural companies. We found major problems in management such precoolers. In
the south we (Jazan) precooling facility do not exist for Mango, which is the main
crop, there is Mango. On 2005 the area lost 6000 carton because of the lack of proper
cold chain. Table (7) lists all what is exist in the Kingdom of precooling capacity.
This is a very limited capacity compared by the production of fruits and vegetables in
the kingdom. Most of the units are not efficient for many reasons. Since most of the
facilities are classified as wet deck system, so it has the following disadvantages:
• Limited temperature drop since water freezes at zero
• The minimum temperature can be achieved is about 2-3°C. This temperature is
not appropriate for grapes and stone fruits.
• All water used are not treated so cross contamination is a major risk.
• Carton gets wetted since the sprayed water is not fully vaporized.
• Limited airflow rate due to its higher density because of water.
Recommendations:
1)
Changing industry from production based to quality and export based.
2)
Infrastructure development (Physical: Farm to market roads, railway, airways
and shipping: cold chain: packing, storage, reefer containers) and involving private
sector investment in cold chain infrastructure
3)
R & D capacity building in the area of fresh produce cold chain, by
establishing a Centre of Excellence in Postharvest Science and Technology.
4)
Human resource development and strengthening R & D and linkage between
the institutions and industry
785
5)
Intelligent Marketing: Improving quality for compliance, exploring new
markets and developing Market Intelligence System.
6)
Policy making:
Subsidize certification of EUREPGAP, HACCP, etc.,
ensuring credit facilities, and duty free import of machinery of cold chain
infrastructure
7)
To build cold chain associations.
8)
Identify different partners on the cold chain and document the current
practices.
9)
Creation of some media, educational tools and programs.
10) Establishments of a cold chain resource directory
11) Establishment of regional center of Excellency of cold store R&D
12) Encourage the "Green cold chain technology".
13) Get the support of different international organization such as FAO, IIAR,
WFLO, GCCA and IARW
14) Organize the first cold chain conference in the region
Conclusion
The statistics shows a big gap between the existing vacancy of the cold store and in
Saudi Arabia. It is suggested that this gab is to be filled by the high-rise fully
automated cold storage facilities. These facilities offer major benefits compared by
conventional standard height buildings using high lift fork trucks. The economics of
automated racks and software has come down significantly since the inception of
automated storage technology. This has enabled cold storage companies in the USA
and Europe to reduce their staff overhead and increase the efficiency of operations
significantly, something that we should simulate in Saudi Arabia. In high-rise
automated storage facilities, a significant reduction in the infiltration refrigeration load
is occurred. Refrigeration loads inside are optimized since there is no people or lights
inside in addition to the small footprint required. Further advantages are possible
improvements of safety and hygiene. Finally a cold chain enhancement program
should be developed in order to meet all challenges facing the postharvest issues
concerning fresh produce and dates is on the top of them.
786
References
[1] Dohlen, G.V. 2011. Refrigeration Implications of High-Rise Automated
Warehouses. Technical Paper #8. Industrial Refrigeration Conference & Heavy
Equipment Show Caribe Royale. Orlando, Florida.
Table (1): Expected projects to be funded by the Saudi Agricultural bank
#
Project
Capacity (Ton)
Product
Value (Million SR)
1
El-Naghala
30,000
Dates
100
2
Nadec
15,000
Onions, Potatoes
60
3
Enma
10,000
Public
40
4
Hial dates
30,000
Dates
100
5
Qassim cooperative
10,000
Dates
40
6
Whole sale market
30,000
public
100
7
Aniza
5,000
Dates
15
8
Awkaf Elraghi
10,000
Dates
50
9
Ghalid Elrshod
10,000
Public
40
10
Safe storage
10,000
Public
30
11
Others
65
Total
630
787
Table (1a): Refrigerated Capacity by Country
788
Table (2): The most important cold store projects in Riyadh.
#
Company name
Capacity
١
Agility
٢٠,٠٠٠
٢
Arasco
Whole sale market
Safe storage
Maaly
Jarir
Leha
Universal
Ngdian
Ghligia
١٥,٠٠٠
٣
٤
٥
٦
٧
٨
٩
١٠
١٥,٠٠٠
١٠,٠٠٠
١٠,٠٠٠
١٠,٠٠٠
١٠,٠٠٠
٥,٠٠٠
١,٥٠٠
١,٠٠٠
١٠,٠٠٠
Other
١٠٧,٥٠٠
Total
Table (٣): The most important cold store projects Qassim area.
#
Company name
CAPACITY
١
Rashed
٢,٥٠٠
٢
Assaf
El-sheigh
Khalid Eldeer
Saleh Elmiteery
Gahez Elmiteery
Deif Allah
Elodeib
Elfawzan
Elshadi
Asasiat
Ayaff
Elhreish
El-Botian
٢,٢٢٠
٣
٤
٥
٦
٧
٨
٩
١٠
١١
١٢
١٣
١٤
٦,٠٠0
٤,٠٠٠
٢,٠٠٠
١,٥٠٠
٦,٠٠٠
٣,٠٠٠
٣,٠٠٠
٢,٠٠٠
٣,٠٠٠
٢,١٠٠
٣,٠٠٠
١٠,٠٠٠
١٢٠٠٠
Other
Total
56,920
789
Table (4): The most important cold store projects Hial area
#
Company name
CAPACITY
1
Hadco
10,000
2
3
4
5
6
Other
Hayat
El-Massara
Eldeify
Rawnek
Rykhias
5,000
7,000
2,000
3,000
3,000
5,000
35,000
Total
Table (5): Some capacity of the cold store of Jeddah
#
Company name
Storage capacity
(TON)
١
Saudi Cold Storage company Limited
7.500
٢
Shar
15.000
٣
Al Shaheen
10.000
٤
Sharbatly Est
١٠ ٠٠٠
٥
Abbar & Zainy Cold Stores Co
١٠ ٠٠٠
5
Al-Gosaibi Cold Stores
٥ ٠٠٠
6
Al Nakhel Cold Store
٢ ٥٠٠
790
Table (6): Compared cost of the refrigerated storage price with differential price
for wholesale prices of dates Inside and outside the season.
Details
Salag
Initial storage date
Final storage date
Storage period (month)
Price at the initial
storage period (SR/kg)
Price at the end of
storage period (SR/kg)
Price difference (SR/kg)
Total cost for storage
(10 halalah/kg/month)
Net profit (SR/kg)
Profit percentage (%)
Famous date variety at Qassim
Nubot Roziz
Barhi Khalas Sukkary
seif
August August August August August
January January January January January
August
January
٦
٦
٦
٦
٦
٦
٥٦
٩٠
٤٨
٦٣
١٠ ٣
١٠ ٠
٨٧
١٣ ٠
٧٩
١٠ ٠
١٣ ٦
١٣ ١
٣١
٤٠
٣١
٣٧
٣٣
٣١
٠٦
٠٦
٠٦
٠٦
٠٦
٠٦
٢٥
٣٤
٢٧
٣١
٢٧
٢٥
٤٤
٣٨
٥٥
٤٩
٢٦
٢٥
Source: Collected and calculated from the average prices of major agricultural
commodities for the local and imported goods into the kingdom's main market areas.
Ministry of Agriculture - Department of Agricultural Marketing. 1431 AH 2010 AD.
Table (7): Precooling facilities in Saudi Arabia
Company
Type of precooling
Location
Capacity
Tabok (stone fruits and Grapes)
Astra (stone fruits and Grapes)
Hadco (Grapes)
Nadec (Store fruits)
Elgouf (Store fruits)
Elboutain (Genral) not working
Thimar (Genral)
Panda (General) not working
Wet deck system
Wet deck system
Wet deck system
Dry system
Wet deck system
Wet deck system
Dry system
Wet deck system
Tabok
Tabok
Hail
Gouf
Gouf
Qassim
Riyadh
Riyadh
5 ton/hr
2ton/hr
3 ton/hr
2 ton/hr
2 ton/hr
5 ton/hr
3 ton/hr
5 ton/hr
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وا_ق اRو\.
OP 40
Exploiting the potential of freeze technology to improve quality
and yield, and stream line the product supply of dhakki dates.
Shahzada A. Saleem*, Ahmad K. Baloch1, Ambreen A. Saddozai2, and Waqar A. Khan3
*
Agriculture Research Institute, Dera Ismail Khan, Pakistan sasdikpk@gmail.com
1
Department of Food Science & Technology, Gomal University, D. I. Khan.
2
Food Microbiology Labs. National Agricultural Research Council, Islamabad, Pakistan.
3
Food, Agriculture and Environmental Chemistry, University of Glasgow, UK.
Abstract
Dhakki date (Phoenix dactylifera L.) originated from Dera Ismail Khan (Pakistan)
is considered amongst the top world-leading varieties. Small seeded fruit of gigantic
size and weight being astringent at Doka (Khalaal) stage develops fine texture and
relish taste on ripening, and hence fetches high price in national and international
markets. Dhakki date, though being a symbol of nationality, it is facing diversified
problems. Dhakki date is late variety its ripening period coincides with stormy
monsoon season and thereby receiving quality degradation of enormous levels. The
losses go on piling up due to lack of appropriate processing technology, and shortage
in storage & transportation facilities. A mechanism is developed to significantly
reduce post-harvest losses, and to streamline production and supply making freshly
ripened Dhakki dates available year around. Doka of Dhakki fruit was left for storage
at 10°, 5°, 0°, and (-)15°C for one year examining hardness, appearance and mold
resistance with subsequent performance to ripening/curing, quality & yield
improvement of the product. The Dokas stored at 10°C were found mold-infected just
after 3 months period, and those at 5°C resisted to changes at least for 10 months. The
Dokas stored at 0° and -15°C remained free from the infection during the study over
year period, however developed Dong (Rutab)-like characteristics on thawing
changing color of Doka from yellow to golden brown, and acquiring softer texture
with sweeter taste. The Doka stored at 5°C on curing/ drying at appropriate time after
monsoon leads to a product with improved quality and yield with enhanced storage
stability at 40°C. Leaving Dokas at 5° to 10°C offers sound proposition to combat
793
against unfavorable climatic conditions, and further processing assures to continue the
supply of freshly ripened Dhakki date during off-season.
Introduction
Role of Date palm (Phoenix dactylifera L.) has been well established ever since the
birth of human race. The Holy Qur’an commended dates at several places, and hence
the dates became part of Islamic culture. Considering as a virtue Muslims consume
dates at several religious occasions. Of course, the date palm nourishes millions all
over the world and contributes significantly towards their development and prosperity
particularly to those living in the Arabian world. Date fruits being sweet and palatable,
are consumed as a staple diet supplying 2500-3000 K calories /Kg of physiological
energy. The fruit provides several vitamins, a high amount of potassium, and furnishes
overall highest percentage of easily assimilating carbohydrates. Further it contributes a
large amount of cellulose and hemi-cellulose materials necessary for maintenance of
intestinal motility. The date fruit is one of the important cash crops for Pakistan, and a
good source of foreign exchange earnings. The total cultivated area of all type of dates
in Pakistan exceeds 78.1 thousand hectares with its estimated annual production over
630 thousand tons, and constitutes about 11% of total world production [3]. According
to Food and Agriculture Organization (FAO), Pakistan is 4th world largest date
producing country [1], exporting dried dates worth US$ 17.8 million annually [2].
Cultivation of dates in North West Frontier Province (NWFP) is over 1000 hectares
with 6700 tons production, and region of Dera Ismail Khan alone contributes more
than 50% of it [3].
Dhakki being the most promising local variety of Dera Ismail Khan is also one of the
top few world-leading cultivars, and presumably furnishes 60% of the total share of this
area and yet displaying continuous increases in its production. The Dhakki has fruits of
choice and market oriented, and hence is gaining unprecedented importance both in
domestic and foreign markets. The fruit possess large size (5-6 cm long and 3-4 cm
thick) and weight (20-26 gm/ fruit), very fine texture with exceptionally high flesh/stone
ratio (96%), relishing taste, good appearance, and reasonably longer shelf life [5]. In
spite of the fact that the Dhakki date is a crop of national significance, yet it is not
gaining requisite pace for the development. Dhakki being a late variety is facing
794
diversified problems some of them are dependent upon the weather stresses while the
other arising out from resource shortage and lack of know-how. The ripening season of
the date starts during high summer temperature of 40-50°C with peak production in
August-September. This period unfortunately coincides with monsoon season in
Pakistan. While commissioning of the Chashma Right Bank Canal has made the climate
even more variable and unanticipated. Since Dhakki fruit at full mature/ ripened stage is
relatively more susceptible to hot and humid conditions, it receives substantial damages
from monsoon rain and storm. Moreover, the fruit during this period is at eatable (Doka/
Dong) stage and prone to infestation by insects / birds and diseases and hence invaded at
a rapid rate under favorable climate of relatively reduced temperature with high
humidity. The losses continue pilling up so long as fruits stay on-tree for want of Dong
formation until the end of September. The fall in day temperature on account of ending
summer season slows down on-tree fruit ripening; consequently the period between
consecutive pickings as well as the number of pickings is increased amounting to further
infestation and expenses. At the same time, a large quantity of freshly ripened dates
becomes available, which not only gluts local market but pre-occupies orchard space
thus proper handling and processing of Dhakki date becomes difficult, and the surplus
produce is wasted. Under the prevailing detrimental environment that expands over
weeks the Dhakki date inflicts with enormous crop losses and quality degradation. Such
losses cannot significantly be reduced due to lack of appropriate on-farm shelters, or
other facilities for safe keeping of the produce.
The traditional methods for ripening/ curing are still under practice, and the fruits at
late Dong stage is spread on mats and exposed to sun in open air. The sun drying under
dusty environment renders the product non-uniform and substandard. Due to persistent
raining and stormy conditions large amount of the harvested dates gets moldy,
fermented, dusty, and birds/ insects invaded. Thus colossal amount of fruit wastage
wrecks the crop yield devastating ultimately the economy of the growers. In our two
previous independent studies on the application of microwave [14] [7] and ripening
using artificial means [13] a successful conversion of Doka into Tamar has been
demonstrated. Such techniques shortened the period of fruit on-tree by 2-3 weeks,
795
curtailed the curing period appreciably, increased the yield substantially, and enhanced
quality of the product significantly.
In order to maintain current tempo of cultivation and propagation of the Dhakki
cultivar it is imperative to make Dhakki variety a profitable crop. Since date in fresh
form is much liked by the consumers and attracts higher prices, attention therefore is
focused to ensure the availability of fresh dates as and when required. Except the
storage at reduced temperature, no effective long-term preservation methodology is
apprehended for storage at perishable Doka stage of Dhakki fruit. Keeping whole
scenario in view, the present study was therefore carried out to explore the potential of
freeze technology so as to make fresh Doka available year around for on-demand
production of fresh dates, and further to improve curing performance, quality, yield
and stability of Dhakki dates.
Research Methodology
The mature Dhakki fruits of early Doka stage, harvested from the orchard of the
Agriculture Research Institute, Dera Ismail Khan, were hard (295-300 mmHg.cm-2
hardness index), yellow in color, and fairly astringent (Fig. 2). After given thorough
washing with tape water and wiping off excess liquid the Doka was taken in 250g
polyethylene bags and stored at temperatures of 10°, 0° and -15°C for a period of 12
months. For this purpose a space was reserved in the cold storage maintained at 10°C,
whereas the fruits that required storage at 0°C and -15°C were kept in a plate type
house-hold deep freezer (Triplet Deep Freezer, provided by Waves Company, Ltd).
The samples were taken out from the storage periodically to examine hardness,
microbial growth, and surface appearance. Fruit hardness was measured by a hardness
testing device developed previously [7]. Observing slime formation on the fruit
surface, given peculiar fermenting smell or recording other sensory characteristics
ascertained the fruit quality. In order to evaluate performance of the frozen Doka for
successive operations the samples after thawing were cured/ dried at a temperature of
40 ± 2°C using air overflow cabinet drier until the moisture content reduced to 2526%. The period for curing/ drying, the extent of ripening/ yield percentage and other
quality attributes determined. Freshly harvested Dokas of the same stage (with or
without overnight freeze treatment) were also processed side by side to serve as
796
controls. Further studies were conducted to verify the effectiveness of potassium
hydroxide treatment against loosening off fruit skin as observed during preceding
experiments. The Dokas from 6-months storage at -15°C were treated with 0.1 to 1.0
% (g/ml) potassium hydroxide. The fruits were steeped in the solution (1:1 w/v) for 1
m and the temperature maintained at 40°C by switching the power on and off. The
treated fruits after given water rinse were cured/ dried under the prescribed conditions.
To examine shelf stability of the product the cured samples taken in polyethylene bags
were incubated in a thermostatically controlled oven at 40°C for 8 months, and quality
evaluated. Moisture contents were determined by oven method of AOAC [4], and the
extent of ripening/ curing and yield percentage determined according to [7].
Quality of the products was evaluated using standard organoleptic technique for
sensory scoring through trained panelists [10]. A descriptive panel was composed on
individuals to evaluate overall acceptability of the products keeping in view quality
parameters like skin appearance, grit, taste, color, and shine of the fruits. The samples
were scored on hedonic scale (score of 1 not acceptable, 10 highly acceptable). The
data were analyzed statistically using MSTAT-C version 2-10 software package
applying Completely Randomized Design (CRD). The means are separated by LSD
test using the same package.
Results and Discussion
The Dokas showed no signs of deformity on storage at 10°C for at least 3 months;
however, most of the fruits beyond that period gave a dull look with soft patches on
the surface, and indicated increased variation in firmness. The fruits became
shriveled and mold invaded (Tables 1, 2, 3). Whereas, the Doka kept at 0 °C and 15°C on thawing displayed much soft and tender texture with significantly reduced
hardness within 3 months storage. Some of the fruits on further storage to 9 months
developed slightly brown spotting possibly caused by crystallization of soluble
solids. However, no signs of mold growth were evident up to 12-months. There
appeared hardly any difference between fruits stored at 0°C or at -15°C except that
the fruits at the lower temperature turned more soft and juicy on thawing. The fruits
were found sweet in taste, much attractive, completely devoid of astringency, and
had developed desired golden amber color, the characteristics advanced on natural
797
ripening (Fig. 3). This confirms our previous findings that the frozen Doka converts
into Dong-like product on thawing [5].
Yield was improved on storing the samples at each selected temperatures. The
samples for 3 months storage at 10°C exhibited 1.2 times greater yield on subsequent
curing/ drying than from those harvested a fresh (65% yield, Table 4). Whereas the
Dokas stored at 0° or at –15°C rendered a product with even much higher yield up to
95 % (Table 4). A short overnight freeze treatment also proved as useful in
improving product yield. Since the fruits were of early Doka stage possessing thick
and hard skin that on freezing/ thawing loosen off from the pulp and manifested skin
cracking as a result of curing/ drying. The extent of the cracking was higher in the
samples that were left frozen overnight or remained under storage at the lowest
temperature. The study indicates that freezing/ thawing inflicted fruit pulp and its
contents probably greater than its skin, and hence agree with views of. Hussan [9]
who reported a marked activity of ripening enzymes including polyphenol oxidase
and pectin methyl esterase after dates were thawed. Maximum polyphenol oxidase
activity was recorded after 3 hours thawing in Zaghloul and Bent-Aisha, and after 18
hour in Samari date cultivar. Moreover, the bitterness of the Doka became
precipitated, and the astringent fruit tasted sweet. It is speculated that the ice crystals
formed on freezing ruptured the fruit cells which on thawing allowed the ripening
system to continue function at a rapid rate. El-Din, [8] reported that Khalaal stage of
Bent Aisha date by freezing at -18°C developed to Rutab (soft) stage. He further
reported that the freezing method gives the best quality in which the low molecular
weight tannin contents were decreased. In the present study besides improvement in
the quality and yield the freeze technology shortened the requisite operational time
for subsequent curing/ ripening from days to hours reducing the moisture content of
the product to 24-26% within 72 hrs. Whereas the freshly harvested Doka used as a
control took 7-8 days under similar curing conditions (Table 4). The selected level of
moisture contents corresponded to 0.60-0.62 aw at which the samples showed optimal
stability on subsequent storage [6].
However, the frozen Doka after curing unexpectedly were under scored for giving
gritty mouth-feel caused by the broken hard skin pieces. Preliminary study was
798
therefore initiated with a view to weaken the loosened hard skin which became
separated from the frozen fruits on thawing and subsequent curing/ drying. Utilizing
the chewing property of alkali to polysaccharides a treatment consisting of potassium
hydroxide instead of sodium hydroxide was chosen for the purpose so as to maintain
composition of the date product sodium free for natural resemblance. Observing
improvement in quality of the product after preliminary treatment with potassium
hydroxide (0.50g/ 100ml) the study was further conducted using different
concentrations of potassium hydroxide in the range of 0.1-1.0% (g/ml). The fruits
from frozen storage were allowed to steep in the reagent solution for I min at 40°C
controlling the temperature of solution manually. The treated fruits were cured/ dried
to 25% moisture contents, and quality evaluated. In order to find out stability the
product was further stored at 40°C for 8 months at the prevailing summer
temperature, and evaluated for acceptability on the basis of taste, appearance and
mouth-feel considering grittiness as a major factor. Improvement in quality was
noted with increasing treatment concentration up to 0.25%. The product tended to
become dark and the score declined gradually on further increase in the
concentration making the product unacceptable with 1.0g/ 100ml. The treatment
concentration of 0.25 % indicated as optimal for rectifying the irritating feature of
gritty feel as the product received highest score of 9.0-9.8 (Table 5, Fig. 4). The
sample also displayed enhanced storage stability at 40°C, besides giving much
increase yield of 98% (Table 4). The study certainly conceives a great breakthrough
for date processing industry as it furnishes a product with exceptionally high yield of
best quality and stability. Moreover, the process offers 2-3 weeks saving from on tree
hang-on period and prevents expected great damages caused by various
environmental factors. The development further ensures streamlining the supply of a
uniform product to market. The consumption and marketing of quality dates and date
products would become under control, and hence keeping the prices greatly stable.
Moreover, it guaranties off-season and on-demand supply of freshly ripened dates,
and keeps marketing activities progressing round the year. Although the economics
has not been worked out it is highly likely to get much higher net return as a result of
substantial reduction in overall losses, better price recovery and conceding extended
799
market activities. Moreover, the technology offers a sizable breathing space to
manage harvesting, processing and storage of dates while minimizing the date
glutting. The option would certainly help boosting up the economy of the growers/
farmers and the country at large.
Conclusions
The harvested Doka of Dhakki dates sustains varying amount of textural and
biochemical changes on prolong freezing, however it remains safe and free from mold
growth at 0 and –15°C for at least a year. The Doka turns into Dong form by acquiring
characteristics similar to those advancing on natural ripening that lead to tenderness,
astringency precipitated and golden amber color developed. The fruits can be cured/
dried at a faster rate to a cured date-biomass. However the product acquires a chipped
surface due to skin peel off. The undesirable surface feature is adequately corrected
after given mild potash alkali treatment consequently the product improved quality,
yield and storage stability at 40°C. The process envisages great potential to ensure the
availability of fresh dates at off-season. Further it will monitor the production,
maintain price of the product stable and keep economic and marketing activity of dates
flourishing, and possibly sizable reduction in post-harvest losses.
800
References
[1] Anonymous. (2000). Production Yearbook, Food Agriculture Organization.
[2] Anonymous. (2001). 3rd National Exhibition on Dates, Dera Ismail. Khan, Export
Promotion Bureau, Pakistan.
[3] Anonymous. (2002). Agric. Statistics Pakistan, Govt. Pak., Ministry Food, Agric.
Livestock (Economic Wing), Islamabad, p.100.
[4] A.O.A.C. (1984). Official Methods of Analysis, 14th ed. Association of Analytical
Chemists, Washington, DC.
[5] Baloch, A. K. (1999). Enhancement of post-harvest quality and stability of Dhakki
dates using advanced technology. Report, Pakistan Science Foundation, Islamabad.
[6] Baloch, M. K, Shahzada A. Saleem, Ahmad K. Baloch, & Waqar A. Baloch, 2005.
“Impact of Storage Atmosphere on Stability of Dhakki Dates”. J. Food Science &
Technology, LWT Switzerland.39 (6): 671-676.
[7] Baloch, A. K., Saleem, S.A., Dar, N.G., Baloch, W. A. & Baloch, M.K. (2003).
Influence of microwave radiation on ripening of Dhakki dates. Journal of Food
Processing and Preservation, 27 (3): 181-193.
[8] El-Din, A.E.M.M.M.N. (1998). Effect of some technological treatments on
ripening and drying of soft date (Bent Aisha variety). Egyptian Journal of Agri.
Research, 76 (1): 247-259.
[9] Hussan, I.M. (1989). Freeze-thaw biochemical changes in three Egyptian date
varieties. Annals of Agricultural Science (Cairo), 34(1): 205-222.
[10] Jellinek, G. (1985). Sensory Evaluation. VCH Publishers, New York, pp. 275-285.
[11] Manan, Fazal (1993). Compositional changes of date Dhakki variety at various
stages of development grown in D.I. Khan, N.W.F.P. Pakistan. J. Science &
Technology, Uni. Peshawar, Pakistan, 17 (1-2), 37-40.
[12] MSTAT-C. (1987). Russsel D. Fareed,
Director, Crop and Soil Science
Department, Michigan State University, Version 2.10.
[13] Saleem, S.A., Baloch, A.K., Baloch, M.K., Baloch, W.A. & Ghaffoor, A. (2005).
Accelerated ripening of Dhakki dates by artificial means: Ripening by acetic acid
& sodium chloride. Journal of Food Engineering 70 (1) 61-66.
801
[14] Saleem, S.A., Baloch, M.K., Baloch, W.A. & Baloch, A.K. (2002). Effect of
ripening by microwave radiation on quality of Dhakki dates. Pakistan Journal of
Biological Sciences, 2 (4): 238-242.
[15] Sawaya, W.N., Khatohadourian, H.A., Khalil, J.K., Safi, W.M. & Al-Shalhat, A.
(1982). Growth and compositional changes during the various development
stages of some Saudi Arabian date cultivars. J. Food Science, 47, 1489 -1492.
802
Table 1. Hardness index (mm Hg cm-2) of Dhakki Doka during 12-month
storage at reduced temperatures.
Incubation
Storage period (month)
Temperature (°C)
0
10
300-295
295-260
270-190
265-100
240-40
1
1
1
1
0
-15
3
9
12
(298A)
(278A)
(230B)
(183C)
(140D)1
300-295
70-50
30-20
-
-
1
1
1
-
-
(298A)
(60E)
(25F)
300-295
20-10
20-10
1
1
1
(298A)
1.
6
(15F)
(15F)
Mean values bearing different letters (A – F) in each column differ significantly (LSD, P ≤ 0.05).
Table 2. Appearance of mold growth on Dhakki Doka stored for
12 months at reduced temperatures.
Storage period (month)
0
3
6
9
12
10
ND
ND
SD
FD
F
0
ND
ND
ND
ND
ND
-15
ND
ND
ND
ND
ND
ND (not detectable), SD (slightly detectable), FD (fermentation detectable), F (fermented).
Table 3.Visual appearance of Dhakki Doka during 9-month of storage at reduced temperatures.
Incubation
Storage period (months)
Temp
0
3
6
9
10°C
Translucent
yellowish,
hard
Dull yellowish,
slightly wrinkled,
firmness variable
with patches
Dull yellowish,
slightly wrinkled,
firmness variable
with patches,
mushy and slimy
Dull, fairly
wrinkled, firmness
variable, microbe
invaded
Translucent
yellowish,
hard
Yellowish Dong-like,
slightly soft, skin
slightly spotted,
sweet in taste
Yellowish Donglike, soft, skin
slightly spotted,
Sweet in taste
Yellowish Donglike, soft, skin
slightly spotted,
sweet in taste
Golden amber, soft
Dong, very
attractive, bright,
sweet & juicy
Golden amber, soft
Dong, very
attractive, bright,
sweet & juicy
Golden amber,
softer Dong, very
attractive, bright,
sweet & juicy
0°C
-15°C
Translucent
yellowish,
hard
803
Table 4. Effect of freezing of Doka and pot. alkali treatment on period of
curing/drying, yield and quality of Dhakki date.
Samples
period of
Curing/
drying
Ripening/
Yield (%)
Acceptability
(score)
Remarks
Doka 1
7-8 days
65
6.2
Variable quality, less attractive
Doka
80 hrs.
90
8.9
Attractive, sweeter taste, loose skin,
patchy
Doka3
6 days
80
9.0
Acceptable, but variable in quality
Doka
72 hrs.
92
9.2
Attractive, skin slightly chipped
Doka5
72 hrs.
95
8.5
Attractive with luster, skin balloon
like and patchy
Doka6
72 hrs.
96
8.9
Very attractive, bright and skin
mostly intact
2
4
Doka freshly harvested, served as a control.
Doka freshly harvested and remained frozen overnight.
2.
Doka left over for 3 months at 10 °C.
3.
Doka left over for 3 months at 10 °C and remained frozen overnight.
4.
Doka taken out after 6 month’s storage at –15 °C.
Doka taken out after 6 month’s storage at –15 °C and treated with 0.50g/100ml potassium hydroxide.
*Mean of three readings, ranking value ranges from 1 – 10, 10 being highly acceptable.
1.
5.
Table 5. Overall effect of pot alkali on acceptability of Dhakki dates with 25% moisture.
Pot. alkali
(%)
After curing/ drying
After 8-months storage at 40°C
Score*
Remarks
Score*
Remarks
F + 0.0
8.6
Good taste, loosen skin
7.1
Good taste, skin slightly broken
F + 0.1
8.7
Good taste, loosen skin
7.3
Good taste, skin slightly broken
F + 0.25
9.8
Good taste, very bright
amber color, skin intact
8.2
Good taste, bright amber color,
skin mostly intact
F + 0.35
9.0
Good taste, amber color, skin
intact
7.4
Agreeable taste, brown
coloration, skin intact
F + 0.5
8.9
Acceptable taste, slightly
brown color
6.0
Taste like burnt sugar, brown
color
F + 1.0
7.5
Dark brown
3.2
Taste like burnt sugar, Dark in
color
F = Sample left frozen for 6 months at –15°C.
*Ranking value ranges from 1 – 10, 10 being the most acceptable.
804
Doka (Khalaal)
Keeping of the Doka frozen until required
for processing.
Freshly ripened dates (Tamar or
KhajoorͿ͘
Dong (Rutab) on thawing.
Application of Potassium Hydroxide prior
to curing/ drying.
Fig. 1 Proposed scheme to streamline production of Dhakki
dates and to make fresh dates available round the year.
Cured date biomass with 25
% moisture contents.
805
Final Dhakki Dates
Fig. 2. Freshly harvested Dhakki Doka prior to storage for 10, 0 and –15°C.
806
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OP 41
Quality of steamed and microwaved dates
Isameldin B. Hashim, Alia Akram and Hanan S. Afifi
Department of Food Science, Faculty of Food and Agriculture,
United Arab Emirates University, P. O. Box 17555 Al Ain, UAE
ihashim@uaeu.ac.ae
Abstract
Safety and quality of dates is one of the concerns of the dates' processors. During
post-harvest and transportation dates are infested with insects. Traditionally dates are
fumigated with methyl bromide prior storage but unfortunately the use of this fumigant
will be completely banned in 2015 due to its adverse effects on the ozone layer.
Effective alternatives are conventional heating, freezing and γ-rays irradiation.
Microwave is an efficient fast heating method but its main disadvantage is the uneven
heating which causes caramelization of date sugars. A combination of microwave
heating and moist hot air has a potential to provide uniform heating to prevent date
quality degradation. The aim of this study was to investigate the effect of steaming
level (low and high) and microwaving power (50, 70 and 100%) and exposure time on
temperature profiles and quality (moisture, water activity, color and texture) of dates.
Dates' temperature increased with increasing steaming level and microwaving
power. Steaming increased the dates' moisture content while microwaving decreased it
significantly. Steaming the dates then microwaving at 50% power had slight effect on
moisture content without affecting the water activity, color and texture. Microwave
heating efficiency improved with steaming without affecting moisture content, color
and texture of dates significantly.
Introduction
Date is a major agricultural commodity of the United Arab Emirates (UAE). During
post-harvest, storage, and transportation, ripe dates fruits, especially those detached
from strands, are infested by all life stages of insects (adults, larvae, and eggs), e.g.
Apomyelois
ceratoniae,
Plodia
interpunctella,
Oryzaephilus
surinamensis,
Oryzaephilus mercator, Tribolium con fusum, Carpophilus hemipterus, C. multilatus,
809
C. dimidiatus, Urophorus humeralis, Cryptolestes ferrugineus, Heptoncus luteolus, and
Cadra spp. The presence of insects in dates is a major quality problem and insect
infestation of >6% of dates is a reason for rejection as human food according to Codex
Alimentarus Standards (1). Currently, most date processing companies are using
fumigation with methyl bromide (MB) to disinfest dates. MB is very fast and effective
in killing all life stages of insects that may be present in dates without any adverse
effects on quality. However, methyl bromide is an environmental nuisance that
depletes the ozone layer and hence its use has been banned. Non Article 5 (developed)
countries have already phased-out methyl bromide in 2005 while Article 5
(developing) countries were required to complete its total phase-out by 2015.
However, some non-A5 countries are still using methyl bromide, e.g. in dates
disinfestation, under the “Critical use Exemptions” because of no technical or
economical alternatives after authorization by the Parties of the Montreal Protocol
(MBTOC). Traditionally date fruits have been fumigated with methyl bromide prior
storage but unfortunately the use of this fumigant will be completely banned in 2015
due to its adverse effects on the ozone layer. Currently, date fruits industries are
looking for alternatives to methyl bromide use. The alternative method should be
effective for pest elimination at all life stages, safe without affecting dates' quality,
economical and there are no regulatory restrictions preventing its use.
A number of other feasible alternatives for dates’ disinfestations with MB have been
suggested including treatments with heat, heat and carbon dioxide, phosphine (PH3),
sulfuryl fluoride, ethyl formate, modified atmosphere, and phosphine/CO2 mixture (2).
The effectiveness of a chosen method might be dependent on the characteristics of
dates, particularly its moisture content and sugar type. Disinfestations of fresh high
moisture dates (Deglet-Nour) without methyl bromide was conducted in Algeria and
Tunisia. The project compared 5 different treatments and studies their effects on the
disinfestations of the carob Moth (Apomyelois ceratonia , Lepidoptera) as well as their
effects on dates quality (color, smell, and taste). They concluded that the controlled
atmosphere (with 99% of nitrogen, 7 days, in 1 cubic-meter chamber) and fumigation
with phosphine with or without ammonia reduced the quality of the fruit. Post-harvest
heat treatments have been used to disinfest fresh and stored dates but their success
810
relies on the balance between the commodity tolerance and insect intolerance to heat
within the aim of complete killing of insects with minimum thermal impact on product
quality. An effective thermal treatment should provide required quarantine security
with minimum adverse effects on products quality (3). Stored grain pests are killed
within seconds when exposed to temperature above 65 °C (4), Mediterranean fruit fly
on citrus fruits were eliminated at 44 °C for 30 min without impairing fruit quality (5,
6). Experiments in our laboratory showed that conventional heating (55 °C for 20 min)
and freezing (-25 °C for 55 min) were effective alternatives for insect disinfestations
without adverse effects on fruit quality (7).
Microwaves are electromagnetic radiation with varying frequencies (MHz).
Microwave heating is based on the transformation of alternating electromagnetic field
energy into thermal energy by affecting polar molecules of a material. Microwave
ovens are very energy efficient providing the major advantage of fast rate of heating
(8). Microwave treatment can provide a continuous process to allow large quantities of
products to be processed in a short period of time. Microwave treatment is considered
a safe and competitive alternative method to fumigation, and can avoid problems of
food safety and environment pollution (9). Various studies have been reported to
control insect infestation by microwave power. Rice weevil (Stiphilus oryzae L.) was
heated to 55 °C using an industrial continuous microwave oven to obtain 100%
mortality (10). It was shown that eggs and pupa of maggots were destroyed in cabbage
by microwave heating for 10 s by an oven providing 3000 W (11). Microwave heating
was also used to disinfest cherries using a pilot scale oven operated at 5000 W (12).
Microwave-vapor heat treatment was shown to eliminate oriental fruit fly, Bactrocera
dorsalis in mangoes. The acceptable range was temperature ranging from 46 to 55 ˚C
with time ranging from 2 to 20 min (13). An electron-beam and a household
microwave were very effective showing 100% insect mortality rate (13). An industrial
microwave was effective for date's disinfestations but the main disadvantage of
conventional microwave is the uneven heating which causes caramelization (Sriraman
S., Al Foah, 2011, Personal communication).
The objective of this phase of the project was to investigate the effect of
microwave-moist heating (using a combination of microwave and moist hot air) on
811
date quality. This heating combination have potential to heat dates fast and provide
uniform heating to prevent degradation of date quality.
Materials and Methods
Khalas dates were obtained from a local date processing company (Al Foah, Al Ain,
UAE). Dates were stored at refrigerated temperature (5oC).
Treatments
A house hold microwave equipped with steamer (Sharp, Healslo, Sharp Company,
Japan) was used. Dates were steamed using low or high steaming option for (30, 60,
90, 120 sec.) then exposed to microwave heating using (50, 70 and 100% power) for
30, 45, 60, 75, 90 and 105 sec.). The experiment was replicated three times.
Temperature profile
Date's temperature was determined with a thermocouple connected to a data logger
(Model sq 2040 Grant).
Moisture content
The moisture content of dates was determined using an oven at 105oC (AOAC, 2000).
Water activity
Water activity of dates was determined using a water activity meter (DECAGON
PAWKIT, USA).
Color
The color was measured with a colorimeter (Colorfex, HunterLab, Reston, VA). A
white tile was used for standardization. Color properties of date fruits were evaluated
by measuring L, a and b values. L value is a measure of lightness and darkness (100
means perfect white and 0 means perfect black). A positive a value is a measure of
redness and a negative a value indicates green color. A positive b value means yellow
color while negative b value is an indication of blueness.
Texture
Texture profiles of dates were measured using a Texture Analyzer (Model QTS 25,
Brookfield Instruments, Harlow, UK). Texture profile analysis with two compression
cycles was applied and the texture parameters were calculated using the TexturePro
software. The hardness value is the peak force of the first compression of the product.
812
Statistical analysis
Data was analyzed with SPSS statistical software version 18. Analysis of variance
(ANOV) was used to determine if a statistical difference exists (p 0.05) and the least
significant difference (LSD) was used for means comparison.
Results and Discussion
Effects of steaming and microwaving on temperature profiles
Temperature profiles of steamed and microwaved dates are shown on figure 1.
Dates' temperature increased slightly with steaming level and steaming time. Dates
steamed with high steaming had slightly higher temperature than dates steamed with
low steaming. Microwave power level had significant effect on dates' temperature,
increased with increasing the power level. As the exposure time increased dates'
temperature increased at all the power levels. The combination of steaming and
microwaving increased dates' temperature significantly. Results of steaming and
microwaving at higher level (70 and 100%) had significant effect on dates' quality (not
reported). Only the results of steaming (at low or high level) and microwaving at 50%
power level are reported. Dates' temperature increased significantly (80-100 oC)
compared to only microwaved dates (64oC). This indicated that steaming improved
microwave heating efficiency.
Effects of steaming and microwaving on moisture content
Moisture content of steamed and microwaved dates is shown on figure 2. Moisture
content increased with steaming. Low and high steaming had similar effect, date's
moisture increased with increasing steaming time. On the other hand moisture content
decreased with microwaving at all microwave power levels. As expected steaming
increased moisture content while microwaving decreased it. The combination of
steaming (at low or high level) and microwaving at 50% power level had no
significant effect on dates' moisture. The dates maintained its initial moisture level.
This showed that steaming compensated the water loss caused by microwaving.
Effects of steaming and microwaving on water activity
Water activity of steamed and microwaved dates is shown on figure 3. Low
steaming had no effect on dates' water activity while high steaming increased water
activity significantly. The combination of low steaming and microwaving at 50%
813
power level had slight effect on water activity. While, the combination of high
steaming and microwaving at 50% power level reduced dates' water activity to the
original level before steaming.
Effects of steaming and microwaving on color
The color of steamed and microwaved dates is shown on Tables 1 and 2. The
combination of low or high steaming and microwaving at 50% power level had no
effect on dates' color. Dates had similar degree of lightness as control dates. While,
dates steamed (low or high) for 30 and 60 sec then microwaved at 70 or 100% power
level had significant darker color (lower L values) compared to control. Exposing
dates to steam for longer time (90 and 120 sec) preserved the color and produced dates
with lighter color with L values similar to the control dates.
Effects of steaming and microwaving on texture profiles
The texture profiles of steamed and microwaved dates are shown on Tables 3 and 4.
Steaming and microwaving had no effect on dates' texture. Control and steamed and
microwaved dates had similar hardness values.Steaming improved heating efficiency
of the microwave, maintained dates quality by preventing moisture loss and
developing darker color.
814
References
1. Codex Alimentarius Current Official Standards, Codex Standard for Date, CODEX
STAN
143-1985,
http://www.codexalimentarius.net/web/standard_list.jsp
Accessed 30 April, 2008.
2.
El-Mohandes, M.A. 2010. Methyl bromide alternatives for dates disinfestation,
Acta Hort. 882: 555-562, http://www.actahort.org/books/882/882_62.htm
3. Tang J., Ikediala, J.N, Wang, S., Hansen, R.P., High temperature-short-time thermal
quarantine methods, Postharvest Biology and Technology, 2000; 21: 129-145.
4. Qaisarani , R. Heat: a non-chemical alternative to control of stored grain insects,
CSIRO stored Grain Research Laboratory, August 2001.
5. Lurie S., Jemric, T., Weksler A., Akiva, R., Gazi Y., Heat treatment of
‘oroblanco’ citrus fruit to control insect infestation, Postharvest Biology and
Technology, 2004; 34: 321-329.
6. Michel, L., Ball S., Forbes, S. and Woolf A., Hot water treatment for insect
disinfestation and reduction of chilling injury of Fuyu persimmon, Postharvest
Biology and Technology, 1997; 10: 81-87.
7. Al Dhahery, Shamsa. Methyl bromide alternatives for post-harvest disinfestation
of dates. M.Sc. thesis in Environmental Science, UAE University, 2008.
8. Fellows, P. J., Food Processing Technology: Principles and Practice, Elsevier
Applied Science, Boca Raton, 2000.
9. Vadivambal R., and Jayas, D.S., Wheat disinfestation using microwave energy,
Journal of Stored Products Research, Article in Press.
10. Zhao, S., Xiong, S., Qui C., and Xu, Y., Effect of microwaves on rice quality,
Journal of Stored Products Research, 2007.
11. Biron, D., Vincent C., Giroux, M. and Marie, A., Lethal effects of microwave
exposures on eggs and pupae of the cabbage maggot and cabbage plants, Journal
of Microwave power & Electromagnetic Energy, 1996; 31: 228-237.
12. Ikediala J., Tang, J., Neven, L. G. and Drake, A., Quarantine treatment of cherries
using 915 MHz microwaves: temperature mapping, codling moth mortality and
fruit quality, Postharvest Biology and Technology, 1999; 16: 127-137.
815
13. Varith, J., Sirikajornjaru, W., Kiatsiriroat, T. Microwave-vapor heat disinfestation
on oriental fruit fly eggs in mangoes, Journal of Food Processing & Preservation,
Volume 2007; 31: 253-269.
14. Al-Abid, M., U. Gohs, M. Al-Farsi, M. Al-Amri, F. Al-Rawahi. 2010.
Disinfestation of dates using electron beams in comparison with other treatments.
Proceeding of the Fourth International Date Palm Conference. Acta Horticulturae
882: 569-576.
816
Table 1. The effects of low steaming and microwaving on dates' color [lightness (L)]
Steaming
30 sec.
60 sec.
90 sec.
120 sec.
Microwa
ving
30 sec.
45 sec.
30 sec.
45 sec.
30 sec.
45 sec.
30 sec.
45 sec.
MW 50
36.8±1
.1a
36.3±0
.3a
36.5±0
.7a
36.0±0
.6a
36.2±1
.1a
35.4±0
.5a
35.9±2
.5a
35.3±1
.6a
MW 70
31.7±1
.7b
32.0±0
.1b
32.1±2
.6b
33.5±1
.2b
34.0±2
.7a
34.4±1
.7a
35.0±1
.9a
35.7±0
.4a
MW 100
31.3±1
.2b
29.4±0
.6b
29.4±3
.2b
29.6±1
.3b
32.4±2
.4b
33.2±0
.2b
34.8±1
.4a
35.3±1
.3a
Control
36.4±0
.2a
36.4±0
.2a
36.4±0
.2a
36.4±0
.2a
36.4±0
.2a
36.4±0
.2a
36.4±0
.2a
36.4±0
.2a
Table 2. The effects of high steaming and microwaving on dates' color [lightness (L)]
Steaming
Microwa
30 sec.
60 sec.
90 sec.
120 sec.
30 sec.
45 sec.
30 sec.
45 sec.
30 sec.
45 sec.
30 sec.
45 sec.
35.7±1
35.6±1
35.9±1
35.1±1
36.7±0
36.1±1
36.4±1
35.8±1
.7a
.0a
.6a
.1a
.8a
.1a
.7a
.5a
33.8±1
33.9±1
33.6±1
33.9±1
36.1±1
35.9±0
35.7±1
34.6±0
.6b
.2b
.5b
.6a
.2a
.6a
.4a
.5a
32.5±3
30.7±0
33.2±2
29.0±1
35.8±1
35.4±1
34.9±1
35.6±1
.0b
.5b
.4b
.5b
.4a
.5a
.6a
.9a
36.4±0
36.4±0
36.4±0
36.4±0
36.4±0
36.4±0
36.4±0
36.4±0
.2a
.2a
.2a
.2a
.2a
.2a
.2a
.2a
ving
MW 50
MW 70
MW 100
Control
817
Table 3. The effects of low steaming and microwaving on texture (hardness) of dates
Steamin
30 sec.
60 sec.
90 sec.
120 sec.
g
Microwa
30 sec.
45 sec.
30 sec.
45 sec.
30 sec.
45 sec.
30 sec.
45 sec.
419.8±
421.5±
419.1±
419.7±
418.8±
419.5±
417.6±
418.5±
1.9a
2.0a
0.6a
0.1a
1.1a
0.4a
1.3a
0.7a
421.5±
422.5±
419.9±
420.7±
419.2±
421.5±
418.7±
419.5±
1.1a
0.2a
0.6a
0.5a
1.3a
0.5a
1.5a
0.4a
423.7±
424.7±
421.4±
422.5±
420.5±
421.5±
419.6±
420.5±
1.2a
0.1a
1.1a
0.3
1.1a
0.6a
0.9a
0.2a
441.3±
441.3±
441.3±
441.3±
441.3±
441.3±
441.3±
441.3±
1.5a
1.5a
1.5a
1.5a
1.5a
1.5a
1.5a
1.5a
ving
MW 50
MW 70
MW 100
Control
Table 4. The effects of high steaming and microwaving on texture (hardness) of dates
Steaming
Microwa
30 sec.
60 sec.
90 sec.
120 sec.
30 sec.
45 sec.
30 sec.
45 sec.
30 sec.
45 sec.
30 sec.
45 sec.
417.9±
418.5±
416.7±
417.7±
416.5±
417.5±
416.1±
417.4±
0.6a
0.1a
0.4a
0.1a
1.7a
0.9a
1.4a
0.1a
420.0±
420.4±
419.0±
419.5±
417.9±
418.5±
417.9±
418.8±
4.0a
0.1a
0.6a
0.4a
0.6a
0.1a
1.1a
0.7a
422.3±
422.5±
421.0±
421.6±
420.0±
420.5±
419.3±
419.8±
0.6a
0.1a
0.6a
0.7
0.3a
0.6a
0.5a
0.8a
441.3±
441.3±
441.3±
441.3±
441.3±
441.3±
441.3±
441.3±
1.5a
1.5a
1.5a
1.5a
1.5a
1.5a
1.5a
1.5a
ving
MW 50
MW 70
MW 100
Control
818
120
dĞ ŵ Ɖ Ğ ƌ Ă ƚ Ƶ ƌ Ğ ; Ϳ
100
Steam Low
80
Microwave
60
SL +M 30 sec
40
SL +M 60 sec
20
SL +M 90 sec
SL +M 120 sec
0
0
30
45
60
75
90
105
dŝŵĞ;^ĞĐͿ
140
dĞ ŵ Ɖ Ğ ƌ Ăƚ Ƶ ƌ Ğ ;Σ Ϳ
120
100
Steam High
Microwave
80
SH +M 30 sec
60
SH +M 60 sec
40
SH +M 90 sec
20
SH +M 120 sec
0
0
30
45
60
75
90
105
dŝŵĞ;^ĞĐͿ
Figure 1. Effects of steaming and microwaving on temperature profiles
819
D Ž ŝƐƚ Ƶ ƌ Ğ Ž Ŷ ƚ Ğ Ŷ ƚ ;Őŵ ͬ ϭ Ϭ Ϭ Őŵ Ϳ
18
16
14
Steam Low
12
Microwave
10
SL +M 30 sec
8
SL +M 60 sec
6
SL +M 90 sec
4
SL +M 120 sec
2
0
0
30
45
60
75
90
105
dŝŵĞ;^ĞĐͿ
18
D Ž ŝƐƚ Ƶ ƌ Ğ Ž Ŷ ƚ Ğ Ŷ ƚ ;Ő ŵ ͬ ϭ Ϭ Ϭ Ő ŵ Ϳ
16
14
Steam High
12
Microwave
10
SH +M 30 sec
8
SH +M 60 sec
6
4
SH +M 90 sec
2
SH +M 120 sec
0
0
30
45
60
75
90
105
dŝŵĞ;ƐĞĐͿ
Figure 2.The effects of steaming and microwaving on moisture
content of dates and microwaving on water activity of dates
820
0.49
0.48
t Ăƚ Ğ ƌ ĂĐƚ ŝǀŝƚ LJ
0.47
Steam Low
0.46
Microwave
0.45
SL +M 30 sec
0.44
SL +M 60 sec
0.43
SL +M 90 sec
0.42
SL +M 120 sec
0.41
0
30
45
60
75
90
105
dŝŵĞ;^ĞĐͿ
0.54
0.53
0.52
tĂƚĞƌĂĐƚŝǀŝƚLJ
0.51
0.5
Steam High
0.49
Microwave
0.48
SH +M 30 sec
0.47
SH +M 60 sec
0.46
SH +M 90 sec
0.45
SH +M 120 sec
0.44
0.43
0.42
0.41
0
30
45
60
75
90
105
dŝŵĞ;^ĞĐͿ
Figure 3. The effects of steaming and microwaving on water activity of dates
821
822
OP 42
Effect of X-Ray Irradiation on foodborne pathogens and Sensory
Properties of Dates
Aleid1 Salah, Kirk Dolan2,3, Sanghyup Jeong2 , Muhammad Siddiq3 , Bradley
Marks2, Karim Maredia4
1
Date Palm Research Center of Excellence, King Faisal University, Alhasa, Saudi Arabia.
2
Department of Biosystems and Agricultural Engineering, 3Department of Food
Science and Human Nutrition, 4Department of Entomology, Michigan State
University. East Lansing, Michigan 48824, USA, seid@kfu.edu.sa
Abstract
Food irradiation using X-rays has been identified as an effective means for
eliminating foodborne pathogens and shelf-life extension. However, further
development and commercialization for such system requires quantification of the
efficacy of low-energy X-ray for microorganisms of interest. The objectives of this
project are: 1) to quantify the efficacy of low-energy X-ray for general spoilage
microorganisms on the surface of dates in terms of total plate counting; 2) to measure
microbial efficacy (D10-value: dose required to reduce 90% of target microorganism) of
low-energy X-ray for Eschericia coli O157:H7 inoculated on the surface of dates or in
the puree of dates; and 3) to conduct quality evaluation for irradiated dates using
chemical, physical, and sensory test at the Date Palm Research Center of Excellence,
Saudi Arabia). To achieve 5-log reduction of general spoilage microorganisms (Aerobic
Bacteria), a minimum of 3.5 kGy will be necessary as a conservative measure. A5-log
reduction for E. coli O157:H7 can be possible with 1.85 kGy. No deteriorating effect on
the tested quality attributes (physical, chemical and sensory properties) have been
observed for irradiated dates at 3, 5 and 7 Kgy . It could be concluded that X-Ray
irradiation of dates, up to 7KGy could be regarded as a good practice not negatively
affecting the physical, chemical, textural and sensory properties of dates.
Keywords: Dates, X-ray, irradiation, foodborne pathogens, Sensory Properties.
Introduction
Food irradiation using X-rays has been identified as an effective means for
eliminating foodborne pathogens and shelf-life extension. However, further
823
development and commercialization for such system requires quantification of the
efficacy of low-energy X-ray for microorganisms of interest. The objectives of this
project are: 1) to quantify the efficacy of low-energy X-ray for general spoilage
microorganisms on the surface of dates in terms of total plate counting; 2) to measure
microbial efficacy (D10-value: dose required to reduce 90% of target microorganism)
of low-energy X-ray for Eschericia coli O157:H7 inoculated on the surface of dates
or in the puree of dates; and 3) to conduct quality evaluation for irradiated dates using
chemical, physical, and sensory test at the Date Palm Research Center of Excellence,
Saudi Arabia).
For the pathogen challenge test (obj. 2), a cocktail of six-serovar Escherichia coli
O157:H7 was used to inoculate the date sample. The stock cultures of each serovar
were maintained separately at -80ºC in TSB-YE medium containing 20% glycerol.
Before use, each serovar was cultured separately in TSB-YE using a minimum of two
consecutive 24 h transfers to obtain cells in late log phase. Immediately before
inoculation, equal volumes of each culture were combined, centrifuged (6,000 rpm, 15
min at 4ºC) and re-suspended in sterile peptone water (2.5 mL). Before inoculation,
200 g of dates and 2.5 mL inoculum were ground/mixed three times using a stand
mixer (Kitchen Aid, St. Joseph, MI) with grinder attachment. About 3 g of inoculated
sample was placed in a plastic bag and flattened to 1 mm thickness sample with 3M
Petrifilm™ spreader to maximize X-ray dose uniformity. The samples were hold in a
refrigerator until irradiation (~20 h).
The inoculated samples were irradiated in a pilot scale low-energy X-ray irradiator
(Rainbow™ II, Rayfresh Foods Inc., Ann Arbor, MI), which currently is housed in the
biosafety level-2 pilot plant at Michigan State University. The irradiator consists of an
industrial grade X-ray tube (modified OEG-75, Varian Medical System, Salt Lake
City, UT), high voltage source, and cooling unit. The X-ray tube operates at a
maximum constant potential of 70 kV and a filament current of 57 mA, which gives 4
kW of maximum allowable input power. Five dose levels of X-ray at maximum power
(70 kV/57 mA) were applied to achieve 1 to 5 log reductions with all experiments
conducted in triplicate. For pureed sample, each sample was irradiated on both sides
by flipping the sample halfway through treatment, to achieve a uniform dose. In
824
addition, whole packages of dates were irradiated (3, 5, and 7 kGy) and shipped to
DPRC for further quality assessment. After irradiation, samples were homogenized
with 0.1% peptone water in a stomacher for 180 s. Appropriate dilutions were plated in
duplicate onto E. coli/Coliform Count Plate Petrifilms™ (3M, St. Paul, MN) for E.
coli O157:H7 and Aerobic Count Plate Petrifilms™ for total plate count of general
spoilage microorganisms. The Petrifilm™ plates were incubated at 35 ± 2ºC for 48 h.
As is standard for analysis of pasteurization processes, the outcomes were first
converted to log reductions, which were calculated by subtracting the log of the
survivor counts for each individual observation from the mean log counts on
inoculated, untreated samples. The D10-values (i.e., the inverse of the slope between
the applied X-ray dose and log reduction) were then determined by linear regression.
For the irradiated dates for quality assessment, Dates were also irradiated in vacuum
package with three different doses (3, 5, and 7 kGy @ surface) of X-ray and sent them
to the Date Palm Research center of Excellence at King Faisal University for
physical/chemical/sensory quality evaluation.
Initial spoilage bacterial load was 2.13 log10 CFU/g, which was too low
concentration to conduct inactivation test. Even if so, an inactivation experiment was
conducted to estimate the efficacy of low-energy X-ray on the general spoilage
microorganisms.
.ر ا
! ار
shows the data and its linear regression
results. Microbial efficacy, in terms of D10-value (dose required to achieve 1 log
reduction), was 0.064 ± 0.057 kGy (95% C.I.) based on the surface dose of dates.
However, a higher D10-value (0.70 ± 0.63 kGy) may be possible, if the dose at the
middle of the sample is used to estimate the microbial efficacy. Therefore, to achieve
5-log reduction of general spoilage microorganisms, a minimum of 3.5 kGy will be
necessary as a conservative measure.
Dates were pitted/flattened and then spot inoculated with 100 µL of E. coli
O157:H7 cocktail on the surface of the sample. However, enumeration results were not
usable for analysis because the spot inoculum was not exclusively inoculated on the
surface of the samples but it was absorbed into the samples which create unreasonable
inactivation results. Therefore, mixing inoculum with pureed dates was considered as
825
an alternative and also conservative method which can represent contamination in/on
dates. Initial concentration was 7.42 log10 CFU/g.
shows a linear regression analysis in which the reciprocal of the slope of the regression
line represents D10-value (0.37 ± 0.037 kGy) and the 95% C.I. and P.I. are also
reasonable. Based on this measurement, 5-log reduction can be possible with 1.85 kGy.
The overall sensory acceptability of X-Ray irradiated dates was slightly decreased
at 0.5 and 0.6 KGy (Table 1). These results were in agreements with Al-Khahtani et al
(1998); El-Sayed and Baeshin (1983); and Grecz et al (1986). This was due to low
protein content and low water activity of Khalas dates (Dowson and Aten, 1962;
Hasegawa et al 1969).
No deteriorating effect on the tested quality attributes (physical, chemical and
sensory properties) have been observed for irradiated dates at 3, 5 and 7 Kgy . It
could be concluded that X-Ray irradiation of dates, up to 7 KGy could be regarded as
a good practice not negatively affecting the physical, chemical, textural and sensory
properties of dates.
Acknowledgements
This work was financially supported by the Date Palm Research center of
Excellence, King Faisal University, Saudi Arabia.
826
References
[1] Al-Khahtani HA, Abu-Tarboush HM, Aldryhim YN, Ahmad MA, Bajaber AS, ElShami E, El-Mojaddidi MA. 1998. Irradiation of dates: Insect Disinfestations,
Microbial and
Chemical Assessments, and Use of Thermo-luminescence
Technique. The first International Conference on date Palms. Al-Ain, United Arab
Emirates. 126-148.
[2] Dowson VHW, Aten A. 1962. Dates, handling, processing and packing. FAO
Agricultural Development paper No. 72, FAO, Rome.
[3] El-Sayed S , Baeshin NA. 1983. Feasibility of disinfestations of date fruits
produced in Saudi Arabia by gamma irradiation. Proceedings, First Symposium on
Date Palm. Date Palm Research Center. King Faisal University. AlHassa, Saudi
Arabia. Pp. 342-350.
[4] Grecz N, Al-Harithy R, El-Mojaddidi MA, Rahma S. 1986. Radiation inactivation
of microorganisms on dates from Riyadh and AlHassa areas. Second Symposium
on Date Palm. Date palm Research Center, King Faisal University. Alhassa, Saudi
Arabia. pp.155-164.
[5] Hasegawa S, Maier VP, Kaszyoki HP, Crawford JK. 1969. Polygalacturonase
content of dates and its relation to maturity and softness. J Food Sci 34:527-529.
827
Table 1. Effect of X-Ray irradiation on sensory quality of dates.
X-Ray Dose
0 kGy
0.3 kGy
0.5 kGy
0.7 kGy
Taste
5.4
6.6
6.6
6.4
Odor
7.4
7.2
6.4
7.6
Texture
5.8
7.6
7.4
7.2
Appearance
5.8
5.8
6.2
7.4
Color
7.8
6.4
7.6
6.8
7.4
7.0
6.4
6.4
Overall
Acceptability
Notes:
• Sensory evaluation was carried out using semi-trained panelists with hedonic
scale of 9-1 ranging from "like extremely (9)" to "dislike extremely (1 )".
• A panel of 5 semi-trained assessors, recruited from DPRC, professors, and employees.
• To evaluate external appearance, fruits was presented in polystyrene trays,
covered with transparent film.
828
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829
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Date Pam and Dates processing
831
832
OP 43
Enhancement of Date Palm as a Source of Multiple Products:
Examples from Other Industrialized Palms
Dennis V. Johnson
3726 Middlebrook Ave
Cincinnati OH 45208 USA
Email: djohn37@aol.com
Abstract
Multipurpose palm species development would benefit from a broader consideration
of the varied economic products palms can potentially provide. All economic palm
species have a primary product which accounts for their exploitation or domestication
and industrialization.
A nearly-exclusive emphasis on the primary product has often
obscured the potential value of secondary products. Responsible disposal of residues
from harvesting and processing of primary palm products often has the potential of
being transformed from a disposal expense and potential source of pollutants into
secondary products of value.
Examples from other palms which may have
applicability to date palm production include: 1) In the oil palm industry, empty palm
oil fruit bunches are used as fuel to generate electrical energy and yields a fine ash
with industrial potential. 2) In coconut plantation operations, pruned leaves can be
gathered, dried and burned to generate energy and to produce a suitable fertilizer for
the plantation. 3) Replacement of ageing plantation trees provides an abundance of
woody material requiring disposal, presenting a periodic opportunity for innovative
secondary product harvest. Technical research on the utilization of palm by-products
is typically focused on an individual species, but the results often have broad potential
adoption for other economic palms.
Introduction
The palm family (Arecaceae) is comprised of 183 genera and over 2,400 species,
and has a worldwide distribution between 44° north and south latitudes [5, 6]. Five
major palm species are domesticated fully and are grown as economic species: areca
or betel nut palm (Areca catechu), coconut palm (Cocos nucifera), date palm (Phoenix
833
dactylifera), oil palm (Elaeis guineensis) and peach palm (Bactris gasipaes). The
taxonomic relationship among the five species, which represent three of the five palm
subfamilies, is presented in Table 1.
Although the five palm species are classified into different sub tribes, in terms of
the variety of products they yield are comparable.
The five share certain
characteristics, for they are all pinnate-leafed, large, erect, slender to thick palms. Two
produce basal suckers (date and peach palms) which can be used for propagation. The
date palm’s oasis habitat differs from the other four species which are tropical wet
climate palms. All five were domesticated primarily for their fruits. Within the
subsistence economies where domestication took place, every part of the five palm
trees would have been evaluated carefully for any possible utility or product.
The purpose of this paper is to outline the multipurpose character of the areca,
coconut, oil and peach palms, and to use the information as a possible stimulus to
broader consideration of the date palm, to enhance current secondary products and to
promote potential new uses.
Shared fruit characteristics
Fruit of palms in the Arecoideae subfamily (areca, coconut, peach, oil palm) are
similar.
Each has a well-developed exocarp, mesocarp, endocarp (shell) and
endosperm (kernel).
The terms nut and seed as applied to palms often create
confusion. In the coconut, for example, both nut and seed may refer to either the
entire fruit or to the fruit after the husk has been removed. Generally, use of more
precise botanical terms is preferable.
The arecanut mesocarp is a source of alkaloids. Coconut has a mesocarp fiber (coir),
activated carbon and other products from the endocarp, and oil and other food products
from the endosperm (meat). Peach palm has an oily starchy mesocarp which can be
cooked and eaten or processed into starch. Peach palm also furnishes palm heart
(apical meristem) as a commercial green vegetable. Major products from the oil palm
derive from the mesocarp and endosperm, both yield excellent quality food oils.
Coconut and oil palms, once they reach sexual maturity, produce fruit continuously
over the year, but with month-to-month fluctuations. Spreading of the harvest of these
two tree crops over the entire calendar year is a decided economic advantage.
834
Unique among the five palms, the date palm has intangible value by creating shade
and microclimatic conditions suitable for the growth of other crop plants. Also, the
palm is used in oases as windbreaks and to stabilize earthen irrigation works.
Once established, palm plantations can provide a sustainable and reliable supply of
fruits and other products for decades.
Modern commercial palm plantation
development has focused almost exclusively on a single economic product; as a result,
insufficient attention has been given to secondary products from the fruits and other
parts of the palms.
Profiles of multipurpose palms
An effective approach in this article is to examine the areca, peach, oil and coconut
palms in terms of their harvest and processing practices, focusing on products.
Profiles of the palms are presented in Boxes 1-4, which summarize briefly the harvest
and processing of primary and secondary products; the boxes are in sequence from the
simplest to the most complex palm. The profiles serve as background for the sections
that follow.
Discussion
Because the harvest and most processing of primary and secondary palm products
occurs in two separate locations, field and factory, it is logical to discuss enhanced
utilization in two parts.
Harvest: As with every tree crop, field harvest consists of taking from the plant its
key economic product, commonly the fruit or fruit bunch, and transporting it to
another location for processing. The perennial plant remains in a living state for
subsequent growth and harvest, unless it must be destroyed in carrying out the harvest.
Field harvest may involve on-site primary processing to reduce bulk and weight, such
as removal of fruits from the bunches, leaving the empty bunches in the field. Areca,
peach and oil palm bunches are cut and transported away; coconuts are harvested
individually without bunch cutting.
Dates harvest involves either cutting of entire fruit bunches and removing the fruit
in the field or picking the fruits individually in the case of high-value elite cultivars.
In either instance, the empty date fruit bunches typically are left in the field. In
835
coconut harvest, if coir mesocarp fiber is not to be extracted, the fruits may be husked
just after harvest and the husks left in the field. If the fiber is to be part of the harvest,
the entire fruit is taken to the factory. As part of the fruit harvest of all five palms,
some green leaves may be pruned from the trees to afford easier access to the fruits or
fruit bunches.
Processing.
The processing of palm fruits varies from simple to complex, the
facilities also ranging from small household cottage industries to modern factories. In
all cases, processing generates residue or waste products. These may be empty fruit
bunches, shells, peelings, spent pulp or seeds.
Arecanut palm fruit processing is a relatively simple operation, consisting of
detaching fruits from the bunches and boiling them whole to reduce tannins. Next the
fruits are husked and the endosperm removed and cut up, dried, often mixed with a
pigment and other ingredients, and packaged for sale. There exists some variation in
processing procedures employed. The arecanut husk has utility in hardboard and
paper making, and as fuel.
Peach palm processing involves removing the fruits from the bunches and boiling
the fruit which is sold whole; fruits may also be canned. In these cases, the only
residue is the empty fruit bunch. Processing the peach palm mesocarp into flour
generates waste made up of fruit peelings and endosperm. In the processing of palm
hearts, the incoming palm heart lengths are peeled to remove inedible outer layers, cut
into sections, cooked and preserved. Residues from flour and palm heart processing
can serve as partial livestock rations. Empty fruit bunches are not utilized.
Oil palm fruit bunches arriving at the factory are steamed, the fruit removed and the
oil from the mesocarp and endocarp separately extracted.
The palm oils have
hundreds of food and industrial uses. Empty fruit bunches are employed as fuel and
the ash from it collected for industrial uses. Livestock can consume press cake as part
of their rations and other waste products are returned to the field as fertilizer.
Coconut processing may be simple, involving removal of the endosperm from the
split endocarps if the end-product is dried copra for oil extraction. This is the practice
when coconut production is insufficient to support a local processing facility.
Alternatively, the endosperm may be removed and immediately processed into various
836
fresh coconut products (e.g. shredded coconut, coconut milk) or oil. In some coconut
processing operations, husk fiber is also an important product. Husks must be retted in
water for a period of time to loosen the fiber, after which it is separated, dried and
spun into numerous products.
The coconut shell (endocarp) is a valuable by-product. Without any treatment, as a
direct fuel it has high caloric value, and can be made into excellent charcoal as well as
activated carbon. Splitting mature coconuts to extract the endosperm releases coconut
water, which is commonly allowed to drain away. However, the liquid contains some
carbohydrates and amino acids. If the coconut water is captured, it has use as a raw
material for fermentation processes, and can be added to cattle feed before
fermentation occurs.
Considering these four palm species together, they have the most in common in
terms of some residues being used as fuel, often mixed with other combustible
material, to generate electrical energy and steam.
Depending upon physical and
chemical characteristics, waste products of the four palms also serve as an additive in
animal rations, or are returned to the plantation fields as fertilizer.
Because
conveyances bringing fruits to the factory return empty, transporting the biomass back
to the field is efficient. Utilization of crop and processing waste products is important
because rather than creating environmental pollutants through their disposal, they can
become low-value products themselves.
Significant research has been carried out in recent years on secondary product use
of the four palms discussed in this paper, as well as on the date palm. However, there
has been inadequate evaluation of the results across major economic palm species.
Two selected examples stimulated by this study, applicable to date palm, can be cited.
One, empty fruit bunches of the date palm appear to be little used; yet, evidence from
the oil palm suggests that the ash derived from burning them has exceptional qualities
that has industrial applications. Two, the palm heart (apical meristem) of the date palm
is edible; yet this food item is scarcely mentioned in the literature. In the Philippines,
when coconut plantations are replaced, the palm hearts are extracted and processed
into a preserved product sold domestically and internationally.
837
Conclusion
The date palm has been a multipurpose species since it was first domesticated more
than five thousand years ago. In modern times, significant progress has been made in
the development of direct and derived date fruit products and the utilization of byproducts from packing and processing;
however, comparatively minor attention has
been given to date palm products other than the fruits.
By highlighting the
multipurpose character of four other major economic palm species, this paper attempts
to broaden the thinking about how other nonfruit products can be developed to
contribute to the total value of the date palm to farmers.
One key recommendation is that the excellent study Date Palm Products [1] be
updated in light of the numerous published studies over the past two decades. The
revision should also place greater emphasis on nonfruit products and include relevant
research on other palm species.
838
References
[1] Barreveld WH 1993. Date palm products. FAO Agricultural Services Bulletin
No. 101, Rome.
[2] Bavappa KVA, Nair MK, Kumar TP eds. 1982. The arecanut palm (Areca catechu
Linn.). Central Plantation Crops Research Institute, Kerala, India.
[3] Bhat KS, Nair CPR eds. 1985. Arecanut research and development. Central
Plantation Crops Research Institute, Kerala, India.
[4] Corley RHV, Tinker PB 2003. The oil palm. 4th edition. Blackwell, London.
[5] Dransfield J, Uhl NW, Asmussen CB et al. 2008. Genera palmarum: the evolution
and classification of palms. Kew Publishing, Royal Botanic Gardens, Kew, U.K.
[6] Govaerts R, Dransfield J 2005. World checklist of palms. Royal Botanic Gardens,
Kew, U.K. Updates: www.kew.org
[7] Grimwood BE 1975. Coconut palm products. FAO Agricultural Development
Paper No. 99, Rome.
[8] Killmann W, Chong WW, Shaari K bt 1996. Utilization of palm stems and leaves:
an annotated bibliography. Research Pamphlet No. 103, Forest Research Institute
Malaysia, Kuala Lumpur, Malaysia.
[9] Mora-Urpí J, Weber JC, Clement CR 1997. Peach palm. Bactris gasipaes Kunth.
Promoting the conservation and use of underutilized and neglected crops. No. 20.
Institute of Plant Genetics and Crop Plant Research, Rome, Italy.
[10] Mora-Urpí J, Gainza E J eds. 1999. Palmito de pejibaye (Bactris gasipaes Kunth):
su cultivo e industrialización. Editorial Universidad de Costa Rica.
[11] Ohler JG 1984. Coconut, tree of life. FAO Plant Production and Protection Paper
No. 57, Rome.
[12] Poh K Mm, Yusoff MNM, Choon KK, Nasir NM eds. 1994. Proceedings 3rd
national seminar on utilisation of oil palm tree and other palms. Forest Research
Institute Malaysia.
[13] Villachica L H 1996. Cultivo del pejuayo (Bactris gasipaes Kunth) para palmito
en la Amazonia. Tratado de Cooperación Amazónica, Lima, Peru.
839
Table 1. Taxonomic Relationship among the Five Major Economic Palm Species
Family
Subfamily
Tribe
Subtribe
Genus and
Species/Common Name
Arecaceae
Arecoideae
Areceae
Arecinae
Areca catechu
(areca palm)
Cocoseae
Attaleinae
Cocos nucifera
(coconut palm)
Bactrinidae
Bactris gasipaes
(peach palm)
ElaeIdinae
Elaeis guineensis
(oil palm)
Phoeniceae
Phoenix dactylifera
(date palm)
Coryphoideae
Cryosophileae
Box 1. Profile of areca palm (Areca catechu) and its products
Domesticated: South and Southeast Asia for fruit.
Cultivation:
monoculture (large estates) and in mixed systems (small farmers).
Solitary slender palm, stem 25-40 cm in diameter, seed propagated. Seasonal fruit
harvest. Attractive tropical ornamental.
Primary economic product: endosperm (ripe or unripe) chewed for mild narcotic
effect. Endosperm contains the alkaloid arecoline; chewed alone or mixed with pepper
leaf and slaked lime. About 10% oil in endosperm.
Primary product harvest: fruit bunches cut and taken from field.
Primary product processing: fruits removed from bunch, husked and dried or boiled
(to reduce tannin content); prepared endosperm cut into pieces.
Secondary harvest products:
leaves for thatch; large leaf sheaths made into
biodegradable plates, sandals; also suitable for ply board and panels. Dead pruned
leaves for mulch and fertilizer.
Secondary processing products: husks represent about 70% of fruit. Potential uses
include hard board, latex-bound fabric; pulp and paper; source of furfural. Boiling
immature nuts produces useful tannin-rich liquid. Residues for fertilizer.
840
Replacement planting products (in addition to secondary products from fruit harvest):
palm heart bitter but edible; whole stems as rustic building material; hard, yellow
stem wood can be cut into various articles, e.g. rules, shelves, etc. Stem residue for
fertilizer. Economic life of plantation about 40 years.
Sources: [2, 3].
Replacement planting products: stem wood for lumber, posts, furniture, fuel wood,
etc.; leaves for thatch and weaving; palm heart extracted (Philippines); leaf residue
as mulch/fertilizer; wood residue buried/burned. Plantation life is 60-70 years.
Sources: [7, 8, 11].
Box 2. Profile of peach palm (Bactris gasipaes) and its products
Domesticated: Tropical Latin America for fruit.
Cultivation: (1) small farmer fruit production, often in mixed systems. Multistemmed
palm, slender stems, 12-26 cm diameter, seed and some offshoot propagation. One or
two fruit crops per year; (2) commercial palm heart on larger estates grown at high
density. Propagation by seed; offshoots can be managed for successive harvests.
Attractive ornamental but has a spiny stem.
Primary economic products: (1) fruit eaten after boiling, mesocarp source of flour or
fermented into alcoholic beverage. Mesocarp contains about 16% oil and is energyand vitamin-rich; (2) palm heart eaten as green vegetable. The two products are
mutually exclusive; (3) seed for establishing new plantings.
Primary product harvest: (1) fruit bunches cut and taken from field; (2) stem felled and
palm heart extracted leaving it enclosed in several protective layers. Harvested palm
heart 60-80 cm in length, varying in diameter depending upon stem size.
Primary product processing: (1) fruit bunches or individual fruits sold after boiling or
they can be canned. For starch production entire bunches cooked, fruit removed,
peeled, deseeded, cut into small pieces, ground and packaged; (2) palm hearts peeled,
cut into short lengths and cooked in cans or jars.
Secondary harvest products: (1) edible male flowers; green leaves for thatch, dead pruned
leaves for mulch and fertilizer; (2) green leaves for thatch, stem wood for parquet
flooring, furniture, carved objects, etc. Residue left in field for mulch and fertilizer.
Secondary processing products: (1) starch production by-products suitable for animal feed
and mixed with tropical grasses for ensilage; (2) palm heart peelings suitable as animal feed.
Replacement planting products: (1) and (2), same as Secondary harvest products; (1)
Economic life of plantation 50-75 years.
Sources: [9, 10, 13].
841
Box 3. Profile of oil palm (Elaeis guineensis) and its products
Domesticated: West Africa for fruit.
Cultivation: monoculture (large estates), especially in Southeast Asia; small farmer
subsistence crop in West Africa. Solitary palm, moderately thick, stem 22-75 cm,
propagated by seed and tissue culture. Once mature, continuously flowers and fruits.
Primary economic products: (1) oil from both mesocarp and seed; (2) small-scale
tapping (Africa) of male inflorescence and stem, and from felled stem (replanting).
Primary product harvest: (1) fruit bunches harvested and transported from the field; (2)
sap collected in vessels attached to tree, transferred to larger containers and taken from
the field.
Primary products processing: (1) fruit bunches steam sterilized, fruit stripped from
bunches, mesocarp oil extracted separation of seed, extraction of seed oil. Both oils are
refined and have wide food and industrial uses; (2) sap fermented into palm wine, sold
fresh and bottled.
Secondary harvest products: fresh leaves pruned for thatch, construction etc. by
subsistence growers. Leaves can be added to animal feed. Pruned dead leaves for
mulch and fertilizer.
Secondary processing products: empty fruit bunches burned as oil mill fuel and ash
collected to use as fertilizer, in soap making, mixed with concrete, etc. Dried mill
effluent and palm kernel cake can be added to animal rations at low proportion. Solids
from effluent returned to field.
Replacement planting products: Denser portion of stem can be used as an ingredient
in particleboard, but economic viability unclear. Leaves, trunks, roots recycled as
fertilizer on site. Economic life of a plantation is about 25 years. Sources: [4, 8, 12].
842
Box 4. Profile of coconut palm (Cocos nucifera) and its products
Domesticated: South Pacific Ocean Region for fruit.
Cultivation: monoculture (large estates) and in mixed cropping and grazing systems
(small farmers). Short to tall, solitary palm of moderate diameter, seed and tissue
culture propagated. Flowers and fruits continuously. Attractive tropical ornamental.
Primary economic products: (1) endosperm (meat) for copra and oil, fresh coconut
meat; (2) coconut water (green nuts); (3) palm toddy (sap) from tapping unopened
inflorescence; (4) mesocarp (husk) for coir fiber; (5) endocarp (shell).
Primary product harvest: individual ripe or unripe fruits cut from fruit bunch on tree
(1, 2); fruits husked in field if coir not processed; fruits transported from field; (3) sap
collected in vessels attached to the inflorescence and taken away.
Primary products processing: (1) husk removed, nuts cracked, endosperm removed,
dried to make copra or processed into various fresh products. Oil has many food,
cosmetic, industrial uses and as fuel additive. Copra cake as animal feed or fertilizer;
(2) green nuts sold whole, coconut water drunk through a straw or drained out,
preserved in containers; (3) palm sap is sweet drink or boiled down to palm sugar; sap
fermented into wine or vinegar and distilled into hard liquor; (4) husks retted and
fibers extracted and woven, for multiple uses; (5) endocarp used for drinking cups,
buttons etc., and made into charcoal, activated carbon.
Secondary harvest products: (1) fresh leaves; (2) leaf midrib; (3) pruned leaves, empty
fruit bunches and husks.
Secondary processing products: (1) fresh leaves for thatch, fencing, decoration, leaflets
braided into mats, baskets, hats; (2) midribs fashioned into brooms, animal cages, fish
traps, etc.; (3) residue used as field mulch or buried between rows.
843
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844
OP 44
Effect of biological treatments of date palm on feeding lambs
A.A. Mahrous1 M. A. El-Manylawi2
1
Animal Production Research Institute, Agricultural Research Center, Giza, Egypt.
2
Faculty of Agriculture, Cairo University, Giza, Egypt.
a2boady@yahoo.com and manyalawi @hotmail.com
ȋ ǡ
ȌǤǡȋͳȌ
Ϊ ȋȌǤ ʹ Ϊ
ȋǤ Ȍ ͵ǣ Ϊ ȋǤ
ȌǤ
ȋδͲǤͲͷȌ ȋδͲǤͲͷȌ ǡ ǡ
ǡ ǡ ʹ͵ Ǥ
ȋδͲǤͲͷȌ ǡ ǡ
Ǥ ȋȌ
ȋȌʹ͵ȋδͲǤͲͷȌͳǤ
ʹͲǤͲάͲǤʹ
ͳʹͲ
ǤȋȌ
ȋʹ͵Ȍ
ȋ ȌǤ ǡ
Ǥ
Keywords: date palm, biological treatments, digestibility coefficients, nutritive value, lambs.
Introduction
In Egypt, there is a significant increase in the demand for animal protein due to the
increase in human population. On the other hand, feed supplies has risen but at a lower
845
rate than that needed. Such trend resulted in shortage in feedstuffs. Therefore, the
greater technical and managerial problem in Egypt is the provision of adequate
nutrients to the existing animal population.
Date production in Egypt has been steadily increasing over the last 30 years. In
2005, numbers of palm trees (Phoenix dactylifera L.) were found to be 11 million,
producing almost 900 thousand tons date crop per year [7]. A large quantity of nonfruits components of the date palm (frond basis, frond midrib, leaflets, spadix stalks,
spathes fruit stems) and fruit by-products (cull dates, seeds and extracted pulp from
processing units) are wasted. All parts and by-products of date can be added to the
feed mixtures of ruminants [10].
Several efforts were carried out to use local by-products (such as date seeds) in
animal feeding to reduce feed shortage problem. However, utilization of these byproducts is limited because of their low palatability and low digestibility, as well as
high fiber content. Therefore, several methods are use such as mechanical, chemical
and biological treatments to increase feed intake and feeding values of these
agricultural by-products.
The objective of this investigation was to study the effect of biological treatment on
improvement of date palm nutritive value and its effect on digestibility coefficients
and performance lambs.
Materials and Methods
The fungal strain Trichoderma viride and Penicillium funiculusms was obtained
from the Microbial Chemistry Department, National Research Center, Dokki, Cairo,
Egypt and used to treat 2 tons of date kernels.
Inoculums was incubated in one liter conical flask, in 500 ml medium containing
10.0 (NH4)2SO4, 5.0 peptone, 0.5 MgSO4, 0.3.7H2O, CaCl2 and 10 glucose (g/l). Flasks
were sterilized, cooled and inoculated with 3 days old slant of Trichoderma viride
F.405 (T. viride) and Penicillium funiculusms (P. funiculusms). Then incubated at (30
o
C) in rotary shaker 150 rpm for 48 hrs. These inoculums were used to inoculate 50
liters fermenter containing 40 liters of sterilized medium containing the composition of
the same above mentioned medium by 10% (v/v) then incubated for 72 hrs. to produce
480 gm fungal biomass. Concentrate feed mixture and treated or non-treated date palm
846
was chemically analyzed according to [1] method. Neutral detergent fiber (NDF), acid
detergent fiber (ADF) and acid detergent lignin (ADL) were determined by the
methods of [34].
Three rations were formulated, R1 (the control) concentrate feed mixture +
untreated date palm (DP). R2 consisted of CFM + treated date palm with (T. viride)
and R3: CFM + treated date palm with (P. funiculusms).
Digestibility trial was conducted using nine mature local Rahmany breed rams (3
animals each) weighing on average 50 kg and 3 years old. Animals were housed into
individual metabolic cages for 21 days (14 days as a preliminary period followed by 7
days as collection period), to determine the digestibility coefficients and nutritive
value of the three tested rations. Average feed consumed per animal per day (as fed).
At the end of the collection period, feces samples of each ram were mixed well and
kept in the refrigerator for subsequent chemical analysis. Chemical composition of
feeds, feces and urine were determined according to [1] method. Neutral detergent
fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were
determined by the methods of [34].
Eighteen Rahmany lambs were taken from the station herd and were distributed into
four similar groups, according to their weight. Average initial live body weight was
20.0±0.2 kg/head and animal groups were fed the four respective rations in 2 meals/day
(8 a.m. and 3 p.m.) for 120 days. Blood samples were drawn from the jugular vein and
centrifuged for 20 min at 3000 r.p.m. The supernatant was frozen and stored at -20oC
for subsequent analysis. Plasma total protein was determined according to [9]; albumin
according to [12]; GOT and GPT according to [28] and urea according to [31].
The data were statistically analyzed according to [32] using [30]. The difference
between means was tested by Duncan’s multiple range test [13].
Results and Discussion
Chemical composition:
Digestibility coefficients and nutritive value:
Nutrient digestibility coefficients and nutritive value have been affected by biological
treatments as presented in Table (2). All biological treatments increase the values of
nutrients digestibility coefficients than that of control. Rations treated T. viride showed
847
the highest (P<0.05) digestibility coefficients for all nutrients compared with the other
biological treatments which had quit similar values for all digestibility coefficients.
In the meantime R2 was (P<0.05) better in all nutrients digestibility coefficients and
nutritive value compared with R3 and R1. These remarkably improve in all nutrients
digestibility in rations contained biologically treated date palm compared with the
control. So, it could be attributed to the effect of biological treatment by Trichoderma
fungi in up grading and positive alteration of the chemical composition of date palm as
shown in (Table 1).
The results presented in Table (2) showed that the total digestible nutrients (TDN)
and digestible crude protein (DCP). The overall means of TDN and DCP were
improved by treatment R2, R3 and R1 being 60.00, 58.63 and 53.23% for TDN and
being 9.25, 8.23 and 7.33% for DCP, respectively.
These positive results could be also supported by the earlier investigations in using
even raw date palm in small or large ruminant’s rations, which recorded positive
impact in improving its digestibility coefficients of DM, OM, CP, CF, EE, NFE and
the nutritive value [18]; [22] and [6]. [27] reported an improvement in the digestibility
coefficients of DM, OM, EE, CF and NFE with increasing date seeds level in sheep
ration while CP digestibility decreased, giving a net increase in TDN. [26] reported
that the nutrients digestibility for lambs fed diets containing olive pulp and date stone
and radical were similar to the control group. [17] found no adverse effect on all
nutrients digestibility and nutritive value (TDN and DCP) as a result of substituting
half of the concentrate feed mixture in the control ration of sheep (50% clover hay +
50% concentrate mixture) by date seeds. [23] reported that nutrients digestibility and
nutritive value (TDN and DCP) were significantly higher for buffalo fed control diet
compared with diet contained date seeds. [14] and [15] reported that biological
treatments with different fungal strain decreased cell wall constituents of
different crop residue. Also, [16] found that TDN content increased from 63.93
and 63.35% in untreated rice straw and corn stalk to 72.31 and 72.88% in
fungal treated ones, respectively.
848
Blood parameters:
Results of blood constituents for R1, R2 and R3 as illustrated in Table (3) showed
insignificant differences (P>0.05) among the three tested groups in all blood
parameters. All parameters were found to be within the normal range as reported by
[20]. [17] found no marked effect in blood total protein, albumin, globulin, urea-N,
cholesterol and creatinine as well as GOT and GPT as a result of feeding sheep on date
seeds at levels of 25, 50 and 75% replacing concentrate feed mixture. [22] found
insignificant differences in serum total protein, albumin, globulin and GOT while
significant in albumin/globulin ratio, urea and glucose among goats fed 100% clover
hay, 70% clover hay + 30% raw date seeds and 40% clover hay + 30% date seeds +
30% concentrate mixture. [29] reported that replacing 50 or 100% of yellow corn in
the concentrate mixture of lactating cows by date seeds had no significant effect on all
blood constituents. On the other hand, [8] found that substituting 50 or 100% yellow
corn in the concentrate mixture of Friesian calves diets by date seeds had significant
effect on serum total protein, albumin and urea but insignificant on globulin and
albumin/globulin ratio.
Feeding trial:
The average DM intake expressed as (g/h/d), average daily body gain and feed
conversion of the experimental groups is presented in Table (4). The result revealed that
the average DMI as (g/h/d) of lambs during 120 days of the experimental period was
higher for lambs fed date palm treated with T. viride (1370g/h/d) followed by P.
funiculusms treatment (1200g/h/d) than the control (1000g/h/d). The results of feed
conversion (gDM/ggain) showed that date palm treated with fungus (T. viride) recorded
the best value (7.98) followed by the P. funiculusms treatment (8.23) than the control
(9.20). The present results are in agreement with those published by [3]; [4]; [5] and [25].
Conclusion
The overall results obtained in this study that the biological treatments of date palm
by T. viride and P. funiculusms increased protein content, protein digestibility, fiber
fractions digestibility. The recycling of agricultural wastes is important to raise its
nutritional value and can be used in the ruminants feeding. Biological treatments can
utilize lignin along with cellulose and other components of the substrate; these
849
organisms grow slowly and degrade the structural carbohydrates of crop residues. In
addition, biological treatments as a result of molecular biology are preferable in terms
of being a biological treatment, rather than the other treatments such as (chemical and
physical) treatments for better and clear environment.
850
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851
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853
Table (1): Chemical composition (% on DM basis) of untreated and treated date
palm and concentrate feed mixture.
Item
Untreated date
palm
DM
OM
CP
CF
EE
Ash
NFE
NDF
ADF
ADL
Cellulose
Hemi-cellulose
93.07
95.17
6.33
25.63
3.25
4.83
59.96
70.72
52.78
29.66
17.94
23.12
DP treated
with
DP treated
with
T. viride
P. funiculusms
90.70
90.20
10.50
19.31
2.10
9.80
58.29
60.40
40.20
10.90
20.20
29.30
92.43
92.14
9.00
21.64
2.30
7.86
59.20
66.25
46.80
18.13
19.45
28.67
CFM
88.80
87.34
14.20
13.04
3.24
56.86
12.66
32.75
10.46
3.48
6.98
22.29
Table (2): Effect of biological treatments on digestibility coefficients and nutritive value of lambs.
Item
±SE
Rations
R1
R2
R3
Digestibility coefficients (%):
DM
62.33c
77.00a
75.67b
±1.03
OM
60.33c
68.40a
66.13b
±0.40
CP
69.50c
78.77a
72.00b
±0.70
CF
66.67c
70.33a
78.00b
±0.78
EE
76.67c
81.67a
79.51b
±0.66
NFE
60.67c
69.67a
65.47b
±0.36
Nutritive value (%):
TDN
53.23c
60.00a
58.63b
±0.20
DCP
7.33c
9.25a
8.23b
±0.30
a, b ,c and d Means with different superscripts in the same row differ significantly
(P<0.05).
854
Table (3): Effect of biological treatments on blood parameters for experimental rations.
Item
±SE
Rations
R1
R2
R3
Urea (mg/100ml)
25.20
24.85
24.79
0.40
Total protein (gm/100ml)
7.32
7.40
7.35
0.52
Albumin (gm/100ml)
3.90
3.84
3.78
0.67
Globulin (gm/100ml)
3.60
3.56
3.57
0.04
A/G ratio
1.08
1.07
1.05
0.11
GOT (U/L)
20.46
20.52
20.40
0.12
GPT (U/L)
33.67
33.71
33.59
0.52
Table (4): Effect of biological treatments on feed intake and feed conversion of experimental animals.
Item
Rations
R1
R2
R3
No. of animals
6
6
6
Experimental period (days)
120
120
120
Initial weight (kg)
20.10
20.20
20.30
Final weight (kg)
33.14
40.80
37.80
Total gain (kg)
13.04
20.60
17.50
Average daily gain (ADG) (g)
108.6
171.6
145.8
DMI (g/d)
1000
1370
1200
Concentrate feed mixture
600
750
650
Date palm
400
620
550
Feed conversion (g DM/g gain)
9.20
7.98
8.23
855
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856
OP 45
Study on the effect of the Use of Diets Containing Different Levels
of Crushed Date Seeds on Growing Assaf Lambs
H.A. Al - Shanti 1, K.J. Al -Shakhrit 1,M.F. Al - Banna 2, I.E .Abu Showayb2 and A.M. Kholif 3
1-Animal Production Department , Agriculture Faculty ,Al- Zhar University -Gaza Palestine
2-Al- Ahlaya Society for the Development of Palm and Dates –Der Al-Balah- Palestine
3-Dairy Science Department, National Research Centre, Dokki, Giza, Egypt
h_shanti@hotmail.com
Abstract
Twenty crossbred Assaf lambs (Breed from 5/8 Awassi, 3/8 Freisian) with an
average body weight of 30-32 kg were divided randomly into four equal experimental
groups (5 lambs each group) to investigate the effect of including crushed date seeds
(CDS) in diets on the growth performance of growing crossbred lambs. The feeding
trial lasted for 150 days (1 May-30 September, 2009). The animals fed restricted
roughage of 600 g./day chopped date leaves, while concentrates feed mixture (CFM)
was offered ad bl. (Control). Crushed date seeds was substitutes 50, 75 and 100% of
corn and barely in CFM in diets of T1, T2 and T3, respectively. Soybean meal and urea
was used to adjust the protein content in treated diets, so the experimental rations were
iso caloric and iso nitrogenous. The concentrate: roughage ratio was (85: 15)
approximately. The results showed that,. Daily dry matter intake as kg/day, g/kg W0.75
and kg/100 kg BW were not significant (P<0.05) affected by treatment. Final weight,
total body weight gain, average daily gain and feed efficiency (kg. gain/ kg. intake)
were not significant affected (P<0.05) by inclusion crushed date seeds in the lambs
diets. However, increasing the level of crushed date seeds substitution in the diet tended
to improve final weight gain, total body weight gain, average daily gain and feed
efficiency which calculated as (kg. gain/ kg. intake) of DM compared with the control
diet. From these results, it can be concluded that substitution of corn and barely by
crushed date seeds can be used to improve the growth performance of Assaf lambs.
Keywords: date seeds, Assaf lambs, growth performance, carcass, blood serum parameters.
857
Introduction
The animal production sector play an important role in the local Palestinian
agriculture sector. The contribution of this sector is about 36% (Ministry of Agriculture
(M.O.A. 2000). The importance of this sector comes from the components of the sector.
which are the sheep, goat, dairy cattle and poultry sector. The fattening operations are
among the important activities within animal production sector. Hammad et al (2002).
Such a fattening projects are important in the animal production sector makes about
61% of the animal production value (MOA, 2000). However, The execution of
fattening operation varies widely in Palestine. The intensive systems of livestock
raising lambs under fattening operation of Awass and Assaf breeds. Cereal grains such
as barley and corn are common feed ingredient in local fattening operation barley is
incorporated in formulated feeds at rate of about 25% (Abo Omer, 1992). The amount
of barely used in local fattening operations is estimated to be 100 thousand tons/ year
(MOA, 2000). The Feeding costs in Palestine make up more than 70% of the total
production costs in any livestock operation. This could interpret on the basis that the
most conventional feed stuffs are imported with higher prices and grains are a main
source of energy for livestock due to steady current competition between man and
animal, aggressively led nutritionists to replace part of starch grain by Date Seed (DS)
in ruminant rations aids in reducing the nutritional gab found between animal needs and
local feed resource. In some previous studies (Khamis et al., 1989, Kholif and Abo ElNor, 1998 and Kholif et al, 2001) date seeds proved to be utilize able component in
ruminant diets but it was in need for supplementation with protein. Date by product is
second of the most available by products of local farming in Palestine especially in
Gaza Strip estimated at 100,000 nearly after the olive cake by product. The objective of
the present study was to determine the effective of partially replacing the expensive
concentration feed mixture (CFM) (barely and corn) with crushed date seeds (CDS) on
growth performances, blood parameter and carcass conformation of Assaf sheep. 2.
Materials and Methods
This study was carried out at the Alahlyia Association for the Development Farm at
Dair Al-Balah – Gaza Strip, Palestine. Twenty crossbred Assaf Male lambs, (Breed
from 5/8 Awassi, 3/8 East Frisian) with an average live body weight of 30 – 32 Kg.
858
were divided randomly into four equal experimental group (5 lambs each group)
investigate the effect of including crushed date seeds (CDS) in diets on the growth
performance of growing crossbred lambs.
1. Experimental rations and feeding trial
The basal ration composed Concentrate feed mixture (CFM). The experimental
group received one of four tested rations.
1st group feed ration one: Contains CFM without (CDS) as Control
2nd group feed ration two: CFM contained 50% (CDS) replace of corn and barely (T1).
3rd group feed ration three: CFM contained 75% (CDS) replace of corn and barely (T 2).
4th group feed ration four: CFM contained 100% (CDS) replace of corn and barely (T 3).
Formation of the different ration and their chemical composition are shown in
(Table 1), and proximate chemical analysis (AOAC, 1990).
The animal were feed daily (CFM) and barely straw at 3% and 0.5 % respectively
of their live body weight (LBW) to cover the nutrition's requirements for growth
according to NRC (1985).
Daily ration were offered in two equal meals at 8:00 and 3:00 pm. Fresh water was
available at all the time.
Animal were weight biweekly intervals at the same time of the day before feeding
from the beginning till the end of the experimental which lasted for 150 day.
Feed intake was daily recorded. Mean daily gain and feed conversion was
calculated to evaluate lambs performance and to calculate the profit from fattening
lambs under this study.
2. Blood Analysis:
Blood samples were taken every four weeks before morning feeding from the
jugular vein of male lamb. The sample were directly collected into vacuum tube,
divided into two tube, one collected at EDTA for hematological analysis, red blood
cell count (RBC), packed cell volume (PCV), hemoglobin (HBG) and white blood cell
count (WBC). Using CELL-DYN 3700 blood analyzer. And another tube without
EDTA, these sample centrifuged at 3500 rpm for 15 min. Serum was separated and
stored at 20°C until analysis for total proteins and albumin according to Weich
selbaum, (1946) and Doumas et al (1971), respectively. Globulin value were
859
calculated by the difference between total protein and corresponding value of Albumin
Urea concentration was estimated by the method of Henry and Davielsohn(1974).
Glutamic- oxaloacetic transaminases (GOT) and Glutamic- Pyruvic transaminase
(GPT) were determined as describe by Reitman and Franked (1957) cholesterol was
also determined according to Allain etal (1974). Triglyceride was also determined
according to Fossati and Principle (1962) and creatinine determined according to
Brood and Sirota (1948), serum glucose concentration was determined immediately by
using commercial kits.
3. Carcass quality:
At the end of the experimental period (150 day) 3 animal from each treatment group
were randomly selected weighted and then slaughtered hot carcass body offal's and
internal organs were separately weighed.
The whole carcass was then cut to neck, rack, loin, flank, shoulder and legs. The
measurement and classification of carcass were carried out according to Colomer et al
(1987) and Maharem (1996). Weight of meat, fat and bone of carcass were calculated
according to Field et al (1963) procedures. Weight of bone the eye muscle longismus
dorsi (LD) was recorded. Dressing was calculated to Abou-Ammo (1992).
4. Statistical analysis:
Experimental data were analyzed using by Anova as a completely randomized
design (SAS, 1991) significant differences of means were tested using Duncan's
multiple test (Duncan 1985).
Results and discussion:
Experimental animals showed no major health problems during the feeding period.
These results were confirmed with Mahgoub et al, (1998) and Mahgoub et al, (2005).
Significant differences were not observed among the diets with respect to the
efficiency of feed conversion. (Table 2) revealed that live body weight (LBW)of Assaf
lamb at the end of the experimental, total weight gain and daily gain did not differ
significantly when they fed rations containing different level of (CDS) replacement of
all the component of the commercial one.
These results were confirmed with El-Shaer et al (1996), Eid (1998), Youseef et al
(2001) and Abou ElNaser and El-Kardawy (2003) who reported that body gain of
860
growing sheep and goat did not differ significant in animal, fed organize waste mixture
than the conventional diets.
However results in Table (2) revealed clearly that feed conversion ratio was less
significantly (P < 0.05) than those received control and T1. Abdou (1998) and Eid
(1998) observed a decreased in daily gain when growing lambs were fed diets
containing 75%, 100% (CDS), Also these result s agreed with Youssef et al (2001)
who concluded that the addition of radycelles enhanced the utilization of diets contains
date seed and olive pulp when feed to the growing goat and these result similar with
Mahgoub and Lodge (1994). Also these results agree with Al-Ani and Farhan (2009)
who concluded that cotton seed meal with date stones in fattening diets of Awassi
lamb achieved a better or similar response to soya bean meal as a source of nitrogen
and higher than that from urea. Al-Nakib et al (1996), Gatenby (1986), Abou El Naser
and El –Kerdowy (2003) and Sayeda et al (1999) found these result in using date seed
in fatting Friesian Calves. The results show that replacement of concentrate with
discarded date leads to an increase in the final weight of the animal without
improvement in feed efficiency. These results are consistent with the finding of ElGasim et al (1986), Al-Dabeeb (2005) and Alhomady et al (2011).
Carcass Measurements and cuts
As shown in (Table 3) there were no significant differences due to feeding at the
treatment also in these study were found difference in the percentage cut and organs
but all these difference also not significant these results were supported by the finding
of Vergara and Gallego (1999), Muhils Maci et al (2003), Mahgoub et al (2005) and
El-Ayek et al (2001) found that the effect of roughage on carcass cuts and dressing
percentage of lamb the differences were not significant, and found the effect of tested
diets on offal also no significant.
As shown in (Table 4) the effect of experimental diet on carcass quality of
longismus dorsi (LD) of tested animals – the result show that the higher weight of ribs
sample and longismus dorsi (LD) was in animal fed on (CDS). The differences
between the tested groups were not significant. these results agrees with the result
obtained by El-Ayek et al (2001) and Taie et al (1998) who reported that dressing
percent and dissectible fat both as weight in creased with increasing level of energy.
861
Blood hematology and biochemical parameters
Hematological data (Table 5) was used as an indication of the health status of
experimental animal, during the course of the experiment all of the blood values
recorded were within the normal reference range for animal of similar group. These
results approve with Mahgoub et al (2005).
The effect of experimental ration on some blood parameters are presented in (Table
5). The results in dictated that total protein (g/dl) and globulin (g/dl) concentration
were not affected by (CDS) percentage values of total protein, albumin, globulin in the
normal ranges the result in agreement with that reported by Kholif et al (1996), ElReweny (1999) and Mustafa et al (2009).
All the parameter are present in there study (Table 5) normal and no significant
effect. These study agreement with that reported by Metwally and Mohsen (1997) and
El-Nasr and El-Kerdawy (2003).
Conclusion:
It could be concluded that crushed date stone (CDS) and palm by-product can be
fed up to 50% instead corn and barely success fully and economically as an energy
source in CFM for lambs without any adverse effect on animal performance which are
reflected on feeding cost and economic efficiency.
862
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866
Table (1) Formulation and chemical composition of fattening diets
Control
Diets 1
Diets 2
Diets 3
CFM 0 %
crush date
seed (CDS)
CFM 50 %
(CDS)
CFM 75 %
(CDS)
CFM 100
% (CDS)
Crushed
yellow corn %
30
15
75
0
Barely %
20
15
75
0
Soya bean
meal
7
6.5
6
6
Wheat bran %
30
30
30
30
Crushed Date
seeds % (CDS)
----
30
45
60
Urea
----
0.5
1
1
Limestone
01
01
1
1
Sodium
chloride %
01
01
1
1
Mineral
mixture
0.5
0.5
0.5
0.5
vitamin
0.5
0.5
0.5
0.5
100
100
100
100
Ingredient
Approximate chemical composition (on DM basis)
Dry matter %
(DM)
90.5
91.5
92.0
92.2
OM
93.5
93.0
91.5
90.0
CP
15
15.1
15.4
15.3
CF
12.5
14.6
18.3
22.5
4.0
3.8
3.5
63.28
63.38
63.6
EE
NFE
64.22
867
Table (2) Growth performance and feeding intake performance of Assaf lambs fed ration
supplemented with different levels of crushed date stone (CDS) by-product (Means ± SE).
Item
Control diet 0
T1 50 %
T3 100 %
T2 75 % (CDS)
% (CDS) no (CDS) replace
(CDS) replace
replace of corn
replace of corn of corn and
of corn and
and barley
and barley
barley
barley
No of animal
5
5
5
5
Duration of
trial (day)
150
150
150
150
Initial weight
(Kg)
31.15 ± 1.32
31.23 ± 1.23
30.18 ± 1.45
32.04 ± 1.62
Final weight
(Kg)
63.45 ± 2.01
63.55 ± 1.98
61.92 ± 1.72
63.98 ± 2.12
Total gain (Kg)
32.3 ± 1.16
32.32 ± 1.01
31.74 ± 1.13
31.92 ± 1.42
AV. Daily gain
(g)
215.33 ± 2.21
215.47 ± 2.63
211.6 ± 2.11
212.8 ± 2.41
Total feed
intake (Kg)
203 ± 3.16
200 ± 4.06
205 ± 4.06
203 ± 3.85
Feed
conversion
Ratio
6.28a
6.19a
6.45b
6.36b
a, b Means on the same line, within each row having different letters were
significantly differ at (P<0.05) * Kg feed required for 1 Kg body weight gain.
868
Table (3)Effect of feeding the experiment diet on carcass trail of Assaf Male sheep lambs
Control
T1
T2
T3
CFM 0 % crush
date seed (CDS)
CFM 50 %
(CDS)
CFM 75 %
(CDS)
CFM 100 % (CDS)
Final fasted
body weight
(FBW) (Kg)
63.24 ± 2.05
63.35 ± 2.88
62.12 ± 3.01
62.42 ± 2.88
Hot carcass
(Kg)
32.66 ± 1.23
33.00 ± 1.64
31.92 ± 1.72
32.25 ± 1.90
Dressing %
51.64 ± 1.36
52.09 ± 1.42
51.38 ± 1.53
51.67 ± 1.43
Item
Organ percent % of (FBW)
Head %
5.4 ± 0.52
4.98 ± 0.42
4.14 ± 0.49
4.7 ± 0.42
Pelt %
10.11 ± 0.20
10.62 ± 0.97
10.70 ± 0.94
11.11 ± 0.98
Feet %
2.15 ± 0.20
2.29 ± 0.21
2.15 ± 0.195
2.21 ± 0.172
Offal's percent % of (FBW)
Heart %
0.48 ± 0.02
0.41 ± 0.03
0.4 ± 0.04
0.4 ± 0.04
Liver %
1.48 ± 0.93
1.48 ± 0.08
1.4 ± 0.1
1.42 ± 0.09
Kidney %
0.32 ± 0.01
0.31 ± 0.01
0.36 ± 0.02
0.30 ± 0.02
Lungs %
1.72 ± 0.03
1.75 ± 0.04
1.81 ± 0.04
1.79 ± 0.06
Spleen %
0.15 ± 0.02
0.15 ± 0.02
0.18 ± 0.02
0.17 ± 0.02
Testis %
1.16 ± 0.03
1.5 ± 0.04
1.42 ± 0.04
1.45 ± 0.05
Fat % of (FBW)
Abdominal
fat %
3.58 ± 0.05
3.71 ± 0.05
3.70 ± 0.06
3.56 ± 0.07
Tail fat %
2.44 ± 0.04
2.64 ± 0.03
2.52 ± 0.04
2.63 ± 0.05
Kidney %
1.86 ± 0.03
1.73 ± 0.03
1.81 ± 0.04
1.77 ± 0.04
Whole sale cuts % of (FBW)
Neck
5.54 ± 0.43
5.51 ± 0.41
5.52 ± 0.39
5.61 ± 0.46
Shoulder
9.97 ± 0.52
9.91 ± 0.53
9.92 ± 0.60
10.00 ± 0.51
Lion
6.92 ± 0.43
7.00 ± 0.42
7.01 ± 0.46
7.10 ± 0.44
Flank
3.36 ± 0.23
3.50 ± 0.26
3.51 ± 0.29
3.6 ± 0.31
Hand and legs
15.9 ± 1.34
16.1 ± 1.56
16 ± 1.46
16.2 ± 1.52
869
Table (4) Effect of feeding the experimental diet on ribs 9, 10, 11(Wt. gm) and
percentage of meal, Bone and Fat and eye muscle area (CM2)
Control
T1
T2
T3
Item
CFM 0 %
crush date
seed (CDS)
CFM 50 %
(CDS)
CFM 75 %
(CDS)
CFM 100 %
(CDS)
Ribs 9,10,11
weight (gm)
726.21 ±
12.32
736.13 ± 11.42
728.15 ± 10.61
715.16 ± 13.41
Meat wt
(gm)
430.22 ± 8.21
420.21 ± 8.03
435.31 ± 9.46
425.31 ± 9.52
Meat wt %
59.24 ± 2.21
57.07 ± 2.11
59.78 ± 1.92
59.5 ± 1.96
Bone wt
(gm)
148.15 ± 4.21a
152.16 ± 3.86b
150.21 ± 3.75b
146.21 ± 3.63a
Bone wt %
20.4 ± 2.31
20.67 ± 2.41
20.63 ± 2.35
20.44 ± 2.11
Fat wt (gm)
147.84 ± 3.21a
163.76 ± 2.98b
142.33 ± 3.11a
143.64 ± 3.15a
Fat wt %
20.35 ± 1.96a
22.25 ± 2.01b
19.55 ± 2.11a
20.08 ± 2.11a
Eye muscle
area cm
37.85 ± 1.38
36.92 ± 1.62
37.01 ± 1.41
37.22 ± 1.43
a, b Means on the same line, within each row having different letters were
significantly differ at (P<0.05)
870
Table (5)Effect of feeding the experimental diet on Hematology blood picture and
some blood serum parameters of Assaf male lambs
Control
T1
T2
T3
CFM 0 %
crush date
seed (CDS)
CFM 50 %
(CDS)
CFM 75 %
(CDS)
CFM 100 %
(CDS)
Red blood cells
(RBC) (×106/m)
5.3 ± 0.28
5.4 ± 0.26
5.1 ± 0.28
5.2 ± 0.30
Hemoglobin (Hb)
(g/l)
12.5 ± 0.19
12.5 ± 0.18
12.4 ± 0.2
13 ± 0.23
Packed cell
volume (PCV)
(%)
32 ± 1.21
34 ± 1.24
33 ± 1.41
34 ± 1.35
White blood cells
(WBC) (×106/m)
5.2 ± 0.36
5.4 ± 0.35
5.4 ± 0.32
5.6 ± 0.34
Total protein
(g/100L)
7.24 ± 0.26
7.32 ± 0.24
7.29 ± 0.23
7.12 ± 0.24
Albumin g/100 ml
3.68 ± 0.16
3.69 ± 0.18
3.74 ± 0.19
3.58 ± 0.18
Globulin g/100 ml
3.56 ± 2.8
3.63 ± 1.31
3.55 ± 2.76
3.54 ± 2.79
A/G ratio
1.03 ± 0.71
1.02 ± 0.82
1.05 ± 0.72
1.01 ± 0.76
Glucose mg/100
ml
61.38 ± 3.1
63.3 ± 2.81
64.5 ± 2.65
64.83 ± 3.11
GPT Iu/l
8.72 ± 1.35
8.38 ± 2.31
8.63 ± 2.21
8.78 ± 2.31
GOT Iu/l
61.32 ± 3.4
62.72 ± 3.61
62.83 ± 3.52
64.63 ± 3.42
Cholesterol
mg/100ml
148.7 ± 12.3
150.2 ± 11.61 149.6 ± 10.08 148.8 ± 12.06
Triglycerides
102.3 ± 11.2
115.6 ± 9.89
Creatinine mg/100
ml
1.15 ± 0.05
1.01 ± 0.06
1.06 ± 0.07
1.08 ± 0.05
Urea mg/100 ml
17.95±2.05
18.87±1.98
17.65±2.03
18.53±2.5
Item
871
107.3 ± 10.31 109.2 ± 10.61
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874
OP 46
Composition and functional properties of the date fruit residue
(DFR) a by-product of date syrup (Debis) production
Isameldin B. Hashim*1 and Ali H. Khalil2
1
Department of Food Sciences, College of Food and Agriculture, United Arab
Emirates University, P. O. Box 17555 Al Ain, UAE
2
Department of Food Science and Technology, Menoufiya University, Egypt
Corresponding author's e-mail: ihashim@uaeu.ac.ae
Abstract
Low quality date fruits are processed to produce date syrup. Date fruit residue
(DFR) is the major by-product of date syrup production and used mainly as animal
feed. The aim of this study was to characterize commercial DFR from three date
varieties (Khulas, Barhee and Lulu) produced at a local date processing
establishment. Microbiological quality, proximate composition, sugars, soluble
dietary fiber (SDF), insoluble dietary fiber (IDF), minerals content, color and
functional properties [water holding capacity (WHC), oil-holding capacity (OHC),
emulsifying activity, emulsion stability, foam capacity and foam stability] were
evaluated. The main components of the DFR were dietary fibre, total (50.8-56.5%)
and sugars (27.7-30.4%). DFR had similar color, WHC (1.86-2.00g/g), OHC (0.660.68 g/g), emulsifying activity (56 %) and emulsion stability (71 %). DFR might be
an alternative source for dietary fiber that will ultimately result in adding value to
DFR and benefiting palm dates growers and processors.
Introduction
Date is one of the most important fruits in The United Arab Emirates (UAE). UAE
is the fourth leading country, producing 755 thousand tons of dates annually which
represent 12% of the world production [1]. Dates are good source of dietary fiber [2; 3;
4; 5]. Dietary fiber content of dates ranged from 4.4 to 11.4% depending on date
variety and ripening stage [6; 7; 8; 9]. A serving of dates (five to six fruit dates) can
provide 14% of the recommended daily intake of the dietary fiber [6].
Consumption of foods containing fibers may prevent or decrease gastrointestinal
disorders [10], hypertension, hypercholesterolemia, obesity [11], diabetes [12; 13; 14],
875
coronary heart disease [15; 16] and cancer [17; 18]. The Dietary Guidelines for
Americans published jointly by the U.S. Department of Agriculture and Health and
Human Services recommend eating foods that have adequate amounts of fiber, The
National Cancer Institute recommends 20 to 30 grams of fiber per day with an upper
limit of 35 grams. To meet these requirements, fibers are added to different food
products. Beside the health benefits, fibers are added to increase cooking yield and
water holding capacity, reduce lipid retention, improve textural properties and
structure, or as bulking agent to reduce caloric content [19].
Low quality dates are processed to produce date syrup. In the UAE, there is several
food processing establishments produce date syrup. Large amounts of date fruit
residues (DFR), the by-product from syrup extraction, are available. Currently, the
sole use of DFR is for animals feeding. DFR has hypolipidemic effects [20]. The
addition of 5% DFR to the diet of rats fed cholesterol significantly increased HDL-C,
lessened the rise in plasma LDL-C and increased the HDL-C/LDL-C ratio. Proximate
composition of dietary fiber extracted from date flesh (press cake) of three sun-dried
Omani date varieties (Mabseeli, Um-sellah, and Shahal) were reported [4]. The
chemical composition and physicochemical properties of concentrated dietary fiber
extracted from Tunisian date flesh cultivars (Deglet-Nour and Allig) were reported [5].
Both studies evaluated dietary fiber and concentrated dietary fiber extracted from date
flesh in the laboratories.
To our knowledge, the compositional and functional characteristics of DFR that
produced commercially from syrup extraction have not been previously reported. The
purpose of this study was to evaluate the microbiological, chemical [proximate
composition, soluble dietary fiber (SDF), insoluble dietary fiber (IDF), sugars and
minerals] and functional properties [water holding capacity (WHC), oil-holding
capacity (OHC), emulsifying activity, emulsion stability, foam capacity and foam
stability] of the DFR collected from a date processing factory in UAE. Therefore, the
information would be useful for promoting DFR as a potential fiber source in
developing functional food products with health benefits.
876
Research methodology
Date fruit residues (DFR)
DFR is a by-product produced when sugar is extracted from dates to produce date
syrup. DFR of three date varieties (Khulas, Barhee and Lulu) were obtained from a
local date processing factory (Emirates Date Factory - Al Saad, UAE), grinded and kept
at room temperature in sealed plastic bags until used for analysis or evaluation.
Microbiological analysis
The presence of the total mesophilic bacteria, coliform bacteria, yeast and mould on
DFR were measured. Total mesophilic bacterial (TMB) counts were enumerated on
standard plate count agar [21] and coliform bacteria were determined using MacConky
agar [22]. Yeast and mould counts were conducted with potato dextrose agar [23].
Plates were incubated for 3 days at 30 ± 1 C, 3 days at 37 ± 1 C and 3-4 days at 20-25
C for mesophilic bacteria, coliform bacteria and yeast & mould, respectively.
Colour Evaluation
Instrumental color analysis of DFR samples was conducted in triplicate with a
ColourFlex Hunter Color Lab (model No. 45/0, Reston, VA., USA). The CIE values
L* (measures the lightness, ranging from 0 (black) to 100 (white)), a* value ranges
from -100 (greenness) to +100 (redness) and b* value ranges from -100 (blueness)
to +100 (yellowness).
Proximate composition
Moisture, ash and fat were analysed according to AACC methods 44-16, 08-01 and
30-20, respectively [24]. Protein was determined based on the Kjeldahl Method 46-10.
The protein content was expressed as nitrogen multiplied by a factor (5.7).
Dietary fibre (DF)
Soluble (SDF), insoluble (IDF) and total dietary fibre (TDF) contents were
quantified using the enzymatic gravimetric procedure of the AACC Method 32-07
[24]. Arabinogalactan from Sigma was used as a standard reference for the
determination of total dietary fiber, giving accuracy of 95.3%.
877
Sugars
Sugar profiles were determined by HPLC according to the AOAC official method
977.20 [25]. Sugars were identified by comparing their retention times with the
standards and quantified using their peaks percentage area.
Mineral analysis
Mineral content was determined using the Inductively Coupled Plasma Optical
Emission Spectrometry (ICP-OES) (Varian- VISTA-MPX, Australia) with Coupled
Captured Detector (CCD).
Functional properties
Water holding capacity (WHC)
Water absorption capacity (WHC) was determined following the method described
by [26]. The values are expressed as grams of water absorbed by 1 gm of DFR.
Oil holding capacity (OHC)
The method described by [27] was used for the determination of fat absorption
capacity (OHC). The values are expressed as grams of oil absorbed by 1 gm of DFR.
Foam capacity and stability
Foam capacity and stability was determined following the method described by [28].
Emulsifying activity and emulsion stability
Emulsifying activity and emulsion stability were determined following the method
described by [29].
Statistical analysis
The data were analyzed by one-way analysis of variance (ANOVA) using SPSS
16.0. Mean separations were performed by Duncan’s multiple range test. Differences
at P < 0.05 were considered to be significant.
Results and Discussion
Microbiological Evaluation
Dates retain some of the natural flora while growing plus contamination from soil,
insects, and other sources. During the processing to produce date syrup as well as the
DFR, some microorganisms associated with dates were removed. Therefore, it is
required to determine the microbiological quality of DFR to estimate its suitability for
human consumption and its shelf-life.
878
The microbiological counts of the DFR are presented in Table 1. Coliform bacteria
are used as an indicator for the presence of pathogenic bacteria. Coliform bacteria
were not detected in all the samples. DFR were free from coliform bacteria, absence of
coliforms were due to thermal processing. The total bacterial counts in DFR ranged
from 3.17 to 3.21 log CFU/g. The yeast and mold counts ranged from 2.04 to 2.09 log
CFU/g. Total viable count (1.7, 3.0 and 2.0 log CFU/g), yeasts and mold count (2.5,
3.6 and 2.0 log CFU/g) were reported [30] for Khulas, Barhee and Lulu at tamr stage,
respectively. While, treating date fruits with ozone (5.0 ppm) for one hour eliminated
coliform bacteria and reduced the total mesophilic bacteria as well as yeast and mold
to 3.54 and 3.61 log CFU/g respectively [31]. The low bacteria, yeast and mold count
as well as the absence of coliform bacteria in all DFR are promising to be included in
developing food products.
Colour
Colour is a quality attribute which plays an important role in food acceptability. If
the DFR will be added to different food products, it is important to know its colour
parameter [lightness (L*), redness (a*), and yellowness (b*)]. The CIE Lab values (L*,
a*, b*) of DFR are presented in Table 2. DFR from different varieties had comparable
redness (7.34-8.02) and yellowness (17.78-18.33). Lulu-DFR had a darker color (the
lowest L* value 48.64), while the DFR from other varieties had lighter color (L* values
54.25-55.51). Tunisian date DF concentrates had lighter (L* values 61.92-65.25) and
less yellow (b* values 14.85-16.28) [5] compared to Emirati DFR. This could be due to
the date variety, extraction technique and the composition of the concentrates.
Proximate Composition
Proximate composition of DFR is presented on Table 3. Carbohydrate was the major
component of the DFR of all date varieties ranging from 85.9 to 87.56%. Lower values
were reported for Omani press cake (81.86-83.33) [4] while higher values were
reported for the Tunisian DF concentrates (88.0-92.4) [5]. Moisture content of DFR
ranged from 6.14 to 8.73. Lulu DFR had the highest moisture content and Barhee had
the lowest value. The moisture content of Omani press cakes (8.3-10.59) were higher
[4]. DFR protein ranged from 2.18 (Lulu) to 3.09 (Barhee). Higher valued were
reported for the Omani press cakes ranged from 3.62 to 5.23% [4] and Tunisian DF
879
concentrates 8.89-9.12 [5]. Ash content followed the same profile as the protein, in
which Lulu had the lowest content (2.15) and Barhee had the highest content (2.98%).
Similar values were reported for Omani press cakes (1.68-2.46%) and Tunisian DF
concentrates (2.01%). DFR had comparable fat content ranged from 0.81 (Khulas) to
1.04% (Lulu). Omani press cakes had higher fat content (1.40 – 2.20%). Compositional
differences could be related to the date varieties and the extraction techniques.
Dietary fibre (DF)
Total DF is the main components of DFR ranging from 50.81 to 56.52% (Table 4).
Among the three varieties Barhee had significantly higher SDF (9.15%), and lower
IDF (41.66%) compared to the other date varieties. The Omani press cakes had lower
total DF values ranging from 25.39 to 33.81% [5] while the Tunisian date DF
concentrates had higher values ranging between 88 and 92% [5]. This could be due to
the date variety, extraction technique and the composition of the DF.
Sugars
Sugar content of Khulas, Barhee and Lulu at tamer stage were reported, glucose
ranged from 29.7 to 30.5% and fructose ranged from 26.5 to 27.6 [32]. While, higher
values were reported for Khulas and Barhee dates stored under commercial and
industrial conditions, glucose (33.1 - 37.8) and fructose (35.2 - 38.3) [33].
Sugar content of DFR is presented on Table 4. The results indicated the presence of
equal concentrations of both glucose and fructose in DFR. Again Barhee had the
highest concentration of glucose and fructose (16.4-16.1%), while Lulu and Khulas
had slightly lower values (15.9 -15.5%) and (15.6-15.2%). This indicated that 50% of
the glucose and fructose were extracted during syrup production. Sugar content of
Omani press cakes was not reported [4] while Tunisian date DF concentrates were
sugar free [5]. The presence of simple sugars in the DFR could be an advantage if used
as an ingredient in baked products.
Mineral contents
Table 5 presents minerals content of DFR. All DFRs had similar Mn and Zn
content. Khulas and Barhee DFRs had similar Ca, Fe, Mg and Na content. Lulu DFR
contained the highest amount of Mg, P, Fe and Mn and the lowest amount of K and
880
Ca. DFRs differ significantly on K and P content. Barhee contained the highest K level
and Lulu contained the highest P level.
Functional properties
Functional properties of DFR are presented in table 6. Water absorption
characteristics represent the ability of a product to associate with water under
conditions where water is limiting, like in doughs and pastes [34]. The results showed
that water absorption were similar for all the DFR. This might suggest that DFR would
be useful in baked products that require hydration to improve handling characteristics.
Fat absorption was similar for all types of DFR. Fat absorption capacity of DFR was
ranging between 0.66 g/g and 0.68 g/g which are considered higher than that of soy
flour [34]. The fat binding capacity of DFR would find useful application in ground
beef products such as patties and sausages. Higher values were reported for DF
concentrate (15.5 g/g for WHC and 9.7 g/g for OHC) which have different
composition [5]. DFR did not show foam capacity. This is might be due to amount of
the protein (low content 2-3%) and the effect of heat treatment during processing that
might denature the protein and consequently destructed the foam capacity. DFR
showed emulsifying activity about 56 % and emulsion stability 71 %. Functional
properties results suggested that DFR might have great potential for addition to food,
not only as a nutrient supplement but also as a functional agent in food.
Conclusions
DFR, date by product produced during date syrup extraction, appears as a suitable
source for dietary fiber with functional properties. The results indicated that DFR could
be considered as an alternative dietary fiber source for different food products. This will
provide benefits to the date industry and a solution for disposing this by product.
Acknowledgement
This research was financially supported by the Research Affairs at the UAE
University under a contract no. 01-02-6-12/03. The authors are very grateful to
Emirates Date Factory -Al Saad, UAE for providing the DFR and Mr. Ismail
Abdelhaliem for technical assistance.
881
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884
Table 1. Microbiological quality (given in LogCFU/g) of date fruit residues (DFR)
1
DFR
Total bacterial
Yeast and mold
Total coliform
Lulu
3.20±0.26a
2.04±0.18a
ND
Khulas
3.17±0.12a
2.09±0.15a
ND
Barhee
3.21±0.13a
2.08±0.26a
ND
Means ± SD followed by the same letter, within a column are not significantly different (P> 0.05). ND not detected.
Table 2. Color of date fruit residues (DFR)
Colour Values
DFR
L*
1
a*
b*
Lulu
48.64±0.15b 7.63±0.08a 17.78±0.12a
Khulas
55.51±0.09a 8.02±0.14a 18.33±0.18a
Barhee
54.76±0.18a 7.34±0.20a
18.22±028a
Means ± SD followed by the same letter, within a column are not significantly different (P> 0.05).
Table 3. Proximate composition (%) of date fruit residues (DFR)1
DFR
Moisture
Ash
Protein
Fat
Carbohydrate
8.73±0.23a 2.15±0.06a 2.18±0.06a 1.04±0.05a
85.90±0.76a
Khulas 7.16±0.11b 2.82±0.10a 2.65±0.11a 0.81±0.04a
86.56±052a
Barhee 6.14±0.14c 2.98±0.15a 3.09±0.12a 0.95±0.03a
86.84±068a
Lulu
1
Means ± SD followed by the same letter, within a column are not significantly different (P> 0.05).
Table 4. Sugars, soluble and insoluble dietary fibre (%) of date fruit residues (DFR)
Dietary fibre
1
DFR
Sucrose
Fructose
Glucose
Lulu
ND
15.5±a
Khulas
ND
Barhee
ND
Soluble
Insoluble
15.9±a
6.19±b
48.32±a
15.2±a
15.6±a
6.53±b
49.99±a
16.1±a
16.4±a
9.15±a
41.66±b
Means ± SD followed by the same letter, within a column are not significantly different (P> 0.05).
885
Table 5. Minerals content (mg/100g) of date fruit residues (DFR)
Mineral
1
DFR
Barhee
Khulas
Lulu
Ca
192.92±a
194.39±a
119.45±b
Fe
8.66±b
10.73±b
21.60±a
K
515.7±5a
443.38±b
342.28±c
Mg
92.77±b
97.41±b
170.70±a
Mn
1.30±a
1.26±a
2.02±a
Na
16.61±b
20.77±b
30.71±a
P
99.03±b
77.69±c
163.96a±
Zn
1.12±a
1.00±a
2.47±a
Means ± SD followed by the same letter, within a row are not significantly different (P> 0.05).
Table 6. Functional properties of date fruit residues (DFR)
DFR
Water
Absorption
g/g
Fat
Absorption
g/g
Foam
Capacity
Ml %
Foam
Stability
min
Emulsifying
Activity
%
Emulsio
n
Stability
%
Lulu
1.96±0.05a
0.66±0.07a
0.00
0.00
56.17±1.07a
71.46±
1.28a
Khulas
1.98±0.09a
0.67±0.05a
0.00
0.00
56.17±1.71a
71.46±
1.49a
Barhee
2.00±0.09a
0.67±0.04a
0.00
0.00
56.14±1.90a
71.48±
0.98a
1
Means ± SD followed by the same letter, within a column are not significantly different (P> 0.05).
886
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) .(% ٧١و ?EاFر ر ا ا $ر آ#ر dف ا>Hا .8وه>ا
g h +1إ F Reر ا !' ! 7/#ا ر و ,ET! +$ا ?! P,
ا ا ;$اi :ج.
887
888
OP 47
Treated effect of palm pollen grains extract (Phoenix dactylifera L.)
on the sterility induced by acrylamide in male rabbits
A.A.Sawad
Department of Anatomy, College of Veterinary Medicine University of Basrah , Basrah , Iraq
Abstract
The study was performed to know the effect of ethyl alcohol date palm pollen
grains extract to treated the sterility that induced by acrylamide injected to the male
rabbits. Twenty male rabbits were divided into two equal groups, the first received 35
mg / kg BW of acrylamide intramuscularly, the second group treated by the same dose
of acrylamide plus 200 mg / Kg BW of ethyl alcohol pollen grain extract orally for
four weeks. Histologically; the infertility effect of acrylamide led into a significant
decreased in the number of spermatogonia, Spermatocytes, Spermatides and
Spermatozoa, in addition to the weight of the testes, total sperm count, While the
second group showed a significant increase in the above parameters and this evidence
that the extract was capable of the diminishing of the acrylamide infertility effect
Key words: Sterility, Acrylamides, Spermatogonia, Pollen, Epididymis.
Introduction
The date palm (Phoenix dactylifera) for many centuries has been used as a tonic
maimed foods (Rajiv, 2002). The Arab believed that drinking date palm pollen juice
improves the chance of bearing children and many scientists investigates that the date
pollen grains contains estrogen like hormones ,no fewer than 800 uses are recorded for
the date palm(Ali et.al.,1999;El-Mougy et.al.,1991)
The dates has been used especially at morning in the middle east as a reversed the
actions of the toxic materials in man(Al-Qarawi et.al.,2001).Many investigators shows
that the extraction of date palm prevent the action of carbon tetrachloride in which
induced hepatotoxicity in rat(Al-Qarawi et. al.,2004;2001).
Acrylamide is a highly reactive and water- soluble polymer which is commonly used
in both industries and laboratories(Nordin,et.al.,2003). The formation of acrylamide is
particularly association with high temperature cooking process for certain
889
carbohydrates-rich foods, especially when asparagines reacts with sugars (Mottram
et.al.,2002).Reproductive toxicity of acrylamide has extensively tested in mice including
abnormal morphology of sperms(Sakamoto et. al.,1988). Male rats administered with
acrylamide exhibited significant reductions of mating, fertility and pregnancy indices as
well as reduction of transport of sperms in uterus(Tyle,et. al.,2000)
The present study was performed to evaluate the protective role of date palm pollen
grains extract on the sterility induced by acrylamide in male rabbits.
Materials and Methods
Plant Extraction
The plant materials (Spadix) were obtained from the local Basrah market, the spadix
were separately and minced and extracted with 1.5 liter of 75%ethanol for8 hours, and
then was filtered ,the crude extract was obtained after removed the solvent by vacuum
distillation (Harborne,1984)
Animals
Twenty male rabbits aged 126-140 days and weighing 950-1000g were housed at
room temperature under natural photoperiod and maintained on standard pellet diet
and tap water(Alleva,1968).
The animals were divided randomly into two equal groups, the first group( Control)
injected by 35 mg\kgBw of acrylamide intramuscularly, while the second group
(treated) injected by the same dose of acrylamide with 200 mg\ kg Bw of ethyle
alcohol pollen grains extract orally for thirty days.
Rabbits were sacrificed by decapitation ,and testes were removed and weighed, after
isolation of epididymis from each testes of both animal groups ,the cauda epididymis
were minced and homogenized for 1 min in 5 ml of physiological saline
solution(Oishi,2002), the homogenate was filtered through a nylon mesh and the 0.1
ml of filtrate was diluted with 2 ml of saline solution containing 4% Trypan blue,20 ul
a aliquots were placed on the hemocytometer for counting the number of the sperms.
The excised testes were fixed in Bouins solution and processed using standard
laboratory procedures for histology, the tissue was embedded in paraffin blocks,
sectioned with 5 micrometers thickness and stained with hematoxylene and eosin
,stained sections were mounted with DPX and examined using light microscope.
890
Results
The control group (acrylamide treated) animals exhibited a significant decreased (p
0.05) on the testicular weigh after the injection with 35mg\kgBw of acrylamide, the
comparison of the testicles weigh in control group ( acrylamide treated) with the
treated group (acrylamide and ethyl alcohol pollen palm grains extract) they indicates
a significant increase in testicle weight
Most striking feature of the reproductive toxicity of acrylamide was reduced sperm
reserves in cauda epididymis isolated from the control group rabbits in comparison
with the treated group which led to significant increase at the total sperm counts.
The rabbits in the control group which treated by acrylamide shows some evidence of
morphological changes in the testicular histology when compared with the treated group.
The control group showed histopathological changes in the seminiferous tubules (Fig;1)
.There were thickening and multiple layering of tubular endothelium , degeneration of
germ cells, and formation of many multinucleated giant cells in atrophied seminiferous
tubules (Fig;2) in addition to the decreasing of spermatogonia, spermatocytes ,
spermatides and spermatozoa. While the treated group shows that the testes returns to
their normal tissue structure with increasing at the spermatogonia, spermatocytes and
the spermatozoa that appears at the lumen of the seminal tubules (Fig 3).
Discussion
Acrylamide shows increased at the weight of the testes due to the damages of the
germ cells of the testes and decreased the spermatogenic cells, such notes were also
reported by (McCollister,1964;Al-Dijaylli,2001;Al-Hially,2002). And the ethyle
alcohol pollen grain extract leads into a significant increase at the testes weight in
comparison with the treated group and that explain the effect of the extract to reduced
toxic activity of the acrylamide and decrease the level of (LH) hormones secretion
(Salomi et.al.,1991; Nari et.al., 1991 ).
The significant decreased at the total sperm count at control group due to the effect
at the spermatogenesis and the germ cells at different development stages and inhibited
the secretion of the FSH (follicle stimulating hormones) and LH (Luteinizing
hormones) which have a toxic effects at the sperms and reduced the total sperm counts
(Wyobek,1983) .
891
The interaction between the acrylamide and the ethyl alcohol pollen grain extract
group (treated)increased the total sperm count in comparison with control group and
that explain the ability of the palm pollen grains to reduce the toxic effect of the
chemical materials that cause sterility, many investigators studies the effect of palm
pollen grains on the spermatogenic activity and treated some cases of atocia (Sawad
and Faleh,2006 ;Hossaini,1977;Darby,1959).
In the present study we evaluated reproductive toxicity of acrylamide which showed
several histopathological lesions in the seminiferous tubules. There were thickening
and multiple layering at the tubular endothelium , degeneration of the germ cells and
atrophied seminiferous tubules.(Kumi-Daka,1999) .While the treated group shows a
significant increased at the spermatogenesis due to the pollen grain effects to return
the testicular tissue into their normal state(Zeitous and Neff,1995).
892
References
[1] Al-Dijalli AN.2001.Effect of alkaloid and phenolic extract of Allium cepa L.on the
male and female white mice fertility. Ph.D. Thesis, College of science, University
of Babylon. Iraq
[2] Al-Hially AA.2002.Effect of Nigella sativa L.on the male mice fertility with some
physiological blood parameters. MSc. Thesis. College of Science. University of
Kufa. Iraq.
[3] Ali B.H.,Basher A.K. Al-Hadrami G.1999.reproductive hormonal status of rats
Treated with date pits . Food Chem. 66;437-441.
[4] Alleva ,J.J.;Waleski,M.V.;Alleva,F.R. and umberger,E.J. 1968.Synchronizing
effect Of photoperiodicity on ovulation in hamster. Endocrinology .82:123-127.
[5] Al-Qarawi A.A.,Abdel-Rahman HA,El-Miugy SA. 2oo1.Hepatoprotective activity
of licorice in rat liver injury models.J.Herbs Spices Med. Plants 8:7-14.
[6] Al-Qarawi A.A.,Abdel-Rahman HA,El-Miugy SA.2004.Protective effect of
Extracts from dates(phoenix
[7] Induced hepatotoxicity in rats.J.Appl.Res.Vet.Med.(2) 3:176-180
[8] Darby J,Ghalioungui P,Grivetti L1977.Food: The gift of the Osiris.(2) Academic
press,London.53-77.
[9] El-Mougy, S.A.,Abdel Aziz,S.A. Al-Shanawany M. Omar A.1991.The
[10] Gonadotropic activity of palmae in mature male rats.Alexandria j.Pharmac
[11] Sci.5;156-159.
[12] Harborne
j.B.1989.Phytochemical
methods.2nd
Champon
and
Hall,New
york,U.S.A
[13] Hossaini-Tabib MM.1959.Tohf hakim moemen. Mostafavi Press ,Tehran and
Qum.122-124.
[14] Kumi-Diaka J,Nguyen V,Butler A.1999.Cytotoxic potential of ptytochemical
geisfalvon and certain environmental chemical compound on testicular cells.
Biology of the cell.91:515-523.
[15] Mc Collister DD,Oyea F,Rowe VK.1964.Toxicity of Acrylamide.Toxical
Appl.Pharma.6:172-181.
893
[16] Mottram DS,Wedzicha BL,Doson AT.2002.Acrylamide is formed in the maillard
reaction,nature.419:448-449.
[17] Nair SC ,Salomi MJ,Panikkar B,Panikkar KR.1991.Modulatory effects of
Croucus satvus and nigella sativa extract on
cis platin induced toxicity in
mice.J.Enthopharmacol.31(1);75-82.
[18] Nordin AM,Walum E,Kjellstrand P,Forsby A.2003.Acrylamide-induced effects
on general and neurospecific cellular functions during exposure and recovery.
Cell Biol.Toxicol.19:43-51.
[19] Oishi S.2002.Effect of propyl paraben on the male reproductive system. Food
Chem.Toxicol.40:1807-1813.
[20] Rajiv
M.
2002.
Arabia
is
the
home
of
the
date
palm.Internet;www.hinduonnet.com
[21] /the Hindu/thscrip.2005.
[22] Salomi MJ.Nair Sc,Panikkar KR.1991.Inhibirory effects of Nigella sativa and
saffron on chemical carcinogenesis in mice. Nut.Cancer.16(1)67-72.
[23] Sakamoto J,Kurosaka Y,Hashimoto K.1988.Histological changes of acrylamideinduced testicular lesion in mice.Exp.Mod.Pathol.48:324-334.
[24] Sawad AA,Faleh BH.2006.effect of palm pollen extracts on spermatogenic
activity of male rabbits.J.Palm Date Res.
[25] Tyl RW,Marr MC,Myere CB, Ross WP, Friendman MA.2000.Relashioship
between acrylamide reproductive and neurotoxicity in male rats. Reprod.
Toxicol.14:147-157.
[26] Wyrobek AJ, Gordan LA, Burkhart JG, Francis MW, Kapp JRW, Letz G,Malling
HV,Topham JC,Whorton DM.1983. An evaluation of human sperm as indicators of
chemically induced alterations of spermatogenic function.Mut.Res.115(1):73-184.
[27] Zeitoun MAM, Neff WE.1995.Fatty acid triacrylglycerol, Tocopherol, Sterol,
Phospholipid composition and oxidative stability of Egyptian Nigella sativa seed
oil .OCL.2(3):245-248.
894
Table 1: Testes weight (g)
Groups
Right testes
Left testes
Control group
(Acrylamide)
1.73
1.33
Treated group
(Acrylamide + extract)
2.79
2.03
LSD = 21.632
Table 2: Total sperm count
Groups
Total count (10) 6
Control group
(Acrylamide)
57 ± 2.4 *
Treated group (Acrylamide
+ extract)
399 ± 0.30
*
p < 0.0
895
Fig 1:Control group shows atrophied Seminiferous tubules
Fig2:Control group testes showing thickening and multiple layering of tubular
endothelium(A),and formation many multinucleated giant cells in seminiferous tubules
Fig3:Testes isolated from the treated group shows normal tissue structures ( ) Spermatozoa
896
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897
898
OP 48
Response of growing New Zealand white rabbit to dietary date
stone meal with or without commercial enzyme supplementation
EL-Manylawi, M.A.F.
Animal Production Department, Faculty of Agriculture, Cairo University
manylawi@hotmail.com
Abstract
A total number of Sixty New Zealand White growing rabbits aged 5 weeks with
nearly equal live body weights was randomly allotted to five groups with three
replicates of 4 rabbits each and used in this study to determine the response of growing
rabbit performance to dietary date stone meal (DSM) with or without Alltech ® enzyme
in the diet. Five experimental diets were formulated to be approximately isonitrogenous and iso-caloric using 10 or 20% DSM with or without Alltech® enzyme
(at 100 g/ton of rabbit feed). The results indicated that feeding growing rabbits during
the experimental period on diets containing 10% DSM supplemented with Alltech®
enzyme had significantly (P<0.05) improved body weight, body weight gain, feed
intake and feed conversion efficiency compared to those fed DSM in diets either at 10
or 20% without Alltech® enzyme. Diets containing DSM supplemented with Alltech ®
enzyme increased significantly (P>0.05) the digestibility coefficients of CF, EE, and
NFE and slightly increased DM and CP compared to the other experimental diets. The
obtained results indicated that DSM could be used in rabbit diets supplemented with
Alltech® enzyme to get best performance and economic efficiency.
Key words: date stone meal, Alltech® enzyme, Growth, Performance, Digestion.
Introduction
In Arab world countries, increasing the price of conventional feed ingredients and
their shortage are the major limiting factor for continuous and development of rabbits
and poultry industry. Therefore, there are urgent needs to search for alternative
ingredients which could be used as cheap sources to partial or full replacement.
Meantime, there are large quantities of non- utilized residues. In this respect date stone
899
meal could be considered as a cheaper by–product and could successfully substitute
some feedstuffs in rabbit and poultry diets. Date stone meal has already been
considered as a source of energy in poultry feeding[1][2]. The feed cost of animal
nutrition represents more than 70% of the total production cost. Few experiments were
carried out on the use of date stone meal as an ingredient in rabbit feeding. Most
recommended levels ranged from 5 to 20% in poultry feeding. The higher level of date
stone meal in poultry feeding showed a negative effect on performance of poultry. The
negative effects may be due to the lower digestibility coefficients of nutrients or
estrogenic effect such as estradiol
[3]
or due to the high levels of both cellulose and
pentosanes (non starch polysaccharides NSPs) which were found to constitute more
than 30% of crude fibers of date stone meal
[4]
. Rabbits are herbivores and consume
high fiber diets. They are hind-gut fermenters and are capable of retaining small fiber
particles for digestion
[5]
. A higher intake of fibrous diet is achieved when nutrient
requirements are met by digestibility of the non-fiber component
[6]
strategy of rabbits for the utilization of fibrous diets was described by
. The digestive
[7]
. Rabbits can
separate fiber and non-fiber components and retain non-fiber components for
fermentation in the cecum. Benefits of supplementing non-starch polysaccharides rich
diets with exogenous enzymes are well documented. Enzyme mixture could support
the endogenous enzymes of the poultry (amylase and protease), break down some
components of cell wall, which cannot be broken down into absorbable nutrients by
endogenous enzymes, lowering the gastrointestinal viscosity in digestive tract, reduced
nutrient entrapment and releasing other nutrients like minerals [8][9][10][11][12]. The aim of
this work is to determine the response of growing New Zealand rabbits to dietary date
stone meal in the diet with or without enzyme supplementation.
Material and Methods
The experimental work of this study was carried out at Poultry Nutrition Farm,
Animal Production Department, Faculty of Agriculture, Cairo University, during
winter 2010.
Five experimental diets were formulated to cover the requirement of growing rabbits
according to [13] and to be approximately iso-nitrogenous using (10 or 20%) date stone
meal (DSM) with or without Alltech® enzyme at level of 100 g/ton of rabbit feed (Table
900
1). Alltech® enzyme product was purchased from local market. Each gram contains:
Phytase 300 SPU, Alpha-Amylase 30 FAU, Beta-Glucanase 200BGU, Cellulase 40
CMCU, Pectinase 4.000 AJDU, Fungal Protease 700 HUT, Xylanase 100XU.
Date palm stone was purchased from local market and it was processed according to
[10]
by sun-drying for 72 hours and ground in a heavy-duty high rotation hammer mill to
pass through 1 mm. mesh sieve, producing a fine powder suitable for chemical analysis
Sixty New Zealand white growing rabbits 5 weeks of age having approximate equal
live body weights were randomly allotted to five groups with three replicates of four
rabbits each. Rabbits of each replicate were housed in separate cages and kept under the
same managerial hygienic condition. Diets were offered ad-libitum and fresh water was
available all the time during the experiment. Individual live body weight, feed intake
and feed conversion ratio were recorded weekly. Digestibility trails were carried out
using four rabbits from each experimental group at the last week of the experiment.
Rabbits of each group were housed in metabolism cages and feces were collected for
four consecutive days. Proximate analysis of the date stone meal, diets and feces were
carried out according to the methods of [14]. At the end of experimental period (14 weeks
of age), four rabbits were randomly taken from each group for carcass characteristics
and fasted for 12 hours before slaughter according to [15]. The economic efficiency (EcE)
was calculated according to the following equation: EcE = (A-B/B) X 100.
Where A is selling price of obtained gains and B is the feeding cost for these gains
in Egyptian pound (L.E). All data were subjected to analysis of variance using the
general linear models (GLM) procedure of [16] and differences obtained upon statistical
analysis were compared using Duncan multiple range test [17].
Results and Discussion
Chemical composition of the experimental diets and date stone meal (DSM)used is
presented in Table (2).The effects of the experimental diet on body weight, body
weight gains, feed intake and feed conversion values of New Zealand White growing
rabbits during the experimental period (5-14 weeks of age) are shown in Table (3).
The results indicated that feeding growing rabbits on diets containing 10% DPSM
supplemented with Alltech® enzyme had significantly (P<0.05) improved body
weight, body weight gain, feed intake and feed conversion efficiency compared to
901
those fed DSM in diets either at 10 or 20% without Alltech® enzyme. Although,
feeding growing rabbits on diets T3 and T5 containing DSM and supplemented with
Alltech® enzyme the two levels resulted in an improvement in feed conversion
efficiency when compared to those fed diets without Alltech® enzyme (T2 and T4).
The values were 3.57 and 3.86 for T2 and T4 respectively in which diets contained
DSM without enzyme. However, the values were 3.17 and 3.65 for T3 and T5
respectively in which diets contained DSM with enzyme. The improvement in rabbit’s
performance may be due to the benefit role of Alltech® enzyme in improving the
digestibility of experimental diets. The results here in were supported by those
reported by
[11] [12]
who found that supplemented diets containing DSM with enzymes
improved growth performance of growing rabbits.
The enhancement in body weight gain due to enzymes mixture supplementation
was reported by
[12]
on growing rabbits and
[18] [19]
on broilers and
[1]
on turkey. The
positive effect of enzyme supplementation could be explained on the basis that
enzymes mixture has specific enzymes such as xylanase, 902ealand902e and Bglucanase. These multi-enzymes could exert a partial hydrolysis for some antinutritional factors in DSM (cellulose)
[10]
and hence, increase the availability of
nutrients causing an improvement in live body weight
[12]
. Generally, the best feed
conversion was recorded with rabbits group fed 10% date stone meal diets
supplemented with Alltech® enzyme, compared to the other experimental groups.
Digestibility and nutritive values:
Table (4) showed the nutrients digestibility of New Zealand White rabbits fed DSM
with or without Alltech® enzyme supplementation. Results showed that using DSM in
rabbits diets caused an insignificantly decrease in digestion coefficients as compared
with the control diet without DSM, the reduction in digestion
coefficients and
nutritive values of the experimental diets due to DSM could be attributed to the high
level of non- starch polysaccharides (NSPs) like cellulose and pentosans (arabinoxlans
and glucans). These results were agreed with those obtained by
[3]
. On other hand,
diets contained DSM with Alltech® enzyme increased significantly (p>0.05) the
digestibility coefficients of CF, EE, and NFE and slightly increased DM and CP
compared to the other experimental diets. Generally, the highest digestibility of all
902
nutrients was recorded with rabbits fed diets containing 10% date stone meal and
supplemented with Alltech® enzyme. The obtained results may be due to the
hydrolytic action of enzyme as a mixture including glucanase, gulactosidase,
proteinase and 903ealand903e works synergistically to improve the nutritive value of a
diet. The same were found by
[3]
and were agreed with those obtained by
[12]
who
found that substitution of 50% yellow corn by ground date stone in growing rabbits
diets and supplemented with Kemzyme slightly increased the digestibility coefficients
of DM, CP, EE, CF and NFE. In this respect
enzyme
as
a
mixture
including
[20]
revealed that the hydrolytic action of
glucanase,
gulactosidase,
proteinase
and
903ealand903e works synergistically to improve the nutritive value of a diet.
Economical efficiency:
The economic efficiency (EcE) is shown in Table (5). Results revealed that using
DSM in rabbit diets depends on the price of this feedstuff, assuming that the other
costs are constant. Therefore, the relative economic efficiency of feeding diets at
marketing age (14 weeks) was only higher with feeding rabbits on diet containing
10%. DSM with Alltech® enzyme compared to the other experimental diets.
Conclusively, the obtained results indicated that DSM can be used up to level of 20
% in rabbit diets either not supplemented or supplemented with Alltech ® enzyme but
for best performance and economic efficiency it is recommended to use 10% DSM
with Alltech® enzyme
903
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stone meal without or with kemzyme supplementation on productive performance
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Journal. Of Desert
Research, 55: 1-18.
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Muhle Mischfuttertechnik, 125: 112 – 113.
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of digestion during hard and soft feces formation in rabbits. J. Physiol. 338:75-86.
6- Hintz, H.F.; H.F., Schryver, and C.E., Stevens, (1978) Digestion and Absorption in
the hindgut of no-ruminant herbivores. J. Anim. Sci. 46:1803-1807.
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Edition. The interstate Printers and Publishers. Danville 11.
8- Tawfeek, M.I., (1996). Effect of feeding system and supplemented diet with
Alltech® enzyme on growth, blood constituents, carcass traits and reproductive
performance in rabbits under intensive production conditions. Egyptian Journal of
Rabbit Science, 6 : 21 – 37.
9- Simon, O. (2000). Non starch polysaccharides (NSP) hydrolyzing enzyme as feed
additions. Male of action in the gastro intestinal tract. Lohman Information, 23: 7-13.
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Nutritive Value of Date Waste Meal as a Feedstuff for Lohmann Brown Pullets
and Layers. Japan Poultry Science Journal, 46 (4): 303-312.
904
11- Soliman, A.Z.M., R.E. Khidr, M.A.F. EL-Manylawi and S.E.M. El-Sheikh, (2009)
Chemical and biological evaluation of date stone meal as an untraditional feedstuff
in growing rabbit diets. The 5th International Poultry Conference 10-13 March
2009. Taba – Egypt (852-863).
12- Ibrahim, M.R., El-Banna, H.M. and El-Manylawi, M.A. (2010). Evaluating
utilization of ground date stone with or without kemzyme in the diets of growing
new 905ealand rabbits. IV International Date Palm Conference Abu Dubi –UAE
March
(2010).
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http://www.actahort.org/books/882/882_75.htm
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Washington, D.C.
14- A.O.A.C., (1990). Official Methods of Analysis. 13th ed. Association Official
Analytical Chemists, Washington, DC
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SAS Institute, Inc., Cary. NC, US.
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905
Table (1). Ingredients and chemical analysis of the experimental diets
Experimental diets
T1
T2
T3
T4
T5
control 10% DPS 10% DPS 20% DPS 20% DPS
Without
With
Without
With
Alltech®
enzyme
Alltech®
enzyme
Alltech®
enzyme
Alltech®
enzyme
Ingredients (%):
Clover hay
30.00
30.00
29.99
32.00
31.99
date palm stone meal
-------
10.00
10.00
20.00
20.00
Yellow corn
23.53
15.00
15.00
5.00
5.00
Wheat bran
21.34
18.87
18.87
15.87
15.87
Soy bean meal (44%
CP)
18.70
19.70
19.70
20.70
20.70
molasses
3.00
3.00
3.00
3.00
3.00
Di Ca-Phosphour
2.27
2.27
2.27
2.27
2.27
Salt
0.50
0.50
0.50
0.50
0.50
Vit & Min. Premix*
0.30
0.30
0.30
0.30
0.30
Lime stone
0.22
0.22
0.22
0.22
0.22
Methionine
0.14
0.14
0.14
0.14
0.14
Alltech® enzyme
-------
-------
0.01
-------
0.01
Total
100
100
100
100
100
* Vitamin and mineral premix at 0.3% of diet supplies the following per kg of diet: Vit. A 1200 IU,
; 500.000 IU.D3; 0.67 mg Vit.K3;0.67 mg Vit B1; 2.0 mg Vit.B2; 0.67 mg Vit.B6; 0.0004 mg
Vit.B12; 16.7 mg Pantothenic acid; 0.07 mg Biotin; 1.67 mg Folic acid; 400 mg Choline chloride;
22.3 mg Zn; 10 mg Mn; 25 mg Fe; 1.67 mg Cu; 0.25 mg I; 0.033 mg Se and 133.4 mg Mg.
906
Table (2) Chemical analysis of the experimental diets and date stone meal (DPSM)
T1
control
Determined analysis
(%):
CP
DE**
CF
EE
Ca
Ph
Lys.
Meth.
DM
89.3
OM
96.8
Experimental diets
T2
T3
T4
10% DPS 10% DPS 20% DPS
Without
With
Without
®
®
Alltech
Alltech
Alltech®
enzyme
enzyme
enzyme
17.49
17.44
17.42
2675
2699
2699
11.60
12.60
12.59
2.44
2.86
2.83
1.93
1.95
1.94
0.96
0.91
0.98
0.73
0.76
0.76
0.36
0.35
0.40
Date palm stone meal (DPS)
CP
CF
EE
Ash
6.0
13.9
7.8
3.2
T5
20% DPS
With
Alltech®
enzyme
17.38
2702
14.13
3.25
1.98
0.90
0.79
0.34
17.35
2702
14.00
3.20
1.96
0.96
0.79
0.36
DE**
3468
NFE
69.0
** Calculated analysis
Table (3): Growth performance of New Zealand White rabbits fed DSM with or without
Alltech® enzyme supplementation during the growing period from 5 to 14 weeks of age
T1
control
Item
T2
10%
DPS
Witho
ut
Alltech
®
enzym
e
Initial body weight (gm)
796
796
a
1951
ab
Experimental diets
T4
T3
T5
20%
10%
20%
DPS
DPS
DPS
Witho
With
With
ut
Alltech
Alltech
®
®
Alltech
®
enzym
enzym
e
e
enzym
e
791
2091
786
a
1771
796
c
1884
bc
MSE
1.75
Body weight at 14 weeks (gm)
2046
64.62
Total body weight gain
1250 a
1155ab
1400 a
985 c
1088bc
64.11
Total feed intake
4175ab
4123 c
4438 a
3802bc
3971ab
135.59
Feed conversion
3.34bc
3.57ab
3.17 c
3.86 a
3.65ab
0.24
a,b,c Means values in the same row bearing different letters differ significantly (P<0.05)
907
Table (4). Nutrient digestibility of New Zealand White rabbits fed DSM with or
without Alltech® enzyme supplementation.
Experimental diets
Item
T1
T2
T3
T4
T5
control 10%DPS 10%DPS 20%DPS 20%DPS
Without With Without With
Alltech® Alltech® Alltech® Alltech®
enzyme enzyme enzyme enzyme
MSE
Digestibility
Dry matter (DM)
76.35
76.09
77.61
75.14
76.43
1.26
Crude protein (CP)
79.27
78.69
79.45
77.00
78.35
1.01
ab
40.56
b
47.57
a
38.81
c
45.07
ab
Crude Fiber (CF)
41.21
1.71
Ether extract (EE)
65.50ab
62.13b
69.61 a
63.51c
66.63ab
1.00
Nitrogen free extract (NFE)
86.62ab
85.49 b
86.71 a
79.94 c
87.03ab
1.00
a,b,c Means values in the same row bearing different letters differ significantly (P<0.05)
Table (5). Economic efficiency of the experiment treatments.
Experimental treatment
Item
T1
T2
T3
T4
T5
Number of survival rabbits
12.00
12.00
12.00
12.00
12.00
Average feed intake/rabbit (Kg)
4.18
4.12
4.44
3.80
3.97
Total feed intake (Kg)
50.10
49.48
53.26
45.62
47.65
Price/Kg feed (L.E)
2.96
2.80
2.86
2.64
2.70
Total feed cost (L.E)
148.17
138.45
152.42
120.42
128.80
Average body weight gain (Kg)
1.25
1.16
1.40
0.99
1.09
Total meat yield (Kg)
15.00
13.86
16.80
11.82
13.05
Selling price * (L.E)
525.00
485.10
588.00
413.70
456.96
Economic Efficiency (EcE) **
376.83
346.65
435.58
293.28
328.16
Relative economic efficiency ***
100.00
91.99
115.59
77.83
87.08
*
Selling price of 1 Kg = 35 L.E.
** Economic efficiency (EcE) = Selling total meat yield – total feed cost
*** Relative economic efficiency = (Treatment EcE ÷ Control EcE) X 100
908
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PP 13
Analyzing the Role of Agricultural Extension services in Date
Palm Orchards in Khairpur, Pakistan.
Shakeel Ahmed Ibupoto, Wazir Ali Maitlo and Mushtaque Ahmed Jatoi
Date Palm Research Institute, Shah Abdul Latif University, Khairpur, Sindh, Pakistan
Shakeelibupoto@gmail.com
Abstract
Dates are major horticultural crop in Middle East region. It is the major fruit crop of
arid deserts, in tropical and subtropical areas of the world including Gulf Cooperation
Council of Northern Africa and Southern Asia. In Pakistan date palm are grown in four
provinces. The area under cultivation of date palm is 98.7 thousand hectares with total
production 735 thousand tons. The date palm of Sindh is mainly concentrated in district
Khairpur and it is the biodiversity centre for the date palm having more than 300
varieties, which is also major date producing district of Pakistan. The Agriculture
Extension Services play an important role in agriculture development by improving
agricultural practices and adopting new production technologies in farming. The data
used in this study was collected directly from date palm growers in district Khairpur
using specially designed questionnaire. Data were collected through field survey in date
palm growing area. The 60 growers in the study area were selected for this purpose.
The Field survey was conducted regarding use of various inputs in date palm orchards,
planting method of newly planted offshoots, irrigation, pollination, cultural practices,
protection from pests & pathogens etc. The aim of present study was to conduct survey
and to improve agricultural practices in date palm orchards & to disseminate
information for production technologies to date palm growers. It was concluded from
the survey that awareness level of the respondents varied too much for different aspects
of crop and orchard management. In general not very large percentage of growers was
aware of latest developments in the field. The study therefore recommends agricultural
extension programs to date palm growers, stakeholders, researchers and Government.
The data were analyzed using the statistical software SPSS. A secondary data source
was also used to draw out results and suggestions.
Key words: Extension Services, Date Palm, Level of Information,
911
Introduction
Pakistan’s agro-climatic conditions provide a suitable environment for the
production of various horticultural crops, as well as a strong comparative advantage
in horticulture sector. Citrus, Dates and Mangoes are Pakistan’s most important
export fruits, constituting about 78 percent of the total value of Pakistan’s fruit
exports (Ministry of Food and Agriculture Government of Pakistan, 2007).[9] The
Date Palm is cultivated in all Continents of the World (except Antarctica) but vary in
area and production. The major dates producing countries include;
Saudi Arabia,
Iran, Egypt, Pakistan, Iraq, Morocco, Tunisia, Algeria, USA and Israel. In Pakistan
date palm is grown in all four provinces. The area under cultivation of date palm is
98.7 thousand hectares with total production of 735 thousand tons (FAO, 2009). [4]
Dates are growing in all four provinces of Pakistan. The main dates producing areas
of Pakistan are Turbat and Panjgoor (Baluchistan), Khairpur and Sukkur (Sindh),
Muzaffar Garh, Jhang, Multan and D.G Khan (Punjab) and D.I. Khan (NWFP).As
per figures of Federal Bureau of Statistics of Pakistan (2008-09) [3], year wise area
and production of Dates of Pakistan is given in “000” thousands of hectares and
production in tons.
2006-07
2007-08
2008-09
2009-10*
Area Production Area Production Area Production Area
84.7
426.3
90.1
557.5
90.7
566.4
98.7
roduction
735
Source: Federal Bureau of Statistics of Pakistan, 2008-09. *FAO, 2009
Date Palm is an important crop of Sindh, mainly growing in Khairpur and
Sukkur districts. Almost 85% of the Sindh dates produced only in Khairpur (Jatoi
et al., 2009). [5]
912
2005-06
Pak
Sind
h
8199
2668
(Ha)
1
1
Product
4965
1928
76
10
Area
ion (Tons)
2006-07
Sha
re %
32.
54
38.
83
Pak
Sind
h
8469
2926
5
3
4262
2010
81
20
2008-09
Sh
are
Pak
%
34.
55
47.
16
Sind
h
9070
3240
0
0
5664
2619
00
00
Sh
are
%
35.
72
46.
23
Source: Federal Bureau of Statistics of Pakistan, 2008-09.
Khairpur District is located in northern Sindh. Khairpur is noted for its bountiful
harvest of dates and it is the biodiversity centre of dates having more than 300
varieties (Markhand and Abdul Soad, 2010; [8] Mahar, 2007). [7] The dry, hot climate
makes the fruit very sweet, supple and juicy. It is very hot and sunny during the
summer and cold in winter. Humidity is low. Khairpur gets its water through a web of
canals coming out of River Indus. The average literacy rate for males is 65% and only
25% for females.
Agricultural extension services play an important role in agricultural development
and can contribute to improving the welfare of growers and other people living in rural
areas (Anderson, 2007). [1] It is the entire set of organizations that support and facilitate
people engaged in agricultural production to solve problems and to obtain information,
skills and technologies to improve their livelihoods. Extension services can be
organized and delivered in a variety of forms, but their ultimate aim is to increase
growers’ productivity and income. The agricultural extension can contribute to the
reduction of the productivity differential by increasing the speed of technology transfer
and by increasing farmers’ knowledge and assisting them in improving farm
management practices by the use of various inputs (Birkhaeuser et al., 1991).
[2]
Additionally, extension services also play an important role in improving the
information flow from farmers to scientists.
The production and export goals can be achieved through education and trainings
of
local growers by exposing them to adopt modern technology in date palm
913
plantation and management, which may include selection of high yielding varieties,
proper use of fertilizer and irrigation, proper time and method of pollination,
harvesting, drying, grading, storing, transporting and marketing of produce and to
protect the date palm from harmful Pests & pathogens which destroyed the date palm
trees (Khan et al, 2004). [6]
The date palm being a renewable resource needs to be given proper management
for its sustainability. The object of this research was to highlight the problems of
date palm orchard management by the use of various inputs and make
recommendations for their solutions. And to find out the solution of the following
questions:
What adaptive measures have farmers adopted in the study area effects
or impacts of dates production?
What roles can agricultural extension play in building the adaptive capacities of
vulnerable people in the study area?
Methodology
The study area and sampling techniques
The study was carried out in district Khairpur using specially designed questionnaire.
Data was collected through field survey in date palm growing area. A multi-stage
random sampling technique was used to select respondents. A Focus Group Discussion
(FGD) was held in each zone to obtain information from growers about the use of
various techniques and inputs which are necessary in management of date palm
orchards. The methodology provides a structure and ways for various aspects of the
problems which is under consideration for level of growers in the study area.
Data collection Technique
The Primary data about the cultivation of date palm practices in the Khairpur
district was collected during the field survey through personal interviews at individual
and group discussion level of date palm growers of different farm category to analyze
the weaknesses of Date palm production technology that affects their farming practices
and production. The information collected through questionnaire includes: land
preparation, planting method maintenance, harvesting, pollination and cultural
practices as well as other date palm associated problems. A total of 60 growers of
Khairpur were interviewed.
914
Analytical Procedure
The data were analyzed using the Special statistical analysis software SPSS and
tabulated for secondary data.
Results and Discussion
The results indicated that the majority of respondents belong to small awareness
level in all the components of scientific date palm farming practices. On the basis of
overall awareness Level respondents categorized into small (48.33%), medium
(38.33%) and large level (33.33%) as shown in Table 1.
This study also evaluated the frequency of date palm growers using traditional and
modern cultivation practices in management of orchards. The results presented in table
2 indicated that 55% of the total growers were using both traditional and modern
cultivation methods, 15% had totally converted their date palm farming in an
improved and modern farming practices, whereas 30% were still entirely engaged with
traditional Date palm cultivation methods.
According to the obtained results the overall awareness level about the use of
various inputs and techniques in management of date palm orchards can be divided in
to three categories; high, medium and low as indicated in table 3. The majority of
respondents were well aware about: Picking (95.0%) followed by Pollination
(91.67%), Cultural Practices (85.0%), Intercropping (73.33%), Irrigation (73.3%),
Increased Land Range Date Palm Orchards and change in timing of plantation for
management of orchards (50%) respectively. whereas medium level awareness about:
Use of organic manure (21%), Disease (21.67%), Timely Pruning of orchards
(23.03%), Irrigation schemes (28.3%), Planting method (28.33%), Pesticide
application (33.33%) and Fertilizer applications (41.67%). The lowest awareness level
was recorded on weather forecast (5%) followed by use of cover crops and mulching
(8.3%), Pruning devices (13.3%), Mechanization (13.3), Improved Tillage Practice
(15%), Weed management (16.6), Pests (18.33%) respectively.
Conclusion and Recommendations
It may be concluded that knowledge of improved date palm farming practices in
study area in District Khairpur is unsatisfactory for development of date palm sector.
This failure may be attributed to lack of intensive efforts to transfer the technologies
915
from the scientific institution to the grass root level. Therefore Scientists, Department
of Agriculture Extension (Govt. of Sindh), District Government Khairpur and
Research Institutes with the help of Extension officers and Researchers must
periodically conduct training and awareness programs on the use of improved
production technology for date palm farming.
The key role of extension service is adaptation to use of production technology to
acquire new knowledge and skills. It is therefore recommended that extension should
brace up to the new challenges by transformation technology to study area for
awareness and disseminating proven measures to boost the adaptive/resilience
capacities of various stakeholders and date palm growers, there is a need for increased
research and innovation in date palm farming practices to find out more Sustainable
ways of adaptation of improved technologies.
916
References
1. Anderson, J.R. 2007. Agricultural Advisory Services’, Background Paper for the
World Development Report 2008.
2. Birkhaeuser, D., Evenson, R.E and Feder, G. 1991. The Economic Impact of
Agricultural Extension: A Review’, Econ. Dev. Cultural Change, 39:607-650.
3. Federal Bureau of Statistics of Pakistan, 2008-09, Pakistan (Year book 2010.)
4. Food and Agricultural Organization of United Nation, 2009.
5. Jatoi, M.A., Markhand, Z and Solangi, N. 2009. Dates in Sindh: facts and figures.
Proceedings of the “International Dates Seminar” organized by Date Palm Research
Institute, Shah Abdul Latif University, Khairpur, Sindh, Pakistan on 29 July, 2009.
6. Khan, A., Islam, M and Shaukat, S.S. 2004. Nematodes associated with date-palm
and their management using Marigold. Sarhad J. Agric. 20: 447-452.
7. Mahar, A.Q. 2007. Post-harvest studies of different varieties of Date Palm
(Phoenix dactylifera L.) fruits, their protection, identification, processing and
preservation at district Khairpur, Sindh, Pakistan. Ph.D Thesis, Date Palm
Research Institute, Shah Abdul Latif University, Khairpur, Sindh, Pakistan.
8. Markhand, G. S., Abul-Soad, A. A., Mirbahar, A.A and Kanhar, N. A. 2010. Fruit
Characterization of Pakistani Dates. Pak. J. Bot., 42(6): 3715-3722, 2010.
9. Ministry of Food and Agriculture Government of Pakistan (2007). Fruit Vegetables
and Condiments Statistics of Pakistan, Trade and Investment Wing, Islamabad.
917
Table (1): Farm Categorization of date palm growers
on the basis of overall awareness Level respondents.
Farm Category
Respondents
Percentage%
Small
29
48.33
Medium
23
38.33
Large
8
13.33
Total
60
100
Table (2): Method of cultivation adopted by
date palm growers in Khairpur.
Method of cultivation
Frequency
Percentage%
Traditional
18
30
Improved
9
15
Both
33
55
Total
60
100
918
Table (3): Awareness level on production Technology for orchard Management in the Study area.
low
Medium
High
Awareness
level
production Technology
Respondents
Percentage
Picking
57
95.0
Pollination
55
91.67
cultural practices
51
85.0
Intercropping
44
73.33
Irrigation
41
68.33
Increased range land date palm orchards
35
58.3
Changes in the timing of land
management
30
50
Fertilizer
25
41.67
Stage of Pesticide applications
20
33.33
Varieties
19
31.67
Planting method
17
28.33
Processing of crops to reduce post
harvest losses
17
28.3
Irrigation schemes
17
28.3
Timely Pruning of orchards
14
23.3
Disease
13
21.67
Use of organic manure
13
21.00
Pests
11
18.33
Pulling out weeding
10
16.6
Adoption of improved tillage practices
9
15
Increased mechanization of agricultural
production
8
13.3
Cutting of infected plants to reduce the
incidence of disease
8
13.3
Mulching/ use of cover crops
5
8.3
Use of weather forecasts
3
5
919
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920
PP 14
Development of New Innovative Procedure for Accelerated
Ripening of Dhakki Dates.
Shahzada A. Saleem*, Ambreen A. Saddozai1, Muhammad Asif2,
Muhammad Mansoor3 and Ahmad K. Baloch4
*
Agriculture Research Institute, Dera Ismail Khan, Pakistan
sasdikpk@gmail.com
1
Food Microbiology Labs. National Agricultural Research Council, Islamabad, Pakistan.
2
Directorate of Planning, ALP, PARC, Islamabad, Pakistan.
3
Arid Agricultural Research Institute, Dera Ismail Khan, Pakistan.
4
Department of Food Science & Technology, Gomal University, D. I. Khan.
Abstract
Date palm (Phoenix dactylifera L.) is playing a vital role by providing food & shelter
to millions. A prominent local cultivar “Dhakki” of Dera Ismail Khan is economically
far more important for having jumbo size & heavy weights with small stone, fine
texture & delicious taste, therefore is highly suitable for export targets. However, being
a late variety it is confronted with enormous environmental stresses. Stormy monsoon
season coincidence with the period of dates ripening, unbalanced production/
consumption, & lack of preservation technology are few extremely disturbing factors
causing quality deterioration & excessive post-harvest losses. The aim of the study is to
induce well advance rapid artificial ripening in Dhakki fruits harvested at firm &
astringent Doka stage, & complete curing/drying before the fall of monsoon. Brine &
vinegar acid has been investigated as ripening initiator/ accelerator, applying
individually and/ or in combined form at 0.25 to 3.5 % concentration. The Doka
immersed in a treatment solution for 5 min was ripened/ cured in an aerated incubator
at 38 to 40°C for 72 hrs. Observing changes in color shade, fruit weight, pulp, texture,
total soluble solids, appearance & the extent of ripening assessed the efficiency of the
treatment. All of the treatments induced ripening of varying degree. However, 2 %
brine appeared highly effective introducing accelerated ripening & causing 75 %
excessive yield over the control sample. The process does not require fruits to attain the
Dong (Rutab) stage for ripening initiation, & hence saved 2-3 weeks period.
921
Introduction
Date palm (Phoenix dactylifera L.) is well recognized ever since the birth of the
human race and since then it is playing important role. It nourishes millions all over the
world and contributes significantly towards development and prosperity especially to
those living in the Arabian world. The Prophet Muhammad (PBUH) encouraged his
followers to honor the tree by calling it their “Aunt”. Muslims consider it as a virtue to
distribute and eat dates particularly at occasion of ‘Iftar’ in the month of ‘Ramadan’.
Date fruits being sweet and palatable, are consumed as a staple diet, and provide 25003000 Calories/Kg of physiological energy, high amount of potassium, easily digestible
carbohydrates, and a significant amount of cellulose and hemi-cellulose materials, but are
virtually free from sodium and cholesterol. On the basis of composition and usefulness
the dates are considered a divine gift particularly for heart patients. The dates are one of
our important cash crops, and a good source of foreign exchange earnings. Pakistan is 5th
largest date producing country in the world constituting about 11% of total world
production [4]. Dhakki date of Dera Ismail Khan is the most promising local variety
reckoned amongst the top few world-leading varieties. The date is quite popular for its
large size (5-6 cm long and 2-3 cm thick) and fruit weight (20-25 gm/ fruit) with fine
texture and relish taste [6] fetching high price in the market.
Although the Dhakki date is a crop of national importance yet it faces diversified
problems. Being a late variety harvesting period of the date coincides with the time of
high humidity and stormy monsoon, and the date consequently doesn’t have a chance to
ripen properly, and insect/bird pest problems and physical damages are common.
Moreover, a large quantity of freshly ripened fruits available from early date varieties
gluts the market and the limited available provision with the farmers is preoccupied,
consequently the Dhakki date suffers heavy crop losses and quality degradation. Quality
of the dates is further impaired using traditional processing techniques of exposing the
fruits on mats to sun at open air. Besides dust and other harmful contamination, the
sizeable amount of the product turns out to be fermented due to insufficient drying and
storage facilities. In order to minimize post harvest damages, reduce financial losses
and improve quality it becomes imperative to ripen dates artificially. In our previous
study we have reported ripening/ curing of Dhakki dates using microwave radiation [15]
922
[7]. Information regarding ripening of dates using salt and vinegar acid is inadequate, and
the few reports available are variety specific [14] [5]. The present study is therefore
aimed at investigating the impact of table salt and vinegar acid as ripening promoters
for Dhakki dates.
Research Methodology
The mature Doka of Dhakki fruits of 250-300 hardness, [7] harvested before the
onset of Dong formation were procured from the Agriculture Research Farm, Ratta
Kulachi, Dera Ismail Khan. Healthy and non-infected fruits were selected for
experimentation. The fruits were given cleaning and washing before subjecting to
ripening treatments with solutions of brine (0.25 to 3.5 %) and vinegar acid (0.25 to
2.5 %, Table 1). The fruits were divided into thirteen lots (1 Kg) each of equal size and
maturity. The samples were immersed in the respective solution (1 L) for 4-5 minutes
at room temperature, allowed to drain and spread out separately on stainless steel
trays. The samples were incubated for 72 hours in an aerated incubator at 38 to 40°C.
A control with water dip treatment was run simultaneously to compare the
effectiveness of the ripening stimulants.
Data collection
Ripening parameters were evaluated immediately after harvesting and ripening
treatments. Performance of a stimulant whether used singly or in combination was judged
by following changes in color, texture, average fruit weight, percent pulp and total
soluble solids (T.S.S). The color of the fruit was recorded by visual observations
comparing with a Horticultural Color Chart [13]. Average fruit weight (gm) and pulp
were recorded by averaging weight of the ripened seed-free fruits. A hardness testing
device developed in the laboratory (Fig. 1, [7]) determined the texture of the ripened fruit.
The degree of ripeness (which also represents yield) and the total soluble solids
were determined according to the methods reported earlier [15] [7]. An organoleptic
test for fruit appearance was performed according to Jellinek [12]. For the evaluation the
samples were distributed to ten panel members from graduate students of the Department
of Food Technology. The evaluation was based on a hedonic scale ranging from 1 to 10
where 1 ranks for very poor, and the 10 stands for the best sample. The samples were
evaluated twice and the average values reported.
923
Results and Discussion
There are at least 4 stages of development and ripening for dates. These include
Kimri, Khalaal, Rutab and Tamar (Fig. 2). The fruits at Khalaal being fully-grown
and mature tend to ripen to acquire half-ripened Rutab stage, which then leads to fully
ripened Tamar form of reduced moisture contents. The changeover of stages through
normal ripening process while fruits mostly remaining attached to tree requires 5-6
weeks period, besides 2-3 weeks further for curing/ drying to biomass of reduced
moisture contents necessary to give shelf stability. The idea behind the study is to
artificially induce speedy ripening in Dhakki Doka at firm, astringent and yellow
stage using stimulants. The curing /drying completed rapidly cutting down most of
the period necessary for Rutab and Tamar conversions (Fig. 2). Adapting the
proposed methodology it is highly likely to avoid detrimental monsoon and reduce
fruit losses considerably. The energy, time and expenses are possibly saved, besides
obtaining the product of better quality and hygienically sound. The results are
discussed in the following sections.
Changes in Fruit Color
Color of fruit is an important index, which plays a pivotal role in the marketing
value and quality determination. Similarly, variation in the color is closely associated
with the extent of ripening. However different cultivars display their own color pattern
on ripening. Dhakki Dokas change their color giving different shades from yellow to
brown during ripening/ curing. The color tends to change as a result of biochemical
activities as induced inside the Doka by the chemical stimulants. The range of colors is
presented in Table 2.
All of the treatments exerted a positive effect on this important property. Normally,
the Dhakki date undergoing ripening changes color from light yellow at Khalaal to
golden brown at the Rutab stage. However, the color shade varied with the nature of
the treatment applied and the extent of ripening (Table 2). The fruits while ripening
under the influence of the chemicals changed color much earlier compared to the
control treatment. The effect was much pronounced with brine treatments and that no
matter whether the salt was employed alone or in combination with vinegar acid, gave
an acceptable ripening color. The data further revealed that brine at 2 % concentration
924
in conjunction with the vinegar acid at any ratio imparted a desired reddish brown and
attractive color with good eye appeal. However, 2 % brine treatment applied alone
offers superior color shine surpassing all other treatments, whereas the color produced
by vinegar acid, as a single treatment was not agreeable. The results indicate however,
that both of the chemicals triggered physiological changes activating ripening
enzymes. Brine responded swiftly in bringing forth change in desired color on
ripening. [5] had also reported a similar finding for Khasab dates, however the same
reagents appeared ineffective for Zaidi & Thoory date varieties [14].
Fruit Texture
The effect of stimulants on texture of date fruit is given in Table 3. The texture of the
fruits treated with the control treatment was slightly soft and firm whereas brine singly
as well as in combination with vinegar acid conferred a soft texture to the fruits. Further
the samples treated with T3, T4, T5, T10, T11, T12 and T13 were found to be much softer,
more pulpy and juicy. It is also noted that the fruits cured by these treatments were
more flaccid at the surface; however increased firmness was noticed towards the center
of the fruits. This indicates that the process of ripening initiated from the fruit surface,
and progressed inside to the fruit core. The fruits ripened by the control treatment
showed a uniform softness throughout, though it was to a lesser degree. Previously the
microwave radiation had also caused a uniform ripening throughout the fruit body [7].
[14]Kalra also reported that salt and vinegar acid produced surface ripening of the
dates. Further, wrinkling was highly pronounced in the case of fruits ripened using the
control treatment; however, fruits treated with salt were least furrowed. Wrinkling of
the fruits most probably resulted from collapsing of the weakened tissues with
disproportionate moisture losses from the fruits undergoing ripening.
Fruit Weight and Pulp Contents
Data on average fruit weight and pulp contents are presented in Table 4. Salt treated
fruits gave higher fruit weight and pulp contents than those for water (control) or
vinegar acid treated samples. A slight increase in fruit weight or pulp was seen in the
case of combination treatments, and the amount was further increased on increasing
the concentration of vinegar acid or salt. However, the extent of rise was much
pronounced in case of salt treatment. Nevertheless both of the treatments performed
925
better than the control. The higher weight for the brine treated fruits may be attributed
to high pulp percent and to more juiciness. This finding is dissimilar to the results from
Khasab date variety where weight and pulp percentage was lower in fruits treated with
vinegar acid alone or in combination with salt as compared to the control samples [5].
Slight variation in the results might have been caused by varietal or procedural factors.
Total Soluble Solids (T.S.S.)
Data regarding total soluble solids are presented in Table 4. The data show that the
samples with treatment T4 and T5 had the highest amount of total soluble solids
compared to other treatments, while the effectiveness of treatment T8 and T9 is of a
minimum degree. T.S.S is considered in relation to total weight of the fruit. It is
generally specified as to what extent the T.S.S. is present in relation to water. It means
that the freshly harvested dates are said to contain more water and less total soluble
solids. It shows that a decrease in moisture or water contents results in more
concentrated fruits T.S.S. Further, water evaporates during incubation for ripening/
curing and moreover, the insoluble fruit materials get solubilized simultaneously as a
result of breaking down of polymers through ripening. This is what has presently been
observed from the data (Table 4). It is however, indicated clearly that treatment with
salt singly or in combination with vinegar acid has responded well giving a higher
percentage of T.S.S. The rise in T.S.S. from brine treatment resulted from conversion of
water-insoluble protopectin to pectin on stimulation of the ripening process by the salt.
Moreover, the higher amount of the soluble solids in salt treated samples prevented
moisture from evaporating easily, hence the cured fruits maintained their juiciness.
Fruit treated with vinegar acid singly showed a lower percentage of T.S.S. as against
the control. This is probably due to greater amount of water loss from the control
samples. The present findings showed a similarity with earlier reports [5], [8], [1] &
[19]. However, some of the berries after treatment with 3.5 % brine and those from
combination treatments became over soft and developed a disagreeable taste and flavor.
Fruits Ripening
Apparently all of the treatments helped inducing ripening of Dhakki date to a
certain degree (Fig. 3). However, the extent of ripening varied with the nature of the
stimulant and amount of the stimulant for application. Increasing the concentration of
926
brine resulted in a progressive increase in the percentage of fruit ripeness and color
change. Vinegar acid also showed a similar trend, but a far lesser degree. Combination
treatments also induced considerable amount of ripening. Maximum ripening as high
as 90% was observed for 2 % brine containing 2.5 % vinegar acid treatment, followed
by 88 % & 87 % for 2 % brine with 1.5% vinegar acid and 3.5 % brine treatments
respectively. Only a few fruits from the control treatment approached the required
ripening level, thus displaying very poor performance. The findings agree with the
results of [14] who reported that the increase in salt concentration progressively
increased the Dong formation of Khadrawi and Shamran date cultivars, and addition of
vinegar acid enhanced the effect, but vinegar acid alone was ineffective. They further
reported that none of the treatments induced softening in Zaidi and Thoory varieties. In
our study however, the effect of vinegar acid alone on Dhakki date was not significant,
though more effective compared to the control treatment. Currently, brine at 2 %
concentration has accelerated the ripening process efficiently and yielded an optimum
quality product. The detection of invertase, polygalacturonase, cellulase, polyphenol
oxidase and other ripening enzymes has been reported in dates at the Doka stage [10]
& [11]. It has further been reported that polygalacturonase and cellulase were absent or
present at only a low level in the green date fruit but displayed large increases in
activity during ripening [10] & [15].
Ranking Values/ Organoleptic Ratings
The brine treated fruits appeared superior in ranking to those from the vinegar acid
(Fig. 4). Treatment with vinegar acid caused light to dark brown unappealing spots on
some of the fruits. The effect was intensified with increasing concentration of the acid.
Combined treatments containing acid also affected the appearance, but to a lesser
degree. Some of the fruits from 3.5 % brine treatment and those from combination
treatments with 2% acid appeared over soft. Moreover some of the acid treated fruits
showed signs of fungal growth on the surface. Fruits treated with the highest
concentration of brine (3.5 %) were slightly salty in taste while an acid-taste was not
detectable in acid treated fruits, except for those over softened fruits, which showed a
slightly acidic flavor. The softened fruits were not astringents in taste and flavor, and
the tannins of the fruits are likely to have been precipitated.
927
Conclusion
It was shown that brine and vinegar acid exerted a positive response inducing the
ripening in Dhakki dates and accelerated the ripening/-curing pace. However, brine
used alone proved more effectiveness and the quality changes in terms of color, taste
and appearance were more pleasant. Previously it was demonstrated that the ripening
of Doka at 250 hardness units is triggered by application of an optimum dose of
microwave radiation [6], [7] & [15]. Since at present, considerable ripening also occurs
in Doka with 250 hardness consequent upon the ripening activators, it proves beyond
doubt that leaving the fruit on the tree for want of Dong formation through natural
process, is quite unjustified. It is highly likely that the post-harvest processing can be
got completed at least 2 weeks before the start of the monsoon season, hence avoiding
adverse consequences, provided the Doka with full maturity is treated with the
optimum dose of reagents, and allowed to cure under appropriate conditions. It is
postulated that sufficient amount of the ripening enzyme existed as an immobile form
in unripe yet fully mature fruits. The ripening agents possibly disrupt the epidermal
cell and the protoplasm thereby releasing and activating the enzymes. Ripening by
involvement of enzymes like invertase, polygalacturonase, cellulase, pectin esterase
and polyphenol oxidases etc., causes the structural parts like pectin and cellulose that
hold cells together to became solubilized, and the tannin precipitated. As a result the
fruit manifests the ripeness states of Rutab and Tamar precipitating out tannins, tasting
sweeter, softening in structure and introducing changes in fruit color and other
ripening associated quality parameters. The extent of modification varies at a rate
depending upon the stage of fruit maturity and the environmental factors responsible
for the ripening/ curing of the fruits. The present findings agree with our previous
views that there is no point in waiting till Dong formation on-tree once the fruit attains
full maturity of Doka level. Although treatment with salt involves some extra work
and cost, however, the grower can resort to remedies to save his perishable commodity
at a Khalaal stage. The harvesting period can easily be shortened by the manipulation
of enzyme inducing techniques thus saving the fruits from significant damage.
Addition of enzyme preparations to dates has been reported to hasten desirable
conversions. Various enzyme preparations containing invertase [17] & [9] for texture
928
and appearance of "sugar wall" dates, and pectic enzymes and cellulases [16] & [18]
for quality improvement of mixed green and substandard dates have been reported.
The ripening and curing processes are simultaneously initiated as a result of chemical
applications transforming the perishable Khalaal of Dhakki fruit to its preserved
Tamar form, and hence bypassing the on-tree Rutab phase successfully. The
conversion resulted to a decrease in fruit weight due to moisture loss, increase in
soluble solids, yielding a soft pliable texture, a browning of the mass, and the
development of the taste and flavor of Tamar date. In the present study changes
brought about by the activity of the ripening enzymes were reflected in a reduction in
fruit firmness, color changes from yellow to reddish brown and taste development, and
the treatments possibly activated such enzymes. It was further observed that the
ripening initiated from the surface of the fruit and progressed inwards.
Acknowledgements
The author thanks the support of PAKISTAN AGRICULTURAL RESEARCH
COUNCIL (PARC) Pakistan for providing the funds under AGRICULTURAL
LINKAGES PROGRAMME (ALP) to Agriculture Research Institute for executing
of these studies.
929
References
[1] Ali, L. 1989. Effect of different chemical treatments on physio-chemical
characteristics and shelf life of date fruit (Phoenix dactylifera L). M. Sc. Thesis.
Agric. Uni. Faisalabad, Pakistan.
[2] Anon. 2000. FAO Production Yearbook.
[3] Anon. 2001. 3rd National Exhibition on Dates, Dera Ismail. Khan. Export Promotion
Bureau, Pakistan.
[4] Anon. 2008. Dates. Agric. Statistics Pakistan. Govt. Pak., Ministry Food, Agric.
Livestock (Economic Wing), Islamabad, p.100.
[5] Asif, M. I., & Al Taher, O. A. 1983. Ripening of Khasab dates by brine & vinegar
acid. Date Palm J., 2 (1): 121-128
[6] Baloch, A. K. 1999. Enhancement of Postharvest Quality and Stability of Dhakki
dates using Advanced Technology. Pakistan Sci. Foundation, Islamabad.
[7] Baloch, A. K., Saleem, S. A., Dar, N.G., Baloch, Waqar A., & Baloch, M. Kaleem.
2003. Influence of microwave radiation on ripening of Dhakki dates. J. Food
Process. & Preserv., 27 (3):181-193.
[8] Cheema, W. A. (1986). Effect of different doses of chemicals and gamma
irradiation on
physio-chemical changes and storage behavior of date fruit. M.
Sc. Thesis. Dept. Hort. Uni. Agric. Faisalabad, Pakistan.
[9] Elzoghbi, M. 1994. Biochemical-changes in some tropical fruits during ripening. J.
Food Chemistry, 49 (1): 33-37.
[10] Hasegawa, S., & Maier, V. P. (1980). Polyphenol oxidase of dates. J. Agric. Food
Chem., 28 (5): 891-893.
[11] Hasegawa, S., & Smolensky, D. C. 1971. . Cellulase in dates and its role in fruit
softening. J. Food Sci., 36: 966-967.
[12] Jellinek, G. 1985. Sensory Evaluation. VCH Publishers, New York, pp. 275-285.
[13] Kader, A. A. 1992. Postharvest Technology of Horticultural Crops. Uni. Calif.,
Div. Agric. & Nat. Resources, Spec. Publ. 3311, 2nd ed., pp. 296-301.
[14] Kalra, S. K., Jawanda, J. S., & Munshi, S. K. 1977. Studies on softening of Doka
dates by brine and vinegar acid. Indian J. Hort., 34: 220-224.
930
[15] Saleem, S. A., Baloch, M. K., Ahmad, K., & Baloch, A. K. 2002. Effect of
ripening by microwave radiation on quality of Dhakki dates. Online J. Biol. Sci,
2 (4): 238-242.
[16] Smolensky, D. C., Raymond, W. R., & Maier, V. P. 1973. Pectic enzymes and
their application in the quality improvement of mixed green dates. Date Growers'
Institute, Vol. 50.
[17] Smolensky, D. C., Raymond, W. R., Hasegawa, S., & Maier, V. P. 1976.
Enzymatic improvement of date quality. Use of invertase to improve texture and
appearance of “sugar wall” dates. J. Sci. Food Agric., 26 (10).
[18] Smolensky, D. C., Vandercook, C. E., Raymond, W. R., & Hasegawa, S. 1975.
Improvements of No.2 dry dates with cellulase treatments. Date Growers'
Institute, Vol. 52.
[19] Thatai, S. K., & Kalra, S. K. 1982. Saline water treatments for softening of doka
dates. Punjab Agric. Uni., India, J. Res., 19 (4): 320-23.
931
Table 1.
Symbols for the brine and vinegar acid stimulants.
Symbols
Stimulants
T1
Control (Dipping in distilled water).
T2
0.25 % brine solution.
T3
1.5 % brine solution.
T4
2.0 % brine solution.
T5
3.5 % brine solution.
T6
0.25 % vinegar acid.
T7
0.5 % vinegar acid.
T8
1.5 % vinegar acid.
T9
2.5 % vinegar acid.
T10
Solution containing 2.0 % brine + 0.25 % vinegar acid.
T11
Solution containing 2.0 % brine + 0.5 % vinegar acid.
T12
Solution containing 2.0 % brine + 1.5 % vinegar acid.
T13
Solution containing 2.0 % brine + 2.5 % vinegar acid.
Table 2. Effect of stimulants on color of Dhakki Dokas.
Stimulants
Color of the ripened/ cured fruits
T1
Slightly brownish but not shining.
T2
Amber and attractive color
T3
Shining brownish color developed.
T4
Reddish bright brown color with much attraction
T5
Translucent reddish brown color
T6
Light brown but not attractive.
T7
Dark brownish color
T8
Dirty brownish color
T9
Brown and not attractive.
T10
Darkish brown and not attractive
T11
Darkish brown and acceptable
T12
Darkish brownish and satisfactory
T13
Darkish brownish with little shinning
932
Table 3. Effect of stimulants on the texture of Dhakki Doka.
Treatments
Texture of ripened/ cured fruits
T1
Firm and least soft
T2
Less soft texture without stickiness.
T3
Soft textured and pulpy.
T4
Soft texture, pliable, pulpy and smooth surface.
T5
Very soft and slightly sticky surface.
T6
Compact and sticky surface.
T7
Soft textured and pulpy.
T8
Pulpy, soft and sticky.
T9
Very soft, loose and sticky.
T10
Soft and juicy.
T11
Soft, juicy and sticky.
T12
Soft, loose and sticky.
T13
Very soft and over juicy.
Table 4. Effect of brine, and vinegar acid applied singly and in Combination
on weight, pulp and total soluble solids of Dhakki Doka.
Treatments
Wt of fruit
Pulp
T.S.S
( gm )
(%)
(%)
T1
13.0
95.4
67.2
T2
15.4
95.8
68.8
T3
15.6
95.7
69.2
T4
16.2
95.1
70.0
T5
16.3
95.6
70.4
T6
13.1
94.6
65.6
T7
13.5
94.4
66.4
T8
13.6
94.2
64.8
T9
13.8
93.8
64.0
T10
16.6
95.3
67.2
T11
16.8
95.1
66.8
T12
16.8
95.8
66.0
T13
17.2
95.9
65.6
933
Fig 1. Hardness testing device for Dhakki dates developed by the author (Dr.
Shahzada A. Saleem Khan)
934
Fig. 3 Effect of artificial ripening on ripening
percentage of Dhakki dates
90
80
Fruit Ripening (%)
70
60
50
40
30
20
10
0
T1
T8
T2
T9
T3
T10
935
T4
T11
T5
T12
T6
T13
T7
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936
PP 15
Model Study of Application Total Quality Management (TQM) for Safe
and Good Quality Foods Products by ISO 9000 Quality Standards and
HACCP System Implementation at Date Palm Packaging house
E.EL-Tanboly, M. A. EL-Hofi, and Azza Ismail
Dairy and Food Technology Department, National Research Centre, Dokki, Cairo, Egypt.
e-mail: tanboly1951@yahoo.com
Abstract
The application of TQM by ISO 9000 quality standards and HACCP system will be
essential maintaining and even expanding date palm packaging house export market,
Pressures for quality assured products from the United States and European buyers.
TQM is a broad management concept and log-term business philosophy that stresses
meeting a "right first time, zero defect". Both ISO 9000 quality standards and HACCP
system embody a great part of the TQM. The application of this preventive oriented
approach would give the food producer better control over operation, better
manufacturing practices and greater efficiencies, including reduced wastes. TQM by
ISO 9000 & HACCP were introduced for the date palm line at packaging house for
Preserved Foods , Egypt for safe and good quality foods products.
Introduction
Date processing enjoys a high economic importance in the world. Dates have
nutritive values and are consumed in large quantity in all parts of the country. The
main aim of the date palm packing to produce high quality and safe foods. To assure
the safety of the food, establishing a system based on a continuous management
including total quality management (TQM), Good Hygiene Practices (GHP) and good
manufacturing practices (GMP), is essential. Therefore, Hazard Analysis and Critical
Control Points system (HACCP) should be examined (Bennet et al.,1999). HACCP is
defined as “an effective system based on GHP and GMP, for providing safe and
healthy foods” (Pierson and Corlett, 1992).
HACCP is an effective system because this food safety system is designed to
provide the information flow for preventive and corrective actions and can easily be
937
established on the production lines of all kinds of foods (Unnevehr and Jensen, 1998).
Safe and healthy products can be served to consumers by eliminating the safety risks
after determining the critical control points by hazard analysis and establishing the
necessary preventive and corrective actions (Pierson and Corlett, 1992).
Whole dates are harvested and marketed at three stages of their development. The
three stages are as follows: Khalal, Rutab and Tamar:. Fruit harvested at Tamar stage
is non-perishable, i.e. micro-organisms cannot grow on it, moisture uptake and its
consequences, and changes in color and taste occur during storage. Most of the dates
varieties are harvested after the fruit has undergone the process of ripening and drying
on the palms. Fruit at the Tamar stage is ideal for marketing as "dried" dates. This fruit
is used for preservation and year-round consumption and also for the production of
various types of products, e.g. cakes, sauces and components of granules or date
honey. The first step to establish the HACCP system in date palm packing line should
be to form the flow diagram of the production line. In this way, critical control points
(CCP)can be determined on the flow diagram sample and hazard analysis can be
performed. A sample flow diagram and packing operations for the process of Rutab
and Tamer date palm packing line. The given sample flow diagram must be verified by
the Quality Control (QC) or Quality Assurance (QA) Department of the plant.
This paper focuses on the flow diagrams based on the production line of date palm
packing and hazard analysis can be performed at Date Palm Packaging house in Egypt.
Materials and Methods
Development and implementation of HACCP system.
The steps used to develop and implement the HACCP system as appropriate to
particular industry under consideration as described by Stevenson & Bernard
(1999) as follows.
Prerequisite Programs.
Good Manufacturing Practices (GMP) Good and Hygiene Practices (GHP).
Basic environmental and operating condition as described in the Wallace and
Williams (2001).Application of HACCP seven principles and FAO (2001)
recommended 12 task in development of HACCP plan for date palm packing
processing line based on (Figure 1)
938
Results and discussion
All the stages of implementation were followed stage by stage, all the procedures
necessary of control and checking were established to check and confirm if HACCP /
ISO 9000 system is implemented in accordance with the principles of the codex and
standard ISO 9000/2005. The analysis of the risks was carried out to identify the
hazards which can occur in the cycle of production, the preventive measures were
established, CCPs and OPRP was determined and posted at the factory, the critical
limits for each CCP were defined and validated. A monitoring system is established to
be ensured if the critical limits are respected and OPRP are mastered. The recordings
relating to this monitoring are held up to date. Procedures of checking were established
to confirm if plans HACCP/ISO 9000 are effective (internal audits). Thus
documentation concerning all the processes, the procedures, measurements and the
recordings were appropriate with the nature and the size of the company.
Implementation of HACCP plans and Operational Prerequisite programs (Figure 1).
1. HACCP team.
A multidisciplinary team was composed of seven persons possessing different skills
related to quality assurance, production, engineering, microbiology and so on. Members
of this team have been trained very thoroughly on the HACCP and ISO 9000.
2. Product description.
Whole dates are harvested and marketed at three stages of their development.
Khalal: Physiological mature, hard and crisp, moisture content: 50 - 85 %, bright
yellow or red in color, perishable;
Rutab: Partially browned, reduced moisture content (30 - 45 %), fibers softened, perishable;
Tamar: Color from amber to dark brown, moisture content further reduced (below
25 % down to 10% and less), texture from soft pliable to firm to hard, protected from
insects it can be kept without special precautions over longer periods.
3. Identify Intended Use (Task3)
The normal expected use of the food was described. With regards to possible
acceptable risk level for a food safety hazard it has to be stated for which group of
population the food is intended (Untermann, 1999). The intended use need to be stated
or informed whether the food need to be prepared prior consumption. Besides that
939
sensitive consumers too need to alert which adequate information on allergenic
ingredients if it were used to prepare the product.
4. Flow diagram.
Flow diagrams have been prepared taking into account all aspects of the process in
the scope of the HACCP system. The flow diagrams were checked on site by the
HACCP team (Figures. 2and 3).
5. Onsite confirmation and verification of process flow (Task 5)
The HACCP team shall perform onsite verification on the accuracy and
completeness of the flow diagram. Besides that the team also was trained to check the
conformity of flow diagram is correct for any shift pattern that normally takes place in
processing plant (Slatter, 2003). The onsite assessment normally involves participation
of respective responsible personnel to explain the processing nature and the operation
procedure during assessment (Tables 1 and 2) . During the assessment, any additional
documentation required for on-site review was examined (Motarjemi, 2000).
Each step was checked and to ensure that all relevant information regarding
potential hazards to the process and products are identified. If any modification
required, it were amended immediately and documented. After the five preliminary
tasks have been completed, the seven principles of HACCP are applied to construct the
HACCP plan (Corlett, 1998).
6. Hazard Analysis on the Production Line
After constituting the flow diagram to determine the critical control points (CCP),
hazard analysis can be performed (Scott and Moberg 1995). Possible risks that may
occur during the production must be taken into account and necessary preventive
actions must be determined.
7. Critical Control Points (CCP) on the Production Line
After hazard analysis, determined risks should be considered by decision tree if they
are critical control points or not. Then, factors that constitute the hazard should be
determined. Parameters used during monitoring critical control points, critical limits,
preventive and corrective actions, and production and operation instructions and
responsibilities of the staff should be well defined (Codex Alimentarius Commission,
1993). To monitor these activities, necessary forms and records should be kept as an
940
archive for internal and external audits (Annon, 1998). Inspection and storage of fruits
date (raw materials);
sorting; cleaning; washing; drying; transporting to the
packinghouse, and serving/distributing the markets, are the critical control points in
the packaging lines (Tables 3 and 4).
7.1 Harvesting the fruits date
Harvesting the fruits date entails the use of experienced workers, or investment in
aluminum ladders, in attaching ladders to the palms permanently or in purchasing
mechanical appliance to lift workers to the top of the palm. Rain can cause damage to
the fruit and impair its quality due to rotting, fermentation and insect infestation. On
the other hand, the fruit purchases raw materials from several contractors. The
production requires a stock monitoring program and raw materials should be
purchased as closer as possible to the production time (Bryan, 1992);
Fruits date raw materials that have microbiological loads over critical limits must be
avoided to ensure food safety and quality. Toxins synthesized by microorganisms; and
pesticides, chemical residues and foreign materials found in these raw materials are
also potential risks for consumer health.
7.1.1 Control
The fruits date raw materials must therefore be protected against rain with the help
of wax-covered paper or nylon sleeves. Harvesting must be faultless and clean, since it
significantly affects the rest of the process (packing and marketing). Harvesting the
fruit straight into containers suitable for transport to the packinghouse prevents the
infection of the fruit by the soil and sand under the palm and ensures that the fruit
arrives in good condition, and that it is not crushed.
Fruits date raw materials should be purchased in accordance with the “Raw Material
Acceptance Criteria” determined by QC/QA Department. QC/QA staff members have
to reject unsuitable raw materials. Microbiological, physical and chemical
characteristics that raw materials must have corresponding and critical limits should be
determined in “Raw Material Acceptance Criteria”. Contractor having quality
certificates like ISO Quality Assurance Systems and HACCP system should be
preferred. QC/QA staff member have to control the expiration date of the packaged
foods. Ripped, pierced, damaged and abnormal shaped packages have to be refused.
941
7.1.2. Monitoring and Keeping Records
During monitoring the inspection and acceptance of fruits raw materials,
responsibilities of the department staff and controllers, inspection methods and
instructions have to be clearly brought up for consideration “Raw Material Control
Procedures”. QC/QA staff members should keep the acceptance records and fill the
necessary forms .
7.2 Storage of fruits date Raw Materials In the packinghouse
In the packinghouse there are a number of processes, designed to improve or
maintain fruit quality. These processes are: fumigation, washing, storage, refrigeration,
hydration, dehydration and curing. Fumigation must not be carried out when the fruit
is fresh, harvested at the Khalal stage, or when stored under deep refrigeration. The
substance most frequently used for fumigation is methyl bromide (CH3 Br), which
makes most of the insects come out before they are killed by the gas. The
concentration of the gas is 30 ppm, i.e. 30 g methyl bromide in 1 m3 of air. The time
recommended for fumigation is 12 - 24 hours. The temperature must be above 16oC. It
is important for the air to swirl within the fumigation installation, in order for it to
spread uniformly within the chamber. In the storehouses the produce must be protected
from recontamination by pests (insects and rodents). The surfaces and packages must
be well made in order to withstand being loaded, shaken on the way and unloaded.
Today, the temperature commonly used for long-term preservation of dates of several
varieties is - 18oC (0oF). This temperature decreases possible water loss and also
decreases the sugar crystallization and skin separation phenomena.
Storage under conditions of 26 % humidity or higher requires a temperature of oºC
enabling a storage period of 6 - 8 months; the storage period can be more than 1 -year
if humidity is less than 26 %; if humidity is less than 20 %, dates can be stored at 25ºC
for up to 1- year; and high sugar content coupled to high humidity tends to aggravate
the situation of fruit going bad.
7.2.1. Possible Risks
Because of insufficient and improper storage conditions, rapid microbial growth can
be seen. Cross contamination of the pathogen microorganisms from storage places to
production area is another important hazard (Bryan, 1992).
942
7.2.2. Control
“Storage of fruits date Raw Materials” should be determined by QC/QA department
for proper storing.
7.2.3. Monitoring and Keeping Records
QC/QA staff members are responsible for proper storing conditions. Temperatures
and relative humilities of the storage places should be monitored by thermocouples
and hygrometers continuously. Temperatures and relative humilities of the storage
places, and changes in these parameters should be recorded; when necessary, these
parameters should be reset. Sanitary and hygienic conditions of the stores are very
significant to avoid the contamination. In addition, hygienic barriers might be used and
stores should be cleaned and sanitized periodically, and records mentioned in “Storage
of fruits date Raw Materials”, should be kept for archive and audits.
7.3 Washing Fruits
Dates exposed to various types of contamination of physical, chemical or/and
microbiological nature. Physical factors: Sand and soil - both as a result of sand storms in
many regions where dates are grown, and soil sticking to fruit lying on the ground. Chemical
factors: These are especially remnants of pesticides, some of which can be removed by
washing. Microbiological factors: External cleaning of the fruit by washing removes some
of the microbiological pollution, also excretions of birds, which may spoil the fruit.
Clean water must be used and care taken that all the fruit is washed. Other methods
exist, such as damp toweling attached to sloping mechanical shakers. While the fruit is
still hanging, it can be cleaned by water spray, accompanied by the use of fine
swiveling brushes, but they must be dried before being packed. When the fruit is
packed immediately after washing, it is important to dry it in drying cubicles or by
means of large fans.
Washing and rinsing periods, chlorine concentrations, temperatures and pressures of
washing and rinsing water should be adequate to remove dirtiness and to decrease the
microbial load.
7.3. 1. Possible Risks
An inadequate washing program causes non-removal of physical, chemical and
microbiological hazards present in natural flora of fruits. Potable water should be used
943
for washing process, otherwise, fruits can be contaminated by unclean water. An
effective rinsing is very crucial to remove chlorine from fruits.
7.3. 2. Control
A detailed “Raw Material Washing Program” should be prepared by QC/QA
department for considering parameters such as the concentration of chlorine,
washing and rinsing period, pressure and temperature of water according to the type
of the raw material.
Generally, 50-125 ppm active chlorine is adequate for eliminating the microbial
risks of the fruits and vegetables (Aran et al., 1987). For very dirty raw materials 1-5
ppm active chlorine should be added to the final rinsing water (Aran et al., 1987). To
avoid the contamination from water used for washing, water analysis (chemical and
microbiological) should be performed by authorized laboratories periodically.
7.3. 2. Monitoring and Keeping Records
QC/QA department is responsible for an effective washing and rinsing. “Raw
Material Washing Program” should be applied completely. Water analysis reports
should be kept for archive and audits.
7.4 Washing and Rinsing the Equipment
Dirty equipment are one of the main sources of physical and microbiological
contaminations. Therefore, an effective equipment cleaning program should be
applied (Bryan, 1992).
7.4 1. Possible Risks
Hazards at this step are closely related to the effectiveness of the washing program.
If the washing program is inadequate, it is impossible to remove physical, chemical or
microbiological hazards. On the other hand, inadequate rinsing causes non-removal of
detergent, chlorine and caustic from equipment.
7.4. 2. Monitoring and Keeping Records
Concentration of active chlorine, caustic or detergent used, washing and rinsing
periods, temperatures and pressures of washing and rinsing water should be clearly
determined. General cleaning of equipment used in production should be periodically
done by caustic solutions. Because has toxic effect on health, it should be checked
whether it was removed completely from the equipment or not after rinsing. Presence of
944
caustic on the equipment can be detected by a test in which the colorless phenol phytalein
turns into purple when dropped on the surfaces if caustic is still there (Troller, 1993).
7.5 Metal Detectors
7.5 1. Possible Risks
It is possible that metal particles can contaminate the fruits during production.
These metal particles may come from raw materials that are not properly handled
during harvest and may cause physical hazards.
7.5. 2 Control
Control is done by metal detectors.
7.5 3. Monitoring and Keeping Records
QC/QA department staff should constitute a detailed “Metal Detector Manual”. In
this manual, dimensions of metal particles that metal detector should determine must
be given (Mortimore, 1994). QC/QA staff member, responsible for this operation,
should periodically check the detector by test and should calibrate it frequently.
7.6 Distributing
7.6 1. Possible Risks
Because of unsuitable distributing conditions, microbiological growth and spoilage
of meal may occur.
7.6 2. Control
Distributing of fruits should be performed according to “Distributing Procedure”
stated by QC/QA department. During transportation, temperature of the fruits should
be -20ºC for Rutab and 5ºC for Tamer. To ensure that, fruits should be distributed in
boxes (Bryan, 1992).
7.6 3. Monitoring and Keeping Records
Final product should be placed into boxes and distributed as soon as possible after
production. Lids of the boxes should be closed tightly and checked. Also refrigerator
conditions must be ensured for fruits products. Loading of the boxes into the cars
should be done according to the distributing route.
8. Keeping Records and Verifying
QC/QA department should ensure to avoid the potential hazards in all steps of the
process by stating preventive and corrective actions. Effectiveness of the HACCP
945
system can be stated by verifying. All the activities taken place in HACCP system
should be kept as records and forms, and archived for periodic internal and external
audits. Audits are performed by Production Management Department and government
officials dealing with food safety (Annon., 1998).
Conclusion
HACCP should be considered as a system based on Good Hygiene Practices (GHP)
and Good Manufacturing Practices (GMP). GMP and GHP applications include
building, environment arrangements and personnel hygiene and behaviors. Sanitary
and hygienic conditions of the plant can be improved.
For serving high quality and safe products to the consumers, inspecting the raw
materials purchasing, storing the raw materials at proper conditions, using well
cleaned equipments in all steps of the fruits date packing process, according to receipts
stated by department while storage are determined as critical points. Distribution also
should be performed according to distributing instructions.
946
Reference
[1] Anon.,A . 1998. Food Quality and Safety Systems. Food and Agricultural
Organization of the United Nations, Rome, Italy. 232p.
[2] Aran, N., Alperden, İ., TOPAL, Ş. 1987. Mold control problem in tomato paste
production and hazard analysis system at critical control points. Food Industry,
Turkey. 1987/2: 43-47.
[3] Bennet, William L, Steed, Leonard L. (1999). An integrated approach to food
safety. Quality Progress 32 (2): 37-42.
[4] Byran, F.L. 1992. Hazard Analysis Critical Control Point Evaluations: A guide to
identifying hazards and assessing risks associated with food preparation and
storage. World Health Organization, Geneva. 72p.
[5] Codex Alimentarius Commission. (1993). Guidelines for application of Hazard
Analysis and Critical Control Point (HACCP) system. Codex Alimentarius
Commission of Food Hygiene, WHO/FAO, Switzerland.
[6] FAO. 2001 Codex Alimentarius- Food Hygiene – Basic Text, 2nd ed. Rome. Italy.
[7] Motarjemi, Y. 2000. Regulatory Assessment of HACCP: A FAO/WHO
consultation on the role of government agencies in assessing HACCP. Food
Control, 11 (5): 341-344.
[8] Pierson, M.D., Corlett, D.A. 1992. Principles and Applications. Van Nostrand
Reinhold, New York.
[9] Scott, V.N., Moberg, L. 1995. Biological hazards and controls. “HACCP
Establishing Hazard Analysis Critical Control Point Programs.”
[10] Slatter. J. 2003. Hazard Analysis Critical Control Point. Encyclopedia of Food
Science and Nutrition pp. 3023-3028.
[11] Stevenson K. E, Bernard DT, eds. 1995. HACCP—Establishing Hazard Analysis
Critical Control Point Programs: A Workshop Manual, 2nd ed.
[12] Troller, J.A. 1993. Sanitation in Food Processing. Academic Press Inc., CA. 478p.
[13] Unnevehr, L.J., Jensen, H.H. 1998. HACCP as a regulatory innovation to
improve food safety in the meat industry. American Journal of Agricultural
Economics 78, 764-769
[14] Unterman, F. 1999. Food Safety management and misinterpretation
[15] of HACCP, Food Control 10: 161-167
947
Table (1) Green or ripe dates (Rutab) Packing Operations
Production
steps
Description
1
Fruit Harvesting
2
Fruit reception & Weighing
3
Grading & Selecting
4
Storage at 5oC / fumigation
8
Dry sorting
9
Water soaking
10
Fruit spray washing
11
Fruit sorted on conveyor
12
Fruit spray washing
13
Fruit Freezing
14
Fruit packed into either:
• *Bulk Pack (in cardboard boxes) 5 kg
• **Retail pack (box, placed in two layers, separated by cellophane,
weighing 220 g - 250 g.)
15
Bags or boxes transfer to labeling department
16
Bags or boxes moved on conveyor to the labeler
17
17 (i)
17 (ii)
Bags or boxes labeled
Glue
Label
18
Coding Fruit packed with ink jet printing with date of
production and expiry date
19
Cardboard tray manually
20
Bags placed on tray by hand
21
Trays is labeled
22
Trays is shrink wrapped
23
Fruit frozen and storage at -20oC/ Humidity 70%
24
Transportation for distribution
25
Distribution
Green or ripe dates (Rutab): Partially browned, reduced moisture content (30 - 45 %), fibres softened, perishable;
948
Table (2) Tamer Packing Operations
Production steps
Description
1
Fruit Harvesting
2
Fruit reception & Weighing
3
Grading & Selecting
4
Storage at 5oC / fumigation
8
Dry sorting
9
Water soaking
10
Fruit spray washing
11
Fruit sorted on conveyor
12
Fruit spray washing
13
Fruit Drying
14
• sorting second time
15
Production Dates lines:
15 (i)
Bulk Packing line
15 (ii)
Pitting/Pressing Line
15 (iii)
Thermo pack Line)
15 (iv)
Date juice (Dibs)
16
Fruit packed into either:
• 50g, 100g, 200g and 500g in PET polyethylene bags or
varying sizes boxes (1kg, 2kg, 3kg, 5kg and 10kg)
17
Bags or boxes transfer to labeling department
18
Bags or boxes moved on conveyor to the labeler
19
19 (i)
19 (ii)
Bags or boxes labeled
Glue
Label
20
Coding Fruit packed with ink jet printing with date of
production and expiry date
21
Cardboard tray manually
22
Bags placed on tray by hand
23
Trays is labeled
24
Trays is shrink wrapped
25
Fruit storage at 5oC
26
Transportation for distribution
27
Distribution
Tamar: Color from amber to dark brown, moisture content further reduced (below 25 % down
to 10% and less), texture from soft pliable to firm to hard, protected from insects it can be kep
without special precautions over longer periods.
Tables 3 &4. are not included due to their large sizes and incompatibility
949
Five Preliminary Steps
(Task 1) HACCP team Assembling
(Task 2) Product Description
(Task 3) Identification of products intended use
(Task 4) Construction of flow diagram
(Task 5) Onsite verification and confirmation of flow diagram
Seven HACCP Principles
(Task 6) Conduct a Hazard Analysis
(Task 7) Determine the Critical Control Points (CCPs)
(Task 8) Establish Critical Limits
(Task 9) Establish CCP monitoring procedures
(Task 10) Establish corrective action
(Task 11) Establish Verification Procedures
(Task 12) Establish Documentation and Record Keeping
Figure 1: 12 task sequence steps for HACCP application
Fruit harvesting
⇓
Fruit reception & Weighing
⇓
Grading & Selecting
⇓
Storage at 5oC / fumigation
⇓
Dry sorting
⇓
Water soaking
⇓
spray washing
⇓
Sorted on conveyor
⇓
Spray washing
⇓
Freezing
⇓
packing
⇓
Coding & Labeling
⇓
frozen and storage at -20oC/ Humidity 70%
⇓
Transportation
⇓
Distribution
Figure 2. The main procedures to Green or ripe dates (Rutab) Packing
950
Fruit harvesting
⇓
Fruit reception & Weighing
⇓
Grading & Selecting
⇓
Storage at 5oC / fumigation
⇓
Dry sorting
⇓
Water soaking
⇓
spray washing
⇓
Sorted on conveyor
⇓
Spray washing
⇓
Drying
⇓
Sorting
⇓
Vacuum Packing
⇓
In PET polyethylene bags 50g, 100g, 200g
Boxes (1kg, 2kg, 3kg, 5kg and 10kg)
⇓
Coding & Labeling
⇓
Shrink wrapped
⇓
Storage at 5oC
⇓
Transportation
⇓
Distribution
Figure 3. The main procedures to Tamar dates Packing
951
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952
Economics and Value Addition
953
954
OP 49
A Study on the Economic Feasibility of Date Palm Cultivation in
the Al-Hassa Oasis of Saudi Arabia
A. Al-Abbad, 1; M. Al-Jamal, 1; Z .Al-Elaiw, 1; F. Al-Shreed, 1 and H. Belaifa2
1
National Date Palm Research Centre (NDPRC) , P O Box 43 , Al-Hassa- 31982,
Kingdom of Saudi Arabia
2
FAO Project (UTFN/SAU/015/SAU), NDPRC, P O Box 43 ,Al-Hassa- 31982,
Kingdom of Saudi Arabia
Corresponding Author: Al-Abbad, A. ( Email : abbad9@yahoo.com)
Abstract
The Kingdom of Saudi Arabia with an estimated 25 million date palms (Phoenix
dactylifera L.) produces nearly a million tons of dates annually accounting for about
15 per cent of the global date production. Our study on the economic feasibility of date
palm cultivation in the Al-Hassa oasis of the Kingdom estimated the average annual
yield of dates per palm to be 48.0 Kg per palm with a selling price estimated at SR
4.00 per Kg. The net income from date palm cultivation in the oasis was found to be
SR 5800.00 / ha (SR 38.67 / palm). Significant number of farmers (23.00%) sell their
produce in the farm itself of which 57% is to known customers indicating sizeable
“farmer-consumer” loyalty. Date palm farmers of Al-Hassa were also found to be
quality conscious who adopt diverse measures (pre to post harvest) to ensure quality
production of dates. SWOT analysis indicated spiritual attachment to the land by the
farmers as a strength of the system however, bureaucratic hurdles to obtain subsidies
and lack of exploitation of facilities by traditional farmers as a major threat to date
farming in the oasis. There also exists a good possibility to develop logistics that
support marketing of dates especially through agricultural cooperatives, besides further
enhancing exploitation of state subsidies for date palm cultivation.
Key words : Date palm cultivation, economic feasibility, marketing, SWOT analysis,
Saudi Arabia.
955
Introduction
The date palm , Phoenix dactylifera L is the main fruit crop of the Kingdom of
Saudi Arabia (KSA), covering approximately 72% of the total area under permanent
crops. With an estimated
25 million date, the Kingdom, produces nearly a million
tons annually accounting for about 15 % of the global production. More than 400
different date palm cultivars are reported to exist in Saudi Arabia (Anonymous, 2006a
; Anonymous , 2009).The date palm is a multipurpose tree, providing food, shelter,
timber products .The date fruit is a good source of food providing, fibre,
carbohydrates, minerals and vitamins besides having anti-mutagenic and anticarcinogenic properties (Baloch et al., 2006; Al-Farsi et al., 2005; Ishurd and
Kennedy, 2005; Vayalill, 2002; Mohamed, 2000).
With an estimated three million palms the Al-Hassa oasis in the Eastern Province is
the largest in the Kingdom where El-Baker (1952) listed 15 date palm varieties of
commercial importance, while Asif et al. (1982) listed 25 cultivars from the Al-Hassa
oasis. Further, Asif et al. (1986) grouped the Al-Hassa date palm cultivars based on
the season of production. Khalas a mid-season cultivar is widely cultivated in the AlHassa oasis and considered by many as the best date in the world, with mostly medium
to big sized fruits that are consumed as both fresh (rutab) and dry (tamar) dates which
store well. Although the date cultivar Reziz has traditionally been the ruling date palm
cultivar in Al-Hassa , currently the cultivar Khalas is cultivated in more than 50 % of
the area ( Al -Khatib et al., 2006 and Al-Mahla and Hussein, 2003).
Despite surplus production of dates in the Kingdom, marketing of the produce at the
national level has been weak which may have been influenced by the closing of some
date processing factories in the Kingdom, besides exports have not been to the
expected level. Also, the per capita consumption of dates in the Kingdom has
significantly decreased over the years while the production has increased. This has
impacted production of dates negatively, in spite of all support from the Government.
The Kingdom currently has 64 date processing factories (Anonymous, 2006b,
Anonymous, 2006c; Anonymous, 2009b; Al- Shuaiby and Ismael, 2007)
The objective of this investigation is therefore to study the economic feasibility of
date palm production in the Kingdom by determining the cost of production and net
956
income generated from the cultivation of date palm in one hectare in the Al-Hassa oasis
. This was achieved by carrying out economic analysis of descriptive and quantitative
variables that influence date palm cultivation, through field surveys (questionnaires),
analyzing Ministry of Agriculture, KSA data and taking up SWOT analysis to
determine the strengths, weaknesses, opportunities and threats of date cultivation .
Materials and Methods
Primary data on the cultivation of date palm in the Al-Hassa oasis was collected in the
field through personal interviews of both farmers (producers) and also from the market
(traders) that answered questions in a questionnaire prepared for the purpose. In the first
model, questions pertaining to both fixed and variable costs were addressed and were
based on an average of 150 palms per hectare and the average annual production of dates
per palm was estimated to be 48.0 Kg. The selling price of one Kg dates was estimated to
be SR 4.00 per Kg. In all 30 respondents were interviewed in different locations of AlHassa to gather basic data pertaining to the cost of cultivation, production and income
from date plantation. Data collected for this study is presented in tables 1 .
In the second model the questionnaire of seven questions revolved around
agricultural practices viz. pest control, sorting of produce, storage of dates etc. that
influenced the quality of dates.
The fixed and variable costs were computed based on the formulae presented below
Cost
Formula
Average fixed cost per
palm
Average fixed cost per hectare / Number of
palms per hectare
Average variable cost per
palm
Average variable cost per hectare / Number of
palms per hectare
Total production cost per
hectare
Average fixed cost per hectare +Average
variable cost per hectare
Average cost of
production per palm
Average fixed cost per palm + Average variable
cost per palm
Average cost to produce
one Kg dates
Average cost of production per palm / Average
production per palm
Further, income per hectare =
Number of palms per ha x Average production per palm x selling price per Kg of dates.
957
Net income per hectare = Income per ha - Cost of production per ha.
Subsequently, based on the findings SWOT analysis was carried out to identify the
strengths, weaknesses, opportunities and threats pertaining to the economic feasibility
of the cultivation of dates in the Al-Hassa oasis.
Results
1. Cost of cultivation and income
Data presented in table 2 reveals that the cost of cultivating date palm in Al-Hassa
was estimated to be SR 23000.00 / ha ( SR 153.33 / palm) which includes the fixed (
SR 5193/ ha) and variable costs (SR 17805/ ha). The income generated was estimated
to be SR 28800.00 / ha ( SR 192.00 / palm). The net income from cultivating date
palm in Al Hassa was therefore SR 5800.00 / ha ( SR 38.67 / palm) .
2. Marketing of dates
In Al-Hassa date palm farmers were found to market their produce in three different
ways (Table 3) ranging from disposing the produce directly in the farm (23%) to
selling dates in the market (40%) and factories (37%). It is pertinent to mention that
sizeable number of farmers (57%) who sell the produce in the farm sold dates to
known customers, indicating a great degree of customer loyalty probably due to the
quality of the produce.
3. Quality enhancing practices
Table 4 indicates that 100 per cent of the date palm farmers studied were quality
conscious and took care to incorporate measures in their farming practices (pre and
post-harvest) to enhance and safe guard the quality of their produce. Majority of the
farmers (57%) practiced thinning of fruits. This practice is known to increase the fruit
size and also prevent shriveling of dates. Results presented in table 5 also reveal that
100.00 per cent of the farmers discarded defective fruits from their produce there by
enhancing quality of the produce. However, only 20.00 per cent of the respondents
practiced sorting of dates according to fruit size. Further, 73.00 per cent of the farmers
in this study used chemicals (sulfur) for combating the menace of mites, Oligonychus
afrasiaticus attacking date fruits. Table 4 also shows that 100.00 per cent of the date
palm farmers in Al-Hassa used a plastic film on the ground while harvesting, thereby
preventing damage to the harvested dates and accumulation of foreign particles (sand,
958
palm refuge etc.) in the produce. However, none of the farmers practiced fumigation of
produce to combat the menace of insect pests in stored dates.
4. Strengths, weaknesses, opportunities and threats of date production
Analyzing the strengths, weaknesses, opportunities and threats (SWOT) is
fundamental to the success and profitability of any commercial venture and is a
valuable tool in strategic planning for the future.
Our study (Table 5) revealed that the date palm farmers are spiritually attached to
their land / farm which is a significant strength of the system. However, children of the
farmers do not have such an attachment which is a weakness that may hamper date
palm cultivation in the oasis in the future. Diverse markets to sell the produce with
good infrastructure and Government support for date cultivation is a significant strength
of date cultivation in the Al Hassa oasis. However, poor relationship between the farms
and date processing factories and poor exploitation of facilities / in fracture to enhance
production and marketing of dates is a weakness. Although, the date cultivar Khalas is
popular and suitable for cultivation in the oasis, relying on a single cultivar, can pose a
challenge in the future as a monoculture system of farming can pre-dispose date
cultivation to attack and quick spread of pests and diseases in the future.
Further, we found that there exists a good possibility to develop logistics that
support marketing of dates especially through agricultural cooperatives , besides the
possibility of further enhancing exploitation of state subsidies for date palm
cultivation. However, bureaucratic hurdles to obtain subsidies and lack of exploitation
of facilities by traditional farmers were found to be a threat to date cultivation in the Al
Hassa oasis (Table 5).
Discussion
The Ministry of Agriculture , Kingdom of Saudi Arabia has estimated the cost of
cultivation of date palm in Saudi Arabia to be SR 15179.90 / ha on the basis of a study
taken up in 13 regions of the Kingdom ( Anonymous, 2006a) which includes both the
fixed and variable costs. However, our study has revealed that the total cost of
cultivation to be SR 23,000/ ha in Al-Hassa in 2009. This increase in the cost of
production can be attributed to the rise in the cost of several production factors
including men and material during the last five years.
959
The net annual income per hectare of date palm was estimated to be SR 39,436 by
the Ministry of Agriculture, Kingdom of Saudi Arabia (Anonymous, 2006a) . In
contrast our study indicates a fall (26.90 5) in the net income from one hectare of date
palm which was estimated to be SR 28,800. This decrease in the net income is due to
several factors including increase in production, low storage capacity of dates at
farmers’ level and decline in the per capita consumption of dates in the Kingdom.
With regard to marketing of dates our study indicated that several farmers sold their
produce to known customers, indicating a great degree of customer confidence and loyalty
probably due to the quality of the produce. This also assured the farmer a better price.
Further, only 23 per cent of the date palm farmers in Al Hassa sold dates to the date
factories indicating lower prices offered by the factories for the produce. There is an
urgent need to develop an export strategy for Saudi Arabian dates that would assure sale
of surplus dates from the Kingdom which in turn would result in better prices for the
farmers which were estimated to be only SR 4.00 per Kg in our study. El-Sabea, 2010
proposed of enhancing exports of Saudi Arabian dates through marketing cooperatives.
Quality production of dates was found to be important criteria for the date farmers
of Al-Hassa as 100 per cent of the respondents were quality conscious and took care to
incorporate measures in their farming practices (pre and post-harvest) to enhance and
safe guard the quality of their produce. These practices included thinning of fruits
(57%). This practices has been recommended by several workers to improve the size
of dates and also to prevent shriveling (Tahaer, 1983 ; Al-Darwish and Ben Abdallah,
2010).100.00 per cent of the farmers discarded defective fruits and used a plastic film
on the ground while harvesting which assured quality production . These practices
have been recommended by the Ministry of Agriculture, Kingdom of Saudi Arabia to
ensure quality production of dates in the Kingdom (Anonymous 2009b; Anonymous
2006a). Mites, Oligonychus afrasiaticus damage to date fruits reduces fruit quality
considerably (Dhouibi, 2005). Date palm farmers (73%) of Al-Hassa were aware of
this problem and used chemicals (sulphur) for combating this pest. However, none of
the farmers practiced fumigation of produce that is known (Al-Zumaiti, 1997) to
combat the menace of pests in stored dates.
960
SWOT analysis indicated strong attachment to the land as strength of date farming
in the Al- Hassa oasis. Further, bureaucratic hurdles to obtain subsidies and lack of
exploitation of facilities by traditional farmers were perceived as major threats to date
farming in the oasis. There also exists a good possibility to develop logistics that
support marketing of dates especially through agricultural cooperatives , besides the
possibility of further enhancing exploitation of state subsidies for date palm cultivation
in the oasis.
Acknowledgement
Institutional support provided by the National Date Palm Research Centre, AlHassa, Saudi Arabia to take up this study is gratefully acknowledged. Authors also
wish to thank Dr Abdallah Ben Abdallah (CTA, FAO Project UTFN/SAU/015/SAU)
for assisting in manuscript preparation.
961
References
[1] Al-Darwish ZS and Ben Abdallah A (2010). Effect of bunch removal and fruit
thinning on shriveling of mature dates in Ghar Cultivar . Fourth International Date
Palm Conference, Abu Dabhi, United Arab Emirates 15-17, March 2010. Acta
Horticulture, In Press .
[2] Al-Farsi M, Alasalvar C, Morris A, Barron M
and Shahidi F
(2005).
Compositional and sensory characteristics of three native sun-dried date (Phoenix
dactylifera L) varieties grown in Oman. J. of Agril. and Food Chem., 53, 7586-7591.
[3] Al- Khatib AA, Al-Jaber MA and Al-Jabber A (2006). Date palm in the Kingdom
of Saudi Arabia (Ed. National Agriculture Development Company (NADEC),
Saudi Arabia) 136p.
[4] Al- Malha J and Husseini AA (2003) . Cost of cultivation of Khalas dates in the
Al- Hassa oasis. ( Ed. King Saud University, Riyadh, Saudi Arabia. 843-853p
[5] Al- Shuaiby AA and Ismael M ( 2007) . Economic alternatives of dates as animal feed
in Al- Hassa and Al- Qatif . Proceedings, Fourth Symposium on Date Palm in Saudi
Arabia, 20- 22 February, 2007, King Faisal University, Al Hassa, Saudi Arabia.
[6] Al-Zumaiti M (1997). Practices of IPM for agriculture.( Ed. Dar Al-Fajr). 456p.
[7] Anonymous (2009a) . Annual statistical data (Ed .
Department of studies,
planning and statistics , Ministry of Agriculture, Kingdom of Saudi Arabia) .269p.
[8] Anonymous (2009b) . Statistics on processing of dates in the Kingdom of Saudi
Arabia
(Ed .
Department of studies, planning and statistics, Ministry of
Agriculture, Kingdom of Saudi Arabia) .6p
[9] Anonymous (2006a) . The famous date varieties in the Kingdom of Saudi Arabia (
Ed. Ministry of Agriculture, Kingdom of Saudi Arabia and Food and Agriculture
Organization of the United Nations). 245 p.
[10] Anonymous (2006b) . A review of the processing of dates in the Kingdom of Saudi
Arabia : 1997to 2005. ( Ed. Ministry of Agriculture, Kingdom of Saudi Arabia) . 25p
[11] Anonymous (2006c) . Dates of Saudi Arabia: Status and Prospects ( Ed. Ministry
of Agriculture, Kingdom of Saudi Arabia) . 129p
962
[12] Asif MI, Al-Tahir OA and Al-Kahtani MS (1982). Inter-regional and inter-cultivar
variations in dates grown in the Kingdom of Saudi Arabia. In, proceedings of the
first symposium on date palm. King Faisal University, Al- Hassa.
[13] Asif MI, Al-Ghamdi AS, Al-Tahir OA and Latif RAA (1986) Studies on the date
palm cultivars of Al-Hassa oasis. In, proceedings of the second symposium on date
palm in Saudi Arabia. King Faisal University, Al Hassa Saudi Arabia.pp 405-413.
[14] Baloch MK, Saleem SA , Ahmad K , Baloch AK and Baloch WA (2006). Impact
of controlled atmosphere on the stability of Dhakki dates .Swiss Society of Food
Sci. and Tech.,39, 671-676.
[15] Dhouibi MH (2005) . Experimental results( 2004-5005) of the Integrated Pest
Management Laboratory in Riyadh ( FAO Project, Ministry of Agriculture, Saudi
Arabia). 96p.
[16] El-Baker AJ (1952). Date cultivation in Saudi Arabia. Report number 31. FAO
,Rome, Italy.
[17] El-Sabea AMR (2010). A comparative study for competitiveness of dates from
the Kingdom of Saudi Arabia and the United Arab Emirates in the world
markets. Proceedings ( Ed. Zaid A and Alhadrami G) Fourth International Date
Palm Conference, Abu Dabhi, United Arab Emirates 15-17, March 2010. 715p
[18] Ishurd O and Kennedy JF (2005). The anti-cancer activity of polysaccharide
prepared from Libyan dates (Phoenix dactylifera L). Carbohydrate Polymers,
59,531-535.
[19] Mohamed AE (2000). Trace element levels in some kinds of dates. Food Chem.,
49, 107-113.
[20] Tahar KH (1983) Date palms and dates in the Kingdom of Saudi Arabia, Ed.
Ministry of Agriculture , Saudi Arabia.
[21] Vayalill PK (2002). Antioxidant and anti mutagenic properties of aqueous extract
of date fruit (Phoenix dactylifera L. Arecaceae ). J. of Agric. Food Chem.,
50,610-617.
963
Table 1. Estimates on cost of cultivation, production and price of produce of
dates in the Al-Hassa oasis in Saudi Arabia
A. Estimates on cost of cultivation
Size of
No. of
plantation palms
( m2)
Depreciation
Cost of
Total cost
of fixed
production
with
assets
(SR)
depreciation
(SR)
(SR)
Total
754200
9889
3427937
1175155
4603092
Mean
25140
330
114265
39172
153436
N= 30
B. Estimates on production and price of produce
Size of
No. of
plantation palms
( m2)
Value of
Production
produce sold
(Kg)
(SR)
Yield per
palm (Kg)
Mean
Price
per
Kg
(SR)
Total
754200
9889
2029410
440790
1451
129.00
Mean
25140
330
67647
14693
48.38
4.00
N=30
Table 2. Cost of cultivation and income (per hectare and per palm) from date
palm in Al-Hassa oasis of Saudi Arabia (2009)
Cost / Income
Per hectare( SR)
Per Palm (SR)
Fixed cost
5193.86
34.63
Variable cost
17805.48
118.70
Total cost
23000.00
153.33
Income
28800.00
192.00
Net income
5800.00
38.67
Table 3. Method of selling the produce (dates) in Al-Hassa oasis of Saudi Arabia
Method of selling dates
Percentage of overall sale
In farm
23.00
In the market
40.00
Directly to factory
37.00
964
Table 4. Importance attributed to quality date production at farmers level in AlHassa oasis of Saudi Arabia ( Model –II)
Quality improvement related functions
Sr.
No.
Care Thinning
for
of fruits
quality
Removal
of
defective
fruits
Use of
chemicals
Plastic film Sorting of Fumigation
on ground
fruits
against
during
storage pests
harvesting
1
Yes
Yes
Yes
Not known
Yes
No
No
2
Yes
Yes
Yes
Sulphur
Yes
Yes
No
3
Yes
No
Yes
Not known
Yes
No
No
4
Yes
Yes
Yes
No
Yes
Yes
No
5
Yes
Yes
Yes
Sulphur
Yes
No
No
6
Yes
Yes
Yes
Sulphur
Yes
No
No
7
Yes
Yes
Yes
Sulphur
Yes
Yes
No
8
Yes
No
Yes
No
Yes
No
No
9
Yes
Yes
Yes
Sulphur
Yes
Yes
No
10
Yes
No
Yes
No
Yes
No
No
11
Yes
Yes
Yes
Sulphur
Yes
No
No
12
Yes
Yes
Yes
Sulphur
Yes
No
No
13
Yes
Yes
Yes
Mixed
Yes
No
No
14
Yes
No
Yes
-
Yes
No
No
15
Yes
No
Yes
Not known
Yes
No
No
16
Yes
No
Yes
Not known
Yes
No
No
17
Yes
No
Yes
No
Yes
Yes
No
18
Yes
No
Yes
Sulphur
Yes
No
No
19
Yes
Yes
Yes
Sulphur
Yes
Yes
No
20
Yes
Yes
Yes
Not known
Yes
No
No
21
Yes
No
Yes
Sulphur
Yes
No
No
22
Yes
Yes
Yes
Sulphur
Yes
No
No
23
Yes
Yes
Yes
Not known
Yes
No
No
965
24
Yes
Yes
Yes
Not known
Yes
No
No
25
Yes
No
Yes
Sulphur
Yes
No
No
26
Yes
Yes
Yes
No
Yes
No
No
27
Yes
No
Yes
No
Yes
No
No
28
Yes
Yes
Yes
No
Yes
No
No
29
Yes
No
Yes
No
Yes
No
No
30
Yes
No
Yes
Not known
Yes
No
No
Yes 100.00
( %)
57.00
100
73.00
100.00
20.00
0.00
No
(%)
43.00
0.00
27.00
0.00
80.00
100.00
0.00
Table 5. Strength, opportunities, weaknesses and threats (SWOT) related to date
palm cultivation in Al Hassa oasis of Saudi Arabia
Strengths
Weaknesses
Spiritual attachment to the land (farm)
regardless of the economics.
Children of the farmer do not feel the
same way ( children lack attachment to
the farm).
Diverse ways to market dates in the
oasis.
Poor relationship between the farms and
date processing factories.
The cultivar Khalas is suitable for
cultivation in Al Hassa and other
regions of the Kingdom.
Significant increase in the production of
the cultivar Khalas resulting in lower
prices for this premier date .
Good infrastructure for date production
and marketing in terms of irrigation,
electricity, motor able roads,
agricultural research centers ,
Government support for date palm
cultivation and marketing etc.
Poor exploitation of facilities / in
fracture to enhance production and
marketing of dates.
Opportunities
Threats
Possibility to develop logistics that
support marketing of dates viz.
agricultural cooperatives.
Lack of awareness among farmers to
join such associations / cooperatives.
Possibility of exploiting state subsidies
to enhance date cultivation in the oasis.
Bureaucratic hurdles to obtain subsidies
and lack of exploitation of facilities by
traditional farmers.
966
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967
968
OP 50
Improving the Market profit distribution effected date palm
production in Yemen
Mohamed Abdulrahman Hashem Al-Hebshi
Po. Box. 23368, Sana'a, ROY, Tel: 967-1-405103, Mob: 967-77177011
Email: malhebshi@hotmail.com or proff_alhebshi@yahoo.com
Abstract
Farmers in Yemen are facing many problems related to natural resource
endowments, availability of water and in the marketing of their products. Despite the
difficult natural circumstances the farmers produce agricultural commodities,
including Date palm. This product is sometimes sold in local markets and sometimes
the product is sold to middlemen who transport the commodities to the markets or
directly to the consumers. The value added in the chain will be distributes over the
various actors, the farmers, the transportation sector and the middlemen. As a result of
the actions of the middlemen only a small part of the value added in the chain is to the
benefit of the farmers. The prices that are offered to the farmers do not allow
compensating for the costs and this result in low profits or no profits at all to the
farmers. As results the farmers are not able to expand their activities and are facing
very low incomes and the risk of complete bankruptcy. This is a limiting factor for
agricultural development and hampers modernization of the Date palm trees in Yemen.
Based on the feasibility study of Net Revenue ($)/ha for farm in Wadi Hadramout
during 2002 to 2006 the net revenue is negative for the farmers. In 2006 farmers lost is
233-($)/ha. While the middlemen gain profit of about 4,256 ($)/ha, for the same Year
that means the net marketing margin is 244% for the middlemen profit. Marketing is
frustration for small farmers in Yemen. Farmer’s production increase but their income
didn’t. Key words: Inefficiency in Market Profit Distribution Effected Date Palm
Production in Yemen. Farm Gat Price, producer surplus, Consumer price, Market
marginal, Middleman marketing profit, Fair prices.
969
Introduction
Food security is one of the most important aspects of human security and it is a vital
factor for political, economic and social stability. Food security has become a national
issue in Yemen particularly with the country increasing dependence on food imports.
Yemen is importing more than 85% of food annually for domestic need. Date Palm
Production and consumption are important for food security.
Purpose
Yemen is classified as a low-income / food deficit country (LIFDC) and imports
over 75% of its main staple wheat. Some 2.7 million people live below the food
poverty line, consuming less than 2,200 calories per capita per day, it is pointed out
that about 40 per cent of the Yemeni population is living under the poverty line and the
unemployment rate is about 37 per cent, in addition to the poor distribution of
wealth.1.
The food security status of households is also threatened by natural
resources degradation. Traditional agricultural practices are still prevailing. Therefore,
there is a real need for revising and modernizing the prevailing agricultural system
being adopted for date palm trees, in light of Sustaining Local Food Systems,
Agricultural Biodiversity and Local Livelihoods, because it is rich, cheap and locally
produced food. Dates have proved to be the best resource to ensure food security
during food shortages and crises. Improve marketing efficiency will sustain local food
system.
Agriculture plays an important role in Yemen’s economy: although it
contributes only to 15 percent of national GDP, it employs more than half of the labour
force and provides livelihood to more than two-thirds of the population. In fact, about
50 percent of cultivated land is rain fed, while 31 percent is irrigated from
groundwater, 10 percent from floods (spate) and the rest from dams, streams and water
tankers. The introduction of a more equitable system of pricing water has high
importance in order to save the environment and to redesign crops profitability. As the
predominant cash crop, being more profitable of any other cash crop by 10 to 20 times,
production plays a key role in rural economies: it accounts for about 6 percent of
970
national GDP and one-third of agricultural GDP, with about 15 percent of employment
in the country. Livestock represents about 20 percent of agricultural GDP.
Methodology
This study is based on data collected from the Ministry of Agricultural & Irrigation
Yemen, Department of Monitoring & Evaluation, for the cost of Date Palm producers,
market intermediaries in production. The Retail price of Date Palm was calculated
from Sana’a City market. Wadi Hadramout was selected for this study because it is
considered the main Date Palm growing area in Yemen.
The successful use of advanced methods of marketing analysis is heavily dependent
on the availability of data which is not the case in Yemen , secondary data like
national statistics and surveys conducted by different organizations. Especially with
the latter it is likely that they have followed different standards and procedures and
hence may vary significantly in quality, validity and representativeness. Hence there is
a need for a thorough validation and repeated discussion of the results. Such discussion
will yield the need for additional data collection especially when dealing with specific
operational decisions such as intervention targeting and support intensity. In order to
empirically advance for this case the existing data had used in the input –output
analysis (Table 1) for Date Palm marketing analysis in Wadi Hadramout.
Market margin analysis
Marketing margin are the differences between prices at two market levels farm get
price and consumers price. Marketing margins have been examined on the basis of
data obtained on prices at different stages of the marketing chain. Marketing margins
have been calculated through computing the absolute margins or price spread, which is
essentially the same as the difference between the prices, paid and received by each
specific marketing agency. The following formula has been used to compute
percentage marketing margins as earned by each market intermediary in the marketing
of farm products.
1- Farm profit = Gross Revenue ($)/ha - Total costs ($)/ha ( see table 2)
2- Marketing Margin = Farm get price – Consumer price. (See table 3)
3- Percentage marketing margins= Farm get price – Consumer price/ Farm get
price * 100 (See table 4)
971
Table (2) show that Farm profit = Gross Revenue ($)/ha - Total costs ($)/ha = 1,744
- 1,977= -233($)/ha. That means farm has received less prices then the real cost of
production The average period lost is -806 ($)/ha (2002 to 2006)
Table (3) show that middlemen have received high profit it is 1,109 ($)/ha in 2002
up to 4,256 ($)/ha in 2006, with average period 2,307 ($)/ha
Table (4) Percentage Marketing margins earned by the middlemen in Sana’a is
172% 2002 to 244% in 2006 with average period 238%
Breakdown of consumer’s one USA Dollars
Breakdown of consumer's Dollars is a phrase applied to the manner in which a
consumer's one Dollars expenditure on a particular commodity is divided among the
producer and marketing agencies. It shows from the table (4) that portion of a
consumer's Dollars which goes to the producer is 0.44 cent and 1.06 is earned by
various marketing agencies such as contractors, commission agents, wholesalers and
retailers. This was calculated by expressing the net margin of a specific agency as
proportion of the retail price.
Marketing costs
The marketing margin indicates the amount received by the different marketing
agencies for providing their services, from the time when commodity leaves the farm
until it reaches the consumers. Such costs are not known and it is not include in the
analysis.
Table (5) explain the market situation, Retail price in Sana’a City increased from
1.5 US$ in 2007 to 3 US$ in 2008 this means the inflation rate is 200% for date palm.
Also Family Budget Survey 2008 indicted that Yemen had import date palm from
Saudi Arabia by 30 Million USA Dollar, these indicted demand is higher than supply.
Conclusion
Like farmers throughout the world, but especially in developing countries, Yemeni
farmers work hard throughout the year to produce high quality crop and livestock
products in sufficient quantities to reach profitable levels. However, also like farmers
everywhere, Yemeni farmers lack marketing information, alternatives, knowledge,
skills, tools, and institutions to make the most of selling the products they worked so
hard to produce. Marketing issues are particularly frustrating for farmers because they
972
often perceive that the ‘middle man’ or the broker gets more of the consumer dollar
than the farmer does. Marketing is frustration for small farmers in Yemen. Farmer’s
production increase but their income didn’t
Recommendation
It is strongly recommended that policy for equity and normally margin profit for
both Farmers and middlemen are applying inside the retail market areas in Yemen.
Efforts to Improve Date Palm Production in Yemen and link it with food security and
poverty elevation. Farmers want new marketing principles to enhanced marketing
capacity and policy for small farmers to get fair prices for their productions.
973
References
[1] Agricultural Year Books – 2002 to 2006
[2] Ali Muhammed Khushk & M. Ibrahim Lashari. Marketing margin analysis of
flowers in Sindh Aug 27 - Sep 02, 2001
[3] CAB International, agro. food marketing, 1997.
[4] Dr. Mohamed A. Al- Hebshi, Economics of Poverty, Environment and Natural
Resource Use, Does Small Farmer Investment In Bananas Jeopardize
Macroeconomic Stability In Yemen, International Conference, 17- 19 May 2006,
Wageningen International Congress Canter, Wageningen, The Netherlands.
[5] Daniel H. Pick, Jeffrey Karrenbrock, Hoy F. Carman, Price asymmetry and
marketing margin behavior: An example for California - Arizona citrus
[6] Food and Agriculture Organization of the United Nations (FAO); United Nations
World Food Programme (WFP), Special, Report - FAO/WFP Crop and Food
Security Assessment Mission to Yemen, Date: 09 Dec 2009
[7] Groundwater & Soil Conservation Project, Sauyen Hadramout December 2007
[8] Ministry of Planning and International Cooperation (MoPIC), YEMEN
HOUSEHOLD BUDGET SURVEY 2008, CSO, Sana’a Yemen, 2009
[9] Ministry of Agriculture and Irrigation, (MoAI), The Cost of production of mean
crops in Hadramout, Department of Monitoring & Evaluation, Sana’a Yemen, 2007.
974
Table (1) Crop budgets of the means cropping patron in Wadi Hadramout 2006
Cropping
Averag
Date
Alfaf
Man
Bana
Onio
Tom
pattern
e
s
a
go
na
n
ato
Garli Potat
c
o
Gross
Revenue
4,469
1,744 6,380 7,356 4,905 3,834
-
7,200 4,336
1534.7
1,977 2,265 2,372 2,221 1,150
359
1,060
2,935
233-
359-
6,140 3,463
($)/ha
Total costs
($)/ha
873
Net
Revenue
4,115 4,984 2,684 2,684
($)/ha
Table (1) shows that all crops net revenue is positive expect the Date Palm and
Tomato are negative by 233and 359 USA$.
Table (2) Net Revenue ($)/ha for farm Date Palm in Wadi Hadramout 2002 to 2006
2006
2005
2004
2003
2002
Yield(kg)/ha
4,000
2,200
2,080
1,384
1,350
farm gate price($/kg)
0.44
0.4427 0.421053
0.34
0.479
Gross Revenue($)/ha
1,744
974
876
473
646
Total costs ($)/ha
1,977
1,959
1,583
1,706
1,521
Net Revenue ($)/ha for farm
233-
985-
707-
1,232-
875-
Calculated from tables annex 1
975
Table (3) Net Revenue ($)/ha for middlemen in Sana’a City 2002 to 2006
2006
2005
2004
2003
2002
Consumer Price (retail price in Sana’a City)
1.5
1.5
1.5
1.3
1.30
Gross Revenue($)/ha
6,000
3,300
3,120
2,075
1,755
Total costs ($)/ha
1,744
974
876
473
646
Net Revenue ($)/ha for middlemen
4,256
2,326
2,244
1,602
1,109
Calculated from tables annex 1
Table (4) Percentage Marketing margins earned by the middlemen in Sana’a 2002 to 2006
2006
2005
2004
0.4427 0.42105
2003
2002
0.34
0.479
farm gate price($/kg)
0.44
Consumer Price($/kg)
1.5
1.5
1.5
1.3
1.30
Market Margin ($/kg)
1.06
1.06
1.08
0.96
0.82
% Market Margin ($/kg) for middlemen
244.12
238.82
256.25
280.00 171.56
Middlemen profit = Gross Revenue ($)/ha - Total costs ($)/ha = 6,000 -1744=4256 ($)/ha
From the table (4) the marketing margins earned by the middlemen in Sana’a was
(1.06/.44*100) = 244($/kg)
(Retail price in Sana’a City)
Table (5) date palm prices in Sana’a City 2007- 2008
Kind
2007
2008
Inflation
price(YR/kg) price(YR/kg)
Rate
2008
price($/kg)
Al-Ngrani (Saudi )
300
600
200
3
Al-Hadhramy(Saudi )
300
500
167
2.5
Al-Bashy(Saudi )
300
550
183
2.75
976
The agricultural sector2
Food and Agriculture Organization of the United Nations (FAO); United Nations World Food Programme
(WFP), Special, Report - FAO/WFP Crop and Food Security Assessment Mission to Yemen, Date: 09 Dec 2009
2
Food and Agriculture Organization of the United Nations (FAO); United Nations World Food Programme
(WFP), Special, Report - FAO/WFP Crop and Food Security Assessment Mission to Yemen, Date: 09 Dec 2009
977
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978
OP 51
A pilot study to evaluate the use of date palm residues (leaves,
Leaf, Kornav) such as organic fertilizer in the in the desert areas
Oustani Mabrouka
Assistant professor at the University of Kassdi Merbah Ouargla (Algeria)
E-mail: belsam.oustani@yahoo.fr
Abstract
This study aims to shed light on the possibility of exploiting some of the organic
material of local origin, date palm wastes (leaves, Leaf, Kornav) in improving the
microbiological activities of the sandy soil strength characteristics of the light areas of
desert in southern Algeria, especially the process of carbon mineralization, something
that would create ways to materials in the form of a permanent and usable by plants.
Thus, after the incubation period of 42 days under conditions of moisture and
temperature of an adequate (80% of saturation, 28 C°) turns out to provide the soil
residues of date palm would raise the metal carbon clearly compared to soil that had
not provided and due to role of the organic matter in the revitalization and
multiplication of microorganisms relevant in the process of the metal and the central
Itherigina incubated energetic materials essential to the creation of microbiological
activity in soil. Analysis of the results obtained shows the positive effect of the legacy
of the date palm on the microbiological activities. Concerning the measurement of
carbon dioxide released from decomposition of organic matter accumulated the
quantities obtained at the end of incubation, respectively:
• Residues in the soil treatment of date palm was recorded
• In the soil changed for the treatment of (witness) was recorded 14.37mg/25g soil.
With respect to estimate the density of microbes in treatment and non-treatment of
date palm residues has been shown that the addition of articles from this topic would
raise the density of microbes in the soil and clear compared to that have not provided
as they were recorded:
• Total Microflora: 50.107germs / gram of dry soil l in the soil treatment residues
palm against 32.107germs / gram of dry soil in the non-soil treatment.
979
• Fungal microflora: 31.103 germs / gram of dry soil in the soil treatment residues
palm of charge. 22.103 germs / gram of dry soil in the non-soil treatment.
• Cellulolytics: 3.103
germs / gram of dry soil in the soil treatment residues palm
against 1.103 germs/ gram of dry soil in the non-soil treatment.
• Amylolytics : 5.104 germs / gram of dry soil in the soil treatment residues palm
against 3.104 germs / gram of dry soil in the non-soil treatment.
Overall results obtained show the importance of adding organic waste palm of the
soil in desert areas this topic last complain of the many obstacles reclamation and
where the physical processes of transformation of organic matter around
microbiological operations.
Key words: Residues palm, Organic fertilizer, Sandy soil, Microorganisms,
Mineralization, desert areas.
Introduction
Sandy soils are widely distributed in the most of arid and semi-arid areas. For
instance, the total estimated extent of Arenosols is 900million hectares (WRB and
FAO/Unesco soil map of the World). It is well known that these “problem soils” are
characterized by a low soil organic carbon, a low cation exchange capacity (CEC), a
high risk of nutrient leaching, a low structural stability, and a high sensitivity to
erosion and to crusting [1,2].
In these soils, in which the clay content is low (3 to 15 % by mass), organic matter
is the main determinant of fertility, nutrient storage, aggregate stability, microbial and
enzymatic activities [3].
However, cultural practices or land uses aimed at increasing organic matter stocks
have a minor impact if compared with the potential storage of organic matter in clayey
soils. Nevertheless, this stock increase is possible in sandy soils and is mainly linked
with the increase of the “vegetal debris” functional pool.
The role of organic matter on the properties of these soils, on their potential of
productivity and on the sustainability of agricultural systems is thus fundamental [1, 2].
The control of soil organic matter on chemical (CEC, pH, some cations such as
calcium and magnesium), and physical (porosity, structural stability) properties has
often been demonstrated [4]. Any time, little work has been done regarding the
980
influence of organic inputs on soil microbial activities in arid sandy although the latter
remains an organic component of soil fertility. It intervenes by acting in part on the
stock of minerals treated, obtained by mineralization of organic matter (control C and
N fluxes), and secondly to preserve the environment [5, 6, 7, 8].
Thus, the management of organic residues represents has means to control the
activity of soil microorganism. Year adequate management of organic matter (through
fallows, improved fallows, pastures, external organic inputs) through its consequences
one soil bio functioning, largely given the agronomic (seedling production) and
environmental (carbon sequestration) potentials off Sandy soils. However, the
maintenance d' an optimum organic matter rate in the grounds, imposes regular
organic matter restitutions, traditionally they come d' a share of the crop waste
products and d' another share of l' incorporation of the varied organic matters (manure;
compost…). On the basis of this point of view, the recycling of the organic matter in
the arid regions is limited, even impossible because of weakness of the production of
vegetable biomass and l' insufficiency of l' breeding, which limits the d' possibilities
considerably; supply of manure and other organic matters [9].
However, according to [10], the Algerian palm growing wealth is estimated at about
10,475,150 of many feet over an area occupied by date palms de154 372 ha of which
almost all of this acreage is located in arid. The palm cultivation by its place in
agriculture in arid areas is a high ecological value and economic. It is therefore
important both for the financial product that it generates by the continuity of life it
allows. Fitting to the agronomic allowed to play its full role in creating, maintaining
and developing economies as a basis for oasis. Nevertheless, the expansion of palm
growing areas generates more and more biomass partially used (hedge, shelter), which
is bulky waste, accumulated, become a source of disease compromising both the
environment and date production.
Taking into account all these considerations and in order to meet growing food
demand of these populations in a context of limited resources, organic, farmers have
developed various techniques to increase crop production, including the use of date
palm residues. The use of these residues fits perfectly into a strategy to improve the
organic status of sandy soils and in the fight against pollution [11, 12].
981
The objective is to design a product to both improving soil fertility and enhancing
these residues (Fins, Lif, Cornafs) and furthermore share the biological fact that it is a
transformation and utilization of byproducts of date palm. In sandy soils whose
fertility is naturally low, optimization of local resource management cycle of organic
matter in agricultural systems is probably the key to increasing agricultural production.
In the present paper, We provide information On the sandy soils in bio functioning,
Interactions between existing organic matter, microbiological activities including the
activity of carbon mineralization of these components, and the determination and
enumeration of microbial density in relation to these components.
Material and methods
1. Pedological material
In order to clarify the incidence of certain types of organic residues from plants and
local soil microbiological activities in arid, our choice fell on a sandy soil in this case
for what contributions organics are of considerable importance and that due to the
improvement of all soil properties. Pedological material so selected was taken from the
experimental farm of the University of Ouargla located in the South East of Algeria.
Thus, the average samples obtained from a mixture of several samples were collected
up to 20 cm depth after discarding the first three inches of soil and they are collected
in sterile glass vials. The moisture content of samples was measured on arrival at the
laboratory. An aliquot of these samples was dried in laboratory conditions is reserved
for the physicochemical characterization of the soil, while the one reserved for
microbiological measurements, was kept before his employment with initial moisture
at 4 C °, as water stress can disrupt biological measures [13].
The soil studied was subjected to physicochemical characterization with particle
size (pipette method of Robinson), capacity (Method Boyoucos). Bulk density
(cylinder method), pH (water suspension in a soil: 1/2.5), Limestone total (Method
calcimeter Bernard), the organic carbon (Anne method), organic nitrogen (Kjeldahl
method) Electrical conductivity (EC at 25 ° C) measured by a conductivity of a
suspension of a report: soil/water
1 / 5, Na+ and K+: measured with a spectrophotometer flame, Ca
++
and Mg
++:
measured with a spectrophotometer to atomic adsorption. CEC (Method of POWER).
982
The analysis methods adopted for the characterization of organic material, are the
same as those used for hardware edaphic, apart from some specific analysis of organic
substances, Thus, organic carbon was determined by the method of calcination, the
dosage crude fiber (Conventional method Weender) Determination of Ca ++, Mg++,
Na+, K+ EAWAC method, Determination of Phosphorus by chromatography (spectral
analysis). The physico-chemical properties of soil (Table 1) are illustrated in Table 1.
2.Organic Material
For economic and ecological reasons and to enhance our local potential in organic
matter, we used an organic substrate of plant origin, it is the mixture of residues of
date palm (Leaves, Leaf, Kornaf). These residues were taken from a palm grove in the
operation of the University of Ouargla. This is a carbon-rich material, costs are
relatively low, humic acid, whose C/N ratio equal to 31.1. Prior to use these residues
were sieved to 2 mm to obtain a homogeneous material of agronomic point of view.
The dose of organic contribution made to the two soil types was dictated by references
to numerous studies, most of which are in agreement as to the existence a linear
relationship between soil texture (especially the percentage of clay) and organic matter
content [14,15].
Thus, samples of both soils were reported at a level organic (as residue intake date
palm) deemed desirable. According to the chart prepared by the INRA laboratory [15],
this level corresponds to a dose of 3%. The chemical composition and Biochemical
palm residues is illustrated in Table 2.
Methodology for studying the biodegradation of both organic matters to soils.
The easiest way to assess the overall activity of the micro flora is to measure carbon
mineralization under controlled conditions, close to the optimum organic. To make
two different types of incubations were conducted under the same conditions of
humidity (80 percent of holding capacity) and temperature (28 degrees) with the
following operations:
1.Minéralization organic carbon (Respirometry Technique: Measurement of CO2
evolution: A method of Guckert)
Incubations were conducted with fresh soil weight equivalent to 25 g of dry soil.
The samples were moistened to 80% capacity retention, and enriched shredded date
983
palm residues at 3% of their initial concentrations of organic matter. The principle of
the method is based on sequestration of CO2 from the respiration of microorganisms
by telluric sodium hydroxide solution NaOH (0.5 N), excess sodium hydroxide is
titrated with hydrochloric acid (HCl 0.2 N) in the presence of a color indicator
phthalein phynol after the precipitation of carbonates in BaCO3 by barium chloride
(BaCl2 20%). The simple system consists of the non-enriched soil (control treatment).
The complex system consists of soil enriched with shredded date palm residues.
It was an empty set containing no soil, serving as a control to determine the CO2
fixed by the atmosphere (Figure1a).
Frequency of dosing CO2
The dosage of CO2 released is done daily; it was the Tale of a period of 42 days.
Three repetitions were performed for each treatment and values expressed are the
mean of three replications.
2.Measurement of telluric micro flora (Figure1b,c)
Our study will focus on developing the total micro flora and fungi, and certain
functional groups such as cellulolytic, amylolytic and related to the different organic
inputs made. Counts of germs are produced either on solid medium (agar) for the total
micro flora (extract agar earth) and fungal micro flora (OGA), or in liquid medium for
cellulolytic and amylollytics germs.
The enumeration of bacteria on solid medium was performed using a colony counter
after one week of incubation at 28 °C (from dilutions of the suspensions due to dilution
by 3 boxes). As for the enumeration of bacteria on liquid medium, it was performed by
the method of most probable number (MPN GRADY MAC) after one month of
incubation at 28 °C (from dilutions of the suspensions due to 3 tubes per dilution). This
is a qualitative and quantitative characterization of the telluric micro flora.
Results and discussion
1. Potential mineralization
The curves of cumulative carbon mineralization (Figure 2) show the intensity and
timing of mineralization (respiratory activity) during 42 days of incubation at 28 °C.
Thus, the release of carbon dioxide observed in the enriched soil is higher than the
control soil. Indeed, we record a release of 14.37 mg/25g dry soil in soil un enriched
984
(control). However, we note a higher value in soil enriched palm date palm waste
material of around 40.02 mg/25g dry soil. In other words, it was under the influence of
mineralized organic substrates palm date palm, a nearly equivalent amount of carbon
to three times that generated by the non-enriched soil.
These results thus show that the recent contribution of organic matter and setting
optimum conditions of the soil favors the development of an active micro flora resulting in
a strong release of CO2 from compounds rich in soluble (amino acid, sugar etc. ... ..) and
are used very quickly by microbes, it reflects in part the work of [16, 17], showing that
microbial attack is favored by the presence readily biodegradable organic compounds.
Indeed, increased microbial activity in the presence of organic substrates may be due
to a lowering of pH caused by decomposition of date palm, for it seems that the alkaline
pH of the soil study (relatively high in limestone) would have been a factor in partial
inhibition of microbial activity [18], this medium-enriched residues of date palm allows
micro-organisms to multiply and inactive increase their activity mineralization.
In addition, a low native organic matter in soil without additives clearly explains the
low release of CO2, this is attributed to factors which act on microbial activity
depressed that this is particularly sandy soils where loss of organic matter is very
important because they are too airy and organic matter decomposes more easily there.
They contain less clay and are then less protected, which could induce physical
protection of this organic matter vis-à-vis the mineralization process [19].
Regarding the rate of release of CO2, we note a strong release during the first weeks
of incubation either for soil enriched or not, this is related to the development of
microorganisms that are easily metabolized foods mineralizable energy to
microorganisms which is consistent with the work of [20,21], which show this phase
is evolved much CO2.
In the last weeks of incubation (the 5th week in the 6 th week) we note a decrease of
carbon mineralization as this and as the incubation time is prolonged, there is a phase
mineralization characterized by slow and steady release of CO2 a low but still higher
than that reached by the non-enriched soil. The decrease in mineralization rate observed
during this phase is attributed to a depletion of easily mineralizable carbon which leads
to a relative enrichment in fractions more or less resistant to the mechanisms of
985
mineralization. In this context we found that the soil enriched by the date palm residues
relatively rich in substances hardly mineralizable (cellulose, lignin,..) To C/N ratios are
far from being completely broken down in the first phase, their secondary
mineralization runs parallel to the phase of primary mineralization [22].
Moreover, the statistical study based on analysis of variance 1a (CV) shows a
significant influence on the biological activity of sandy soil, the latter estimated by the
respiration rate.
2 Influence of palm residues on microbial density
After a week of incubation at 28C °, we find on the agar medium that Enrichment of
organic substrates has a positive effect on soil microbial populations. This translates in
terms of density by a steady increase in number of seeds of non-enriched soil (control
treatment) to the ground enriched by date palm residues.
1.Effect
concerning
of
date
palm
the
total
micro
residues
flora,
on
we
total
recorded
and
the
fungal
micro
following
flora
densities:
50,107 germs/gss for soil enriched by the residues of date palm.
• 32,107germs/g.s.s for non-enriched soil. (Figure 3).
This population growth in the presence of organic substrates (palm date palm) is
explained by the richness of this compounds in essential nutrients and fermentable
material and favorable to growth and development of germ-microbial. Indeed, [23],
considers that the density of telluric micro flora is directly influenced by organic
matter. While [24], shows that the stimulatory effect may be explained by the positive
role exerted by organic matter on the physicochemical properties of soil.
In addition, the balance of micro flora is altered after burial of organic matter,
leading to an increase in the number of microorganisms in the soil.
For the fungal micro flora developed on the middle elective OGA we noted a
growing microbial density consist of:
• 31,103 germs / gss for soil enriched with chased out of date palms.
• 22,103 germs/g.s.s for non-enriched soil (Figure 4).
These results reveal the importance of the contribution of organic matter with
respect to the fungal micro flora whose development requires above all a warm acid;
the highest density was recorded under the effect of palm residues.
986
The greatest effect in stimulating the contribution of fins is certainly due to the
enrichment of the soil relatively rich in lignin substrates relatively acidic, which
consequently promotes strong growth of fungi lignolytics, this is consistent with the work
[24, 25],showing that fungi play an important role in the decomposition of liginolytics.
But pedological terms, we attribute the increase in fungal micro flora, being that it
does not seem to be strongly inhibited by the sandy texture. In fact, mushrooms are
unlike bacteria are favored by the sandy texture.
At this point, [21], showed that a bacterium or another mixed population prefer a
fine texture, whereas Aspergillus prefers a coarse texture. [26], show that a carbon
substrate in a soil is sandy-loam degraded primarily by fungal activity, then assisted by
bacterial activity, whereas in the case of a clay soil (60%) the substrate is
simultaneously decomposed by the action of fungi and bacteria.
There is indeed a negative correlation between clay content and fungal biomass,
whereas
this
correlation
is
positive
with
bacterial
biomass.
The 90% of fungi are preferentially localized at the surface of aggregates, whereas
bacteria (90%) are located within [7].
2. Effect of date palm residues on the physiological groups
The study of cellulolytic germs in liquid media shows that after four weeks of
incubation at a temperature of 28°C, it develops a population of approximately:
• 5 .103 germs / gss for soil enriched residues of date palm
• 1103 germs / g.s.s for non-enriched soil (Figure 5 ).
We can think a priori that the observed increase in non-enriched soil (control
treatment) is due solely to the low native organic matter, since the action of the sandy
texture appears weak and is masked in the nature of other factors in particular aeration
and organic matter content.
Also, the initial poverty of our soil nitrogen is in part responsible for the low density
of sprouts celllulolytics which confirms the findings of [21], who attributes the
slowdown cellulolysis to insufficient soil nitrogen. The relatively lower density counts
for soil enriched by the waste material is certainly due date palm substances which
cellulose is associated (especially lignin).
987
Indeed, cellulolytics are slowed by the presence of encrusting substances such as
lignin, which hinders the production of microbial cellulase [21]. Regarding the
amylolytics germs, the functional group of amylolytics is quantitatively higher in soil
enriched by the residues of date palm in the control soil. The observed values are:
• 5.104 germs/ gss for soil enriched residues of date palm.
• 3.104 germs/gss for unenriched soil (Figure 6).
Analysis of these results shows that the microorganisms involved in the process of
amyloid found in the presence of palm residues the conditions most conducive to their
growth and proliferation. This high amylose may be due to the diversification of
people involved in the metabolism of starch. This does not seem to stimulate a specific
micro flora [14].
Finally, the stimulation observed in this functional group under the effect of organic
substrates, confirms once more our results for the respiratory activity of our soil.
Conclusion
This study on the recovery of organic waste from local waste represented by the
date palm (palms, lif, kornaf) in improving the microbiological properties of sandy
soils in arid regions, we identified a number of points concerning the evolution of this
type of organic substrate in the ground and is thus a modest contribution to address the
chronic deficit in soil biology studies in these areas.
The analysis of overall results allowed to demonstrate a positive and significant
residues
of
palm
trees
on
the
density
and
soil
biological
activity.
Indeed, it was noted that the incorporation of residues of palm causes after 42 days of
incubation a significant increase in carbon mineralization (through respirometric tests:
measurement of CO2 released) compared to non-enriched soil.
This clearly shows that these residues improve soil biological activity, and yet it
exerts its positive effects for energy, nutrients and elective components, it offers
organisms a great atmosphere with its soil biology ability to improve certain physical
and chemical properties of sandy soils.
One the other hand, concerning the impaction the density microbian, this favorable
study confirms extensively the effect off residues palms dates and upon the biological
actions conducted one the mineralization off carbon. Consequently, this has allowed
988
custom to stress one the fact that all the physiological groups cuts been largely affected
by the adjunction off adequate substrates in the soil. This proves that inspite off the
inadequacy off to their activities; several individuals remain within those biotopes, in
which they preserve the species. It edge be thought that they cuts subjected to has
process off selection gold adaptation to the medium. At the mean time all our results
suggest the economic and edaphic advantage off using the palms dates in dry areas,
where tremendous problems off evaluation (low concentration off organic matter, high
salt concentration….extract agar of land and where the process off natural evolution
off organic matter overcome of biological process.
989
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[12] OUSTANI M.2006- Contribution à l'étude de l'influence des amendements
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salés dans les régions Sahariennes (Cas de Ouargla) .Thèse Magister.
Uuniversité .Ouargla. 187p
[13] FARDOUX J., FERNANDES P., NIAN-BADIAN A., COTT J.L., 2000-Effets
du séchage d'échantillons d'un sol ferrugineux tropical sur la détermination de la
biomasse microbienne,comparaison de deux méthodes biocidales de référence.
Etude et gestion du sol N°7, Vol 4, pp: 385-394.
[14] MALLOUHI N, JACQUIN F., 1986- Influence of certain ions the biological properties
of a salt enriched pelosol. Agrokémia es Talajtan, Tom 35 n01-2, pp 105-111.
[15] SOLTNER D., 2003- Les bases de la production végétale, Tome I, le sol et son
amélioration. éd collection science technique agricoles., 472p.
[16] DOMMERGUES Y., 1962-. Contribution à l'étude de la dynamique microbienne
des sols semi aride tropicale sèche. Thèse Doct., Paris. p178.
[17] CHAREAU C., 1975 - Organic matter and biochemical properties of soil in the
dry tropics of West Africa, Bul, FAO Soils, n0 27, Rome, pp 305-323.
[18] TOUTAIN F., 1981- Humus forestiers, structures et modes de fonctionnement,
rev, forest, Franç (6), pp 449-464.
[19] NAMAM F., SAOUDI B., CHIANG C., 2001- Impact de l'intensification agricole
sur le statut de la matière organique des sols en zones irriguées semi –arides au
Maroc: Etude de gestion du sol, AFES, Vol 8,4, pp 269-277.
[20] GUCKERT A., 1973 - Contribution to the study of polysaccharide in soils and their
role in the mechanisms of aggregation. Thesis in October Es Sci Univ, Nancy, 124p.
[21] DUCHAUFOUR PH., 1997 - Short of Soil Science. Soil, vegetation, environment
.5 th Edit. Al. Teaching Earth Sciences. Masson, Paris P. .291
[22] DOMMERGUES Y et MANGENOT F., 1970-. Ecologie microbienne du sol
.Masson et Cie Editeurs, Paris, 796
991
[23] CALIBREX R., 2005. Impact des intrants organiques et conduites culturales sur
la biomasse microbienne
et la diversité des bactéries telluriques .Thèse
Doctotat .Univer Rouen p167.
[24] PARR, J. F. (1973): Nature and signification of inorganic transformation in
tiledrained soils and fertilizers. n° 32, pp 411-415.
[25] MEKHAZNI D., 1990- Activité cellulolytique et évolution des microorganismes
aérobie dans un sol salé enrichi en paille. Thèse Magistere ., INA, Alger, 55 p.
[26] ADU J K., OADES J.M., 1978- Physical factors influencing in aggregates: Soil
Biol. Biochem 10, pp: 109-115.
992
Table 1.Physico-chemical and biochemical characteristics of the soil (0-20 cm).
Parameters
Sandy Soil
Granulométrie
Clay (%)
05.94
SILT (%)
2.34
Sand (%)
٩١٧٢
Total limestone (%)
.94١٢
Global saltiness EC dS/m
01.00
Soil reaction (pH of water: 1/2.5)
08.30
Characteristic Biochimics
Exchangeable cations
Cmmol/kg
OC (%)
00.38
N (%)
0.068
OM (%)
00.66
Mg++
01.20
Ca++
04.04
Na+
00.75
K+
00.06
CEC (T)
06.05
Table 2. Chemical and biochemical composition of palm residues
(% shredded date palm residues)
CE (1/2.5)
pH
Nitrogen
total
C/N
Hemi
cellulose
Cellulose
Lignin
(1/2.5)
C.
Org
dS/m
5.58
5.05
47.3
1.5
31.1
17.4
19.0
58.4
993
C
1
2
Fig.1. b. Dispositif of
incubation of fungal
microflora and total (solid
Fig. 1.a. Dispositif of
incubation of the samples of
the mineralization of organic
carbon
media culture)
Fig.1.c. Dispositif of
incubation of physiological
groups (Culture on liquid
medium)
45
40
35
Unenriched
soil
released into the
soil (mg/ gramme
Amount of CO2
30
25
Soil
enriched
bythe
residues of
date palm
20
15
10
5
0
1
2
3
4
5
Jours d'incubation
6
7
Fig 2. Evolution of cumulative release of CO2 ( mg /25 of soil) during
incubation in the sandy soil enriched and unenriched by residues of date
994
50
45
40
35
Number of 30
germs /g 25
(x10 7) 20
15
10
5
0
soil enriched by
residues of date
palm
Unenriched soil
Number
of germs/g
dry soil (
10 3)
30
20
10
0
1
Treatment
1
Treatment
Soil
enriched
by the
residues of
date palm
Uneriched
soil
Fig.4 Influence of date palm residues on the
fungal microflora
Fig 3.Influence of residues of date palm on
total microflora
3
2.5
Number of
2
germs /g dry soil 1.5
1
(10 3)
0.5
0
40
Soil enrichedby residues of
date palm
Unenriched soil
5
4
Number of 3
germ/ g
2
drysoil (x104)
1
Soil enriched by the
residues of date palm
Unenriched soil
1
Treatment
0
1
Fig 5. Influence of date palm residues on
germes cellulolytic
Treatment
Fig 6 .Influence of date palm on germs amylolytic
995
The photomicrographs below show some colonies obtained by culture on solid
medium sandy soil enriched by the date palm residues.
Fig.7Bacterial Colony obtained to leave
the culture of the total microflora on solid
medium (Extract agar of land)
Fig.8 Bacterial Colony obtained to
leave the culture of the total
microflora on solid medium (Extract
agar of land)
Fig 9.Colony of yeasts obtained to
leave the culture of the total
microflora on solid medium (OGA)
Fig. 10. Mycelial filaments of
Aspergillus obtained to leave the
culture of the fungal microflora on
solid medium (OGH) (Gross x 10)
996
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998
OP 52
Preparation of novel product by using mixture of meat and dates
*Hanaa A. Abdel Aziz and **El-Kafrawy, T.M.H.
* Meat and Fish Technology Research Department, Food Technology Research
Institute, Agriculture Research Center, Giza, Egypt
** Central Laboratory for Date palm Research and development, Agriculture Research
Center, Giza, Egypt
http://www.gitfood.com/Date-Spread-Dept/58/
Abstract
In this research carried out production of new product from meat and date, whereas
the three beefkofta samples were prepared with replacement 15% from the meat by 15
% ground whole date mixture, by15 % the fiber and epicarps mixture and by15 % the
date spread, then these samples were evaluated. From the evaluation for the three
samples, it was found that the first and second samples were un preferred for the
present of the epicarps, while the third sample was preferred, but with appear of the
sweet taste for the three samples. Therefore, the preferred third sample was prepared
with low various percentages of the date spread. Beef kofta samples contained date
spread were prepared by addition of following concentrations (2.5, 5, 7.5 and 10.0 %
date spread of beef) and compared with the control sample (0.0 % date spread) by the
sensory evaluation and the five sample were physically and chemically analyzed. From
results it was noticed that the addition of date spread improved the sensory, physical
and chemical properties of beef kofta.
Keywords: Beef, Dates, Date spread and Beefkofta,
Introduction
The use of the whole of date or date product after removing the drupe have the best
uses of the date as well as other types of fruit, because it returns the highest benefit to
the producer. However, there are some conditions that need to use part of the fruit
flesh in order to raise the utilization rate of the crop output. Date fruit products derived
to: 1-Date juice, 2- Juice products, 3- Juice concentrates (a. Date spread - b. Date
syrup - c. Liquid sugar) (Barreveld, 1993).
999
Date spread, though it has reached a far advanced stage of product development, is
not a commercial product. It is reviewed first in the range of juice concentrates
because as a product it fits between date paste made of the whole date flesh, and date
syrup, from which all non-solubles have been removed. The idea of date spread
originated in Libya, when after having initiated date syrup production on a pilot scale
and planning for a commercial factory, it was felt that increasing the product range
would benefit the economics of the commercial date syrup factory as well as give
some more scope to creating outlets for a second quality fibrous raw material, almost
always contaminated with dust and other foreign matter including insect fragments
(Shaari, et al., 1966). To make date juice, water has therefore to be added to dissolve
and dilute the soluble solids of the date, after which the non-soluble solids are
separated out (Chemap, 1984).
The chemical composition of date fruit is of prime importance to the user of dates,
in particular the packer, processor or trader, because it affects the possibilities and
limitations of the raw material for the intended end-use. The date goes from one
extreme of moisture content (85% at the early kimri stage) to another (5-10% in dry
desert dates). Sugars contribute the most prevalent single component and the date has
been used more as a sugar source than as a fruit. For practical purposes all sugars in
dates consist of a mixture of sucrose, glucose and fructose of which the latter two are
the derivations of sucrose after inversion. Total sugars (at the tamr stage) on a dry
weight basis for the more known varieties in the world do not appreciably differ in
quantity are between 5 – 88 %. Proteins occur in date fruit in the range of 1-3%. Fat
occur in small amounts in the date flesh (2.5-7.5%). Fat is mainly concentrated in the
skin (2.5-7.5%) and has a more physiological importance in the protection of the fruit
than contributing to the nutritional value of the date flesh (0.1-0.4%). Palmitic, capric
and caprylic acid were identified as the major free fatty acids in the date flesh followed
by linoleic, lauric, pelargonic, myristic acid. The crude fibre amounts to 2-6% of the
date flesh. The date contain vitamins A, B1, B2 and niacin in reasonable amounts and a
good source of minerals such as potassium, calcium, iron, phosphorus, magnesium,
sulphur, copper and chlorine. Also, it contain other chemical substances such as
polyphenols, organic acids, flavour volatiles, pigments and sterols (Barreveld, 1993
1000
Al-Hooti et al.(1995) reported that the date had a considerable amount of some
important minerals such as Ca, Mg, P, K, Fe, Cu and Zn. However, the high
nutritional status of dates is due to the considerable quantity of ash or the appropriate
mineral balance.
In many countries, the date palm plays an important economical. The dates are
considered high energy foods containing sugars and fiber thus being suitable for all
people. Dates contain tannins which are made mainly of polyphenols and thought two
groups of them (phenolic acids and condensed tannins) to be important in producing
the astringent sensory response (Myhara et al.,2000).
Dates are produced in hot arid regions of the world and marketed worldwide as a
high value confectionery. It is considered as an important subsistence crop in most of
the world’s desert areas. Worldwide, date production has increased exponentially over
the last three decades from 1.8 million tons in 1963, to 2.6 million in 1983 and 6.7
million in 2003. This increase of 4.9 million tones since 1963 represents an annual
expansion of about 6.8%. In 2001 the top five producing countries - Egypt, Iran, Saudi
Arabia, Pakistan and Iraq (FAO, 2003) – were responsible for 69% of total world
production. If the next five most important countries (Algeria, United Arab Emirates,
Sudan, Oman, and Morocco) are included, then this percentage rises to 90%. This
clearly indicates that most of the world’s date production is concentrated in a few
countries in the same region (Erskine et al., 2003).
Tafti and Fooladi,(2006) determined the physico-chemical properties of Iranian
Shamsaei date at different stages of maturity found that the dates had the highest total
soluble solids (TSS) 65% and reducing sugars (61%) owing to moisture loss (which
decreased to about 15%) at the tamr stage. The tannin content of green dates was
about 1.7% which decreased around 0.24% at the tamr stage. The pH at tamr stage
reached about 7.
In conventional date processing, dry or soft dates are eaten as whole fruit, seeded
and stuffed, or chopped and used in a great variety of ways: as ingredients in cereals,
puddings, breads, cakes, cookies, ice cream, and confectionaries. The pitting may be
done by crushing and sieving the fruit or, more sophisticatedly, by piercing the seed
out of the whole fruit. The calyces may also be mechanically removed. Surplus dates
1001
are processed into cubes, paste, spread, powder (date sugar), jam, jelly, juice, syrup,
vinegar or alcohol (Morton, 2006). Recent innovations include chocolate-covered
dates and products such as sparkling date juice, used in some Islamic countries as a
non-alcoholic version of champagne, for special occasions and religious times such as
Ramadan (Wikipedia, 2006).
Date Spread is one of those sweet fruity spreads that you can use in so many
different ways: for baking (best dates rougalach or date filled cookies), to spread over
toast with butter, fill cakes with and more. The purpose of this study to preparation of
novel product by using mixture of meat and dates.
Materials and Methods
Beef meat was obtained from local market and minced in our laboratory. Dates
(Siwa dates) were obtained from El-Jawhara for drying, packing dates and developing
agricultural crops in Siwa. Also, the other ingredients (such as, egg, dry rusk, onion,
salt and spices) obtained from local market.
Spices mixture (10g): 2g black pepper + 2g Cubeb + 1g cumin + 0.5g nutmeg +
1.5g fennel + 1g cinnamon + 1g cardamom + 1g ginger.
Preparation of date spread (DS):
Two Kg of dates fruit were cleaned by tap water and then the drupes were removed
from the flesh of fruits. The fruits have been cut into small pieces and divided into
two parts, the first part (150gm) was grounded. Meanwhile, the second part was
soaked in water at ratio (1: 0.5, dates: water w/v) for 24 hours in refrigerator, then the
mixture mixed in blender to obtain a date paste. The date paste was filtered through
piece of clean gauze. The filtrate is the date spread (DS), meanwhile the precipitate on
the gauze is the fiber and epicarps.
Note: One kilogram of dates was given 8oo gm flesh without the drupes. After
the filtration the obtained of date spread (DS) was 1150 gm + 50gm (fiber +
epicarps mixture).
Preparation of beefkofta samples
Beef kofta sample was prepared according to the following recipe: minced meat
80 % + egg 8 % + dry rusk 2.5 % + onion 7.5 % + salt 1 % + spices 1 % (control
sample (0.0 % dates).
1002
Treatments:
Beef kofta sample was divided into four groups as following: The first group: beef
kofta mixed with 15% from the ground whole date mixture (A), the second group: beef
kofta mixed with 15% from the fiber and epicarps mixture (B), the third group: beef
kofta mixed with 15% from the date spread (C) and the fourth group: beef kofta
containing different levels (2.5, 5, 7.5 and 10.0 % of date spread). All treatments were
mixed and homogenized by a laboratory chopper and then the analytical methods were
carried out at zero time. Sensory evaluation was carried out for fried beef kofta
samples. The frying carried by using the corn seed oil.
Methods
Sensory evaluation:
Sensory evaluations of appearance, odor, color, texture, taste and overall
acceptability were carried out by aid of 10 panelists according to Molander (1960) and
judging scale was as : 9 – 8 (very good ), 7.9 – 7.0 ( good ), 6.9 – 6.0 ( accepted ) and
less than 6.0 was unaccepted. Conventional statistical methods of sensory properties
were used to calculate means and LSD. Statistical analysis (ANOVA) was applied to
determine significant differences (P < 0.05) according to Snedecor and Cochran,(1980)
Physical analysis
Water holding capacity (WHC) and plasticity of samples were measured using the
method of Golavin (1969). Cooking loss was calculated according to AMSA, (1995).
The Warrer-Bratzler shear force apparatus was used to measure the tenderness of meats,
whereas, low shear values indicated high tenderness as reported by Herring, (1976).
Chemical analysis:
Chemical composition (moisture, protein, fat and ash contents) was determined by
the standard methods as reported in the (AOAC, 2005). Total carbohydrates content
were determined as percentage, by calculating the difference between hundred and the
sum of moisture, protein, fat and ash percentage. Total Soluble solids % (TSS) was
estimated by using the Abbe digital refractometer according to (AOAC, 2005). The
minerals potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), zinc (Zn), iron
(Fe), cupper (Cu) were determined using a Perkin-Elmer HGA-600 atomic absorption
spectrophotometer 3300 (AOAC, 2005). The total phosphorus (P) was determined
1003
according to the method of (Troug and Meyer, 1959). Thiobarbituric acid (TBA) value
was determined as described by Egan, et al., (1981) and Total Volatile Nitrogen (TVN)
was determined according to the method published by Winton and Winton (1958).
Results and Discussion
1-Chemical composition and minerals content of date spread:
Results in Table (1) shows the chemical composition and minerals content of the
date spread (DS). It was noticed that total soluble solids (total sugar, minerals and
soluble vitamins and proteins) were 36 %, this percentage was less than (TSS) for date
as reported by Tafti and Fooladi,(2006), may be due to the addition of water to obtain
the date paste. Also, the moisture content of DS was high, may be due to the addition
of water for dates to obtain the date paste. The protein content was low, may be due to
that the date had low content of protein as mentioned by (Barreveld, 1993). The same
author reported that the fat is mainly concentrated in the skin, therefore, the DS has
low content of fat. From results for the ash and minerals contents, it was found that the
DS considered good source for minerals like whole dates as reported by Al-Hooti et
al.(1995). The carbohydrate content was high, may be due to that the date has high
content of sugar and it has been used more as a sugar source than as a fruit as reported
by (Barreveld, 1993).
2-Sensory properties
a) The sensory properties of beef kofta samples contained 15 % ground whole
date mixture, 15 % the fiber and epicarps mixture and 15 % date spread:
Results in table (2) shows the sensory properties of beef kofta samples with addition
of 15 % ground whole date mixture, 15 % the fiber and epicarps mixture and 15 %
date spread (DS). From the sensory evaluation, the three samples had good
appearance, odor, color and overall acceptability, except the (B) sample had accepted
texture and taste, the (A) sample had good texture and taste, while the (C) sample had
very good texture and taste, but the (A) and (B) samples were un preferred for the
present of the epicarps and the sweet taste was clearly appear for three samples. From
the results of statistical analysis, it could be observed that there was significant
differences in the texture, taste and overall acceptability, but there was not significant
differences in the appearance, odor and color between the three samples, it was found
1004
that, the (C) sample contained 15 % date spread had the higher scores for appearance,
color, texture, taste and overall acceptability than the other samples, but approximately
(A) and (B) samples were resemble to the control sample in scores of odor. Trained
panel sensory evaluations indicated that the (C) sample was preferred to consumers as
the control, but the (A) and (B) samples were un preferred because the epicarps which
were appeared with the chewing. Therefore, the addition of date spread improved the
sensory properties of beef kofta, but the sweet taste was appeared with 15 % addition.
Accordingly, the preferred third sample was prepared with low various percentages of
the date spread.
b) The sensory properties of beef kofta samples contained date spread (0.0, 2.5, 5,
7.5 and 10.0 %):
Data in Table (3) shows the sensory properties of beef kofta samples with addition
of date spread (0.0, 2.5, 5, 7.5 and 10.0 %). It was noticed that the 2.5 and 5 % DS
samples were like the control sample in appearance, while 7.5 and 10 % DS samples
were similar in appearance and the latter samples were slightly best in appearance than
the control, 2.5 and 5 % DS samples, however, there was non-significant differences
between the five sample in appearance. The scores were given for odor increased with
increase the percentage of date spread, however that also there was non-significant
differences between the five sample in odor. The scores were given for color increased
with increase the percentage of date spread from control to the third sample (5 % DS),
but the 5, 7.5 and 10 % DS samples were similar in color and also there was nonsignificant differences between the five sample in color. The scores were given for
texture increased with increase the percentage of date spread for all samples and there
was significant differences between the samples contained date spread and control
sample and between the 2.5 % DS sample and 5, 7.5 and 10 % DS samples, but the
latter three samples were similar in texture. The scores were given for taste increased
with increase the percentage of date spread from control to the third sample (5 % DS),
while the 7.5 and 10 % DS samples had less scores than the 2.5 and 5 % DS samples,
may be due to appearing of sweet taste, but it was higher than control sample, although
the sweet taste was appeared, the 7.5 and 10 % DS samples still acceptable. There was
non-significant differences between the five samples in taste, this indicator that the
1005
samples with addition of date spread up to 10 % were accepted with respect to the
taste. The scores were given for overall acceptability increased with increase the
percentage of date spread and there was non-significant differences between the five
sample in overall acceptability, this indicator that the samples with addition of the
different percentage of date spread up to 10 % were accepted with respect to the all
sensory properties than the samples which showed in Table (2).
3- Chemical composition of beef kofta samples with addition of date spread (0.0,
2.5, 5, 7.5 and 10.0 %):
Results in Table (4) shows the chemical composition of beef kofta samples with
addition of date spread (0.0, 2.5, 5, 7.5 and 10.0 %). It was noticed that the moisture
content slightly decreased from (61.67 %) for the control sample to (61.34 %) for
sample contain 2.5 % date spread, may due to that the moisture content for the meat is
high, then the moisture content increased with the increase of date spread percentage,
due to the addition of water to the dates for prepare the date juice as reported by
Chemapag, (1984). Also protein, ash and carbohydrates contents increased with
increase the percentage of date spread, there was slightly increase of protein content
due to the dates contain low percentage of protein as reported by (Barreveld, 1993),
while there was evident increase of ash and carbohydrates contents, due to the dates
contain high percentages of ash and carbohydrates as reported by Barreveld, (1993),
Al-Hooti et al.(1995) and (Myhara et al.,2000), but the fat content decreased with
increase the percentage of date spread, due to the fat is mainly concentrated in the skin
of the dates as reported by (Barreveld, 1993), also as it is observed from the results of
DS in Table (1). From the statistical analysis, the significant differences between the
five samples for moisture, protein, fat, ash and carbohydrates indicator that the
addition of the different percentage of date spread up to 10 % had good effect on the
chemical composition of beef kofta samples.
4- Minerals content of beef kofta samples with addition of date spread (0.0, 2.5, 5,
7.5 and 10.0 %):
Results in Table (5) shows the minerals content of beef kofta samples with addition
of date spread (0.0, 2.5, 5, 7.5 and 10.0 %). It was noticed the contents of calcium,
potassium, phosphorus, magnesium and iron were increased, while the contents of
1006
sodium, zinc and copper were decreased with increase the percentage of date spread,
due to that the DS had high contents of Ca, K, P, Mg and Fe and low contents of Na,
Zn and Cu as it is observed from the results of DS in Table (1). Therefore, the samples
contained the DS considered a good source of minerals as the dates as reported by
Barreveld, (1993) and Al-Hooti et al.(1995).
5- Physical properties of beef kofta samples with addition of date spread (0.0, 2.5,
5, 7.5 and 10.0 %)
Data in Table (6) shows the physical properties of beef kofta samples with
addition of date spread (0.0, 2.5, 5, 7.5 and 10.0 %). It was noticed that the addition
of date spread led to decrease the cooking loss and increased the water holding
capacity (WHC), plasticity and tenderness of beef kofta samples, the rate of decrease
and increase was low with increase the percentage of date spread, therefore, the
addition of date spread led to improvement physical properties as reported by Tafti
and Fooladi,(2006).
6- Chemical properties of beef kofta samples with addition of date spread (0.0,
2.5, 5, 7.5 and 10.0 %):
Results in Table (7) shows the chemical properties of beef kofta samples with
addition of date spread (0.0, 2.5, 5, 7.5 and 10.0 %). It was noticed that the content of
total volatile nitrogen (TVN) and the values of thiobarbituric acid (TBA) decreased
with increase the percentage of date spread, may be due to the decrease of the
percentage of meat in beefkofta samples, because these materials related to the meats,
therefore, the addition of date spread led to improvement chemical properties as
reported by Tafti and Fooladi,(2006).
1007
Reference
[1] Al-Hooti, S; Sidhu, JS and Qabazard, H (1995): Studies on the physico-chemical
characteristics of date fruits of five UAE cultivars at different stages of maturity.
Arab Gulf J. Sci. Res., 13: 553-569.
[2] AMSA (1995): Research guidelines for cookery, sensory evaluation and
instrumental tenderness measurements of fresh beef. American Meat Sci. Assoc.,
Chicago, USA.
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[4] Barreveld, WH (1993): Date palm products. FAO Agricultural Services Bulletin
No.101. Food and Agriculture Organization of the United Nations Rome, Italy.
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No. 4428969.
[6] Egan, H; Kirk, RS and Sawyer, R (1981):
Pearson`s Chemical Analysis of
Foods.8th ed. Churchill Livingstone. Longman Group Limited U.K.
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Ragy, SM (2003): ‘Date palm in the GCC countries of the Arabian Peninsula’.
Available on: http://www.icarda.org/aprp/Datepalm/introduction/intro-body.htm
[8] FAO (2003): Agro-Statistics Database. FAO: Rom.
[9] Golavin, AM (1969): Control of fish products . Pishcevaio Permish Lemest
Publishers, Moscow (in Russian).
[10] Herring, HK (1976): Evaluation of beef texture, objective methods for food
evaluation, A Symposium, 7, National Academy of Sci., Washington, DC, USA.
[11] http://www.gitfood.com/Date-Spread-Dept/58/
[12] Molander, AL (1960): Discernment of primary test substances and probable
ability to Judge Food . Iowa State University press., Ames, Iowa, USA .
[13] Morton, J. (2006): Date. p. 5–11. In: Fruits of warm climates. Julia F. Morton,
Miami, FL.
[14] Myhara, RM; Al-Alawi, A; Karkalas, J and Taylor, MS (2000): Sensory and
textural changes in maturing Omani dates. J. Sci. of Food and Agric.,80: 2181-2185.
1008
[15] Shaari, A; Barreveld, WH and Bon, J (1966): Report on the development of a
new date product: Date spread. Government Fruit Processing Plant, Ministry of
Industry, Tripoli, Libya.
th
[16] Snedecor, GW and Cochran, WG (1980): Statistical Methods.7 Ed., Iowa State
Univ. Press, Ames Iowa, USA.
[17] Tafti, AG and Fooladi, MH (2006): A Study on the Physico-Chemical Properties
of Iranian Shamsaei Date at Different Stages of Maturity. World Journal of
Dairy & Food Sciences 1 (1): 28-32.
[18] Troug, E and Meyer, AH (1959): Improvement in the Deniges colorimetric
method for phosphorus and arsenic, Industrial and Engineering Chemistry,
Analytical Edition, 3: 136
[19] Wikipedia
(2006):
Date
palm.
Available
on:
http://en.wikipedia.org/wiki/Date_palm.
[20] Winton, AL and Winton, RB (1958): Okoloff Magnesium Oxide Distillation
Volumetric Method for the Determination of Total Volatile Nitrogen . The
Analysis of Foods, P.848.John, Wiley, New York. Chapmann and Hall .London
1009
Table 1: Chemical composition and minerals content of the date spread (DS)
Chemical composition (%)
TSS
36
Moisture
62.10
Protein
Fat
Ash
Carbohydrates
2.32 WW
0.37 WW
1.23 WW
33.98 WW
6.12 DW
0,98 DW
3.25 DW
89.65 DW
Minerals content (mg / 100 gm sample)
Calcium Phosphorus Magnesium Potassium Sodium Iron Zinc
Cupper
(Ca)
(P)
(Mg)
(K)
(Na)
(Fe)
(Zn)
(Cu)
52
86
100
990
21
6.6
1.3
0.21
WW = Wet weight
DW = Dry weight
Table 2: Sensory properties of beef kofta samples with addition of 15 % ground
whole date mixture, 15 % the fiber and epicarps mixture and 15 % date spread.
Sensory Properties
Samples
Appearance
Odor
Color
Texture
Taste
Overall
acceptability
Control
7.63 a
7.83 a
7.27 a
7.55 b
7.74 ab
7.60 ab
A
7.36 a
7.79 a
7.29 a
7.79 b
7.57 ab
7.56 ab
B
7.00 a
7.29 a
7.43 a
6.71 c
6.79 c
7.04 b
C
7.86 a
7.57 a
7.86 a
8.57 a
8.07 a
7.99 a
LSD
0.878
0.983
0.901
0.715
0.838
0.508
1010
Table 3: Sensory properties of beef kofta samples with addition of date spread
(0.0, 2.5, 5, 7.5 and 10.0 %).
Sensory Properties
Samples
Overall
Appearance
Odor
Color
Texture
Taste
Control
7.4 a
7.2 a
7.0 a
6.8 b
7.0 a
7.08 a
2.5 % DS
7.4 a
7.6 a
7.3 a
7.6 ab
7.6 a
7.52 a
5.0 % DS
7.4 a
7.6 a
7.6 a
8.0 a
7.9 a
7.70 a
7.5 % DS
7.8 a
8.1 a
7.6 a
8.1 a
7.2 a
7.76 a
10.0 % DS
7.8 a
8.2 a
7.6 a
8.2 a
7.2 a
7.80 a
LSD
0.895
0.764
1.530
0.860
1.368
0.847
acceptability
Table 4: Chemical composition of beef kofta samples with addition of date spread
(0.0, 2.5, 5, 7.5 and 10.0 %).
Chemical
composition
(%)
Samples
Control
2.5 %
DS
5.0 %
DS
7.5 %
DS
10.0 %
DS
LSD
Moisture
61.67 d
61.34 e
62.39 c
63.17 b
64.00 a
0.036
Protein (w.w)
16.25 e
16.88 d
17.09 c
17.29 b
17.50 a
0.036
Protein (D.w)
42.39 e
43.65 d
45.42 c
46.95 b
48.61a
0.037
Fat (w.w)
13.46 a
9.14 b
6.61 c
4.75 d
2.45 e
0.018
Fat (D.w)
35.12 a
23.65 b
17.56 c
12.89 d
6.82 e
0.038
Ash (w.w)
0.96 e
1.03 d
1.08 c
1.14 b
1.22 a
0.020
Ash (D.w)
2.50 e
2.67 d
2.87 c
3.09 b
3.38 a
0.035
Carbohydrates
(w.w)
7.66 e
11.54 d
12.84 c
13.65 b
14.83 a
0.055
Carbohydrates
(D.w)
19.99 e
30.04 d
34.14 c
37.06 b
41.20 a
0.055
1011
Table 5: Minerals content of beef kofta samples with addition of date spread
(0.0, 2.5, 5, 7.5 and 10.0 %).(mg / 100 gm sample)
Samples
Minerals
content
Control
2.5 % DS
5.0 % DS 7.5 % DS
10.0 % DS
Calcium (Ca)
4.00
16.88
29.75
42.65
55.50
Phosphorus (P)
389
396
402
413
425
Magnesium(Mg)
15.20
19.00
21.80
23.60
25.40
Potassium(K)
350
459
567
646
684
Sodium(Na)
55.20
53.57
51.99
50.39
48.78
Iron(Fe)
2.92
3.25
3.64
3.96
4.28
Zinc(Zn)
3.20
3.11
3.01
2.92
2.83
Cupper(Cu)
0.64
0,63
0.61
0.60
0.58
Table 6: Physical properties of beef kofta samples with addition of date spread
(0.0, 2.5, 5, 7.5 and 10.0 %).
Physical
properties
Samples
Control
2.5 % DS 5.0 % DS 7.5 % DS
10.0 % DS
Cooking
loss (%)
13.50
12.68
11.60
10.94
10.36
WHC cm²
3.5
2.6
2.2
1.8
1.4
Plasticity
cm²
2.4
2.5
2.9
3.0
3.1
Tenderness
(kg/ cm²)
3.9
3.2
2.7
2.3
2.1
1012
Table 7: Chemical properties of beef kofta samples with addition of date spread
(0.0, 2.5, 5, 7.5 and 10.0 %).
Samples
Chemical
properties
Control 2.5 % DS
5.0 % DS
7.5 % DS
10.0 % DS
TVN (mg/ 100g
sample)
13.50
12.68
11.60
10.94
10.36
TBA (mg
malonaldehyde /
kg sample)
3.5
2.6
2.2
1.8
1.4
1013
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ا
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ا-را - 7 ,ة @ ,
** أ-ث و ; 0 f+ا ,Zآ -ا#ث ا-را - 7 ,ة @ ,
ا!BC
,5 \+ه'ا ا b-Hإج 4 % %. c3ا \-وا ` 0 -+ \+ bT ,0ث 3ت 4آ< ا \-
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وا 2Fر و % ١٥ا @?ء ا 0< Fد ) VQ \` (date spreadا ^ث 3ت 4 .#Tا \F
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ا -ا @?ء ا 0< Fد 5]Gا 0آ?ات ا ٧h٥ , ٥ , ٢h٥ ) +zو % ١٠ا @?ء
ا 0< Fد 4ا \-ا 0FHى( و Qر 3 Y Vا Fر ) % ٠h٠ا @?ء ا 0< Fد ( اf
ا \Fا ,#-و V Tا ;{ 3ت H1وآ> 4 .ا c>3أ Lو] Zأن إ] 5ا @?ء ا F
<0د 4#Tا ; e>Bا #-وا Hfوا > 4آ< ا \-ا 0FHى.
ا"!ت ا ا :ا \-ا 0FHى ,ا ر ,ا @?ء ا 0< Fد وآ< ا \-ا 0FHى
1014
4
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