Vainilla
Vainilla
Vainilla
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in fulfillment of the requirement for the degree of
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Doctor of Philosophy
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BIOTECHNOLOGY
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March 2009
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Affectionately Dedicated to
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“PRUTHVI”
– The Mother EARTH, she who nurtures you and me!
Sreedhar R.V.
Research Fellow
Department of Plant Cell Biotechnology
CFTRI, Mysore-570 020
E-mail: rvsree@rediffmail.com
DECLARATION
I, Sreedhar R.V., declare that this thesis entitled “Novel approaches for Molecular
Analyses, Micropropagation and Curing of Vanilla (Vanilla planifolia)” is the result
of research work done by me under the supervision of Dr. Bhagyalakshmi Neelwarne
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at Plant Cell Biotechnology Department of Central Food Technological Research
Institute, Mysore- 570 020, India during the period of January 2004 to February 2009. I
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am submitting this thesis for the award of Doctor of Philosophy (Ph.D.) degree in
BIOTECHNOLOGY of the University of Mysore.
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I further declare that this thesis has not been submitted by me for the award of
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any other degree / diploma of this or any other University.
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CERTIFICATE
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This is to certify that the thesis entitled “Novel approaches for Molecular Analyses,
Micropropagation and Curing of Vanilla (Vanilla planifolia)” submitted by
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Mr. Sreedhar R.V. to the University of Mysore for the award of the degree of
Doctor of Philosophy in Biotechnology is the result of research work carried out by
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him under my guidance in Plant Cell Biotechnology Department, Central Food
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Technological Research Institute, Mysore during the period of January 2004 to
February 2009.
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background. Molecular analysis among different accessions also yielded identical PCR
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band profiles in both RAPD and ISSR analyses. These results clearly indicate that
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V. planifolia cultivated in India appears to share the same genetic background and
therefore, the genetic diversity is either extremely low or non-existing.
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Occurrence of genetic variants during micropropagation is occasionally
encountered when the cultures are maintained in vitro for long period. Through an
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established that micropropagation protocol used in this study can be carried out for a
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considerable length of time without any risk of genetic instability. Vanilla shoot
multiplication in semi-solid (SS), complete immersion system (CIS) and partial
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immersion system (PIS) were evaluated for biomass, shoot multiplication and
elongation aiming at developing an improved micropropagation protocol. GrowtekTM
bioreactor, functioning on the principle of PIS, was found to be most suitable for
micropropagation of vanilla. For automation of the micropropagation system, different
medium contact periods were provided to shoot cultures in CIS in which the 30 min
contact three times a day appeared most congenial. Similarly a bioreactor developed
with intermittent bathing of the cultures with nutrient medium was congenial for shoot
multiplication of vanilla. A combination of red soil: sand: vermicompost in equi-
proportion was found to be the best for greenhouse hardening. Field-evaluation showed
that the micropropagated plants were early to flower and high yielders than the
conventionally propagated ones.
The vanilla shoots cultured under completely immersed condition showed
hyperhydricity syndrome (HHS). A study focusing on unraveling the major structural,
biochemical and molecular changes occurring during HHS was carried out. The HHS
was associated with severe damages at cellular and sub-cellular levels, increase in free
polyamines and accumulation of water, and decrease in quantities of chlorophyll,
protein and drastic changes in reducing and non-reducing sugars. The onset and
progression towards hyperhydricity (HH) showed higher activities of antioxidant
enzymes, indicative of shoots’ defensive efforts against oxidative stress. Thirty one HH-
associated cDNAs identified by DDRT-PCR were cloned and sequenced whose
electronic homology searches using BLASTX analysis resulted in the identification of
23 cDNA clones showing homology with various stress, apoptosis, DNA repair and
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carbohydrate breakdown related proteins expressed differentially during HHS.
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BLASTN analysis yielded 18 fragments having homology with different stress linked
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cDNA clones. A partially characterized transcriptome of hyperhydric condition in V.
planifolia has been developed which paves the way for a better insight into gene
expression during this common physiological disorder.
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Vanilla beans derived from the micropropagated plants along with the beans
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available commercially were cured following different biotechnological approaches for
development of an efficient curing technique. In this study, effects of pre-treatments on
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the flavor formation during accelerated curing at 38 oC for 40 days were studied. Use of
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naphthalene acetic acid (5 mg/L) or ethrel (1%) with blanching pre-treatment resulted in
3-fold higher vanillin on 10th day. All major quality parameters analyzed were found
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I am ever grateful to Dr. G.A. Ravishankar, Head, Department of Plant Cell
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Biotechnology, CFTRI, for his constant encouragement and support during the
pursuit of my research work. C
I wish to extend my gratitude to Dr. M.S. Narayan a lot by way of scientific
discussions, who was more than willing to lend his helping hand during the
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needy hours. I sincerely thank Dr. Maya Prakash, Dr. Varadaraj M.C. and
Dr. Prafulla S.G. for their constant encouragement and valuable suggestions.
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My heartfelt thanks to the staff of PCBT Dr. Rajasekaran, T., Dr. Sarada R.,
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Dr. Giridhar P., Dr. Mahadevaswamy M., Dr. Arun Chandrasekar, Mrs. Karuna,
Mr. Shivanna, Mr. Srinivas Yella, Palaksha, Shashi and Channe Gowda who have
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Deepest thanks to all my dear seniors and colleagues Dr. Thimmaraju, Dr. KNC
Murthy, Dr. Vinod, Dr. BCN Prasad, Dr. Sathya, Dr. Vanitha, Dr. Sandesh, Venkat,
Roohie, Ashwin, Dayananda, Vidhya, Jyothi, Ranga, Rama, Gururaj, Sakthi, Pari,
Kathir, Lokesh, Shibin, Mahindra, Harsha, Manjunath, Imtiaz, Anila, Kumudha,
Ganapati, Gurudatt, Padma, Ramesh, Avinash, Simmi, Santosh, Sridevi, Kavitha,
Akshatha, Kalpashree and many others. I would like to thank all my friends in
other departments Raghunath Reddy, Harish Babu, Kisan, Revannappa, Rachappa,
Ravi, Dr. Uma, Desai, Namitha, Mamatha, Ravikumar, Ramesh, Badri,
Harshavardhan Reddy, Gangadhar, Devaraj, Rajashekar, SriRanga, for their moral
support, affection and encouragement.
I acknowledge the timely help and cooperation of staff of supporting
departments: HRD, Stores and Purchase, CIFS, FOSTIS, Computer Center and
Administration.
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continuous encouragement, it would not have been possible to accomplish this
task. Lastly, but most importantly, I thank my dear fiancée Roopa for her love,
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compassion, understanding and support.
To all others, who had helped me knowing or unknowing wherever they are,
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goes my thanks and with them the assurance that their assistance not be
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forgotten.
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(Sreedhar R.V.)
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TABLE OF CONTENTS
Chapter Page
Content
No.
List of Figures i
List of Tables iii
List of Abbreviations iv
General Introduction and Review of Literature
G1 General Introduction 01
G1.1 The plant 02
G1.2 The bean 03
G1.3 Chemistry of vanilla bean 03
G1.4 Qualitative variations among vanilla species 05
G2 Vanillin 06
G2.1 Natural occurrence of Vanillin 06
G2.2 Site of vanillin synthesis in vanilla beans 07
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G2.3 Biosynthesis of vanilla flavor compounds 07
G2.4 Other sources of Vanillin 09
G3 The vanilla ‘crisis’ 10
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G4 Origin and Dissemination of Vanilla 11
G4.1 V. planifolia in its area of origin and introduction 11
G4.1.1 The history of V. planifolia in Mexico 11
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G4.1.2 V. planifolia in introduced areas 13
G4.2 Diversity analysis 14
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G4.2.1 Genetic Diversity of vanilla 17
G4.2.2 Diversity in Indian vanilla 17
G5 Micropropagation 18
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G6 Hyperhydricity syndrome 19
G6.1 Genetic analysis by Differential display 20
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G7 Curing of vanilla 22
1.1 Introduction 27
1.1.1 Isozymic analysis 28
1.1.2 RAPD 29
1.1.3 ISSR 29
1.1.4 Genetic diversity analyses in vanilla and other orchids 29
1.2 Materials and methods 30
1.2.1 Plant sampling 30
1.2.2 Isozymic analysis of peroxidase (POD) 31
1.2.3 DNA extraction and quantification 32
1.2.4 Primer selection 32
1.2.5 DNA amplification 33
1.2.6 Analysis of PCR product by agarose gel electrophoresis 34
1.2.7 Data analysis 34
1.3 Results 36
1.4 Discussion 38
1.5 Conclusion 40
Chapter II Genetic Fidelity and Advanced Micropropagation
Techniques
Summary 41
2.1 Introduction 43
2.2 Materials and methods 47
2.2.1 Genetic fidelity 47
2.2.1.1 Plant material and establishment of shoot cultures 47
2.2.1.2 Extraction of DNA, PCR analysis, Primer selection, DNA
amplification, Analysis of PCR product by agarose gel
electrophoresis and Data analysis 48
2.2.2 Micropropagation 48
2.2.2.1 Plant material 48
2.2.2.2 Culturing conditions 48
2.2.2.3 Kinetic parameters 50
2.2.2.4 Growth parameters 51
2.2.2.5 Rooting, hardening and green house cultivation 51
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2.2.3 Planting material for field performance study 51
2.2.3.1 Stem cuttings 51
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2.2.3.2 Micropropagated plantlets 51
2.2.4 Statistical analysis 52
2.3 Results 52
2.3.1 Genetic fidelity 52
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2.3.2 Micropropagation 57
2.3.3 Greenhouse hardening and field performance studies 62
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2.4 Discussion 65
2.4.1 Genetic fidelity 65
2.4.2 Micropropagation 68
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3.1 Introduction 77
3.2 Materials and methods 79
3.2.1 Morphological and Biochemical changes 79
3.2.1.1 Plant material and in vitro culturing conditions 79
3.2.1.2 Morphological changes 80
3.2.1.3 Measurement of pH, conductance and osmolarity 81
3.2.1.4 Chlorophyll content 82
3.2.1.5 Carbohydrate content 82
3.2.1.6 Analysis of polyamines 82
3.2.1.7 Protein content and antioxidant enzyme activity 83
3.2.1.8 Statistical analysis 84
3.2.1.9 Isozymic analysis of POD 84
3.2.2 Molecular changes 84
3.2.2.1 RNA isolation 84
3.2.2.2 mRNA Differential display 84
3.2.2.2.1 PCR amplification 85
3.2.2.2.2 Urea formamide denaturing polyacrylamide gel electrophoresis 85
3.2.2.3 Elution of differentially expressed amplicons 86
3.2.2.4 Re-amplification 86
3.2.2.5.1 T/A cloning of isolated differential amplicons 86
3.2.2.5.2 Ligation of A-tailed PCR product to T-tailed vector 87
3.2.2.5.3 Transformation of E. coli with the ligation reaction mix 87
3.2.2.5.4 Preparation of competent cells using CaCl2 90
3.2.2.5.5 Transformation of competent cells 90
3.2.2.5.6 Selection of transformants/recombinants and PCR confirmation
of the transformation 90
3.2.2.5.7 Isolation of plasmid DNA from the transformed colonies 91
3.2.2.6 Sequencing of the clones 91
3.2.2.7 Validation by RNA dot blot 91
3.2.2.8 Quantitative (relative) reverse transcriptase polymerase chain
reaction (qRT-PCR) 93
3.3 Results 95
3.3.1 Morphological and Biochemical changes 95
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3.3.1.1 Morphological and ultra structural changes 95
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3.3.1.2 Changes in medium kinetics in CIS 95
3.3.1.3 Changes in chlorophyll and carbohydrate contents 99
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3.3.1.4 Polyamines content 99
3.3.1.5 Protein content and activities of antioxidant enzymes 102
3.3.2 Molecular Changes 104
3.3.2.1 Cloning 106
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3.3.2.2 PCR analysis to confirm cloning 106
3.3.2.3 Sequence analyses of cDNA fragments 106
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3.3.2.4 BLASTN analysis 106
3.3.2.5 BLASTX analysis 108
3.3.2.6 Verification of the DD cDNAs expression by RNA blot analysis 114
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4.3 Results
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4.3.1 Pre-treatments experiment 141
4.3.1.1 Changes in physical parameters 141
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4.3.1.2 Formation of flavour compounds 143
4.3.1.3 Protein content 148
4.3.1.4 Enzyme activities 149
4.3.1.4.1 -Glucosidase 149
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4.3.1.4.2 Cellulase 150
4.3.1.4.3 Peroxidase 150
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4.3.1.5 E-nose analysis 151
4.3.2 Elicitation experiment 152
4.3.2.1 Total protein 152
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of shoot cultures of Vanilla planifolia 47
2.2 Growtek™ bioreactor used in the present study 49
2.3 Diagrammatic representation of modified bioreactor developed for
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automation of submerged cultivation of vanilla (as TIS) 49
2.4 A representative Randomly Amplified Polymorphic DNA (RAPD)
amplification pattern 55
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2.5 A representative Inter Simple Sequence Repeat (ISSR) amplification
pattern 56
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2.6 Various micropropagation systems used in the present study 58
2.7 pH, osmolarity and conductivity of the medium at various stages of
culturing shoot cultures 59
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bioreactor (PIS) 60
2.9 Scale-up of shoot cultivation in a modified bioreactor 62
2.10 Greenhouse hardening and field cultivation of vanilla 65
3.1 Shoot cultures of Vanilla planifolia cultured on solid and complete
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immersion system 81
3.2 Shoots cultivated in normal and liquid medium showing various
degrees of hyperhydricity 81
3.3 The experimental model adopted for the differential display study 89
3.4 Cross-sections of stem in normal and hyperhydric shoot cultures of
vanilla 96
3.5 Cross-sections of leaf in normal and hyperhydric shoot cultures of
vanilla 96
3.6 Scanning electron micrographs of normal and hyperhydric shoot
cultures (stage H2) of vanilla 97
3.7 Kinetic parameters of the medium used for culture of vanilla shoots 98
3.8 Protein content and activity of antioxidant enzymes in normal
(shoots cultured in solid medium) and hyperhydric shoot cultures 103
3.9 Zymogram on denaturing gel of PODs after activity staining 104
3.10 Differentially expressed bands visualized on polyacrylamide gel 105
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3.11 Formation of blots of different intensities confirmation of
differential accumulation of gene products of VP2 and VP9 114
3.12 Expression and relative transcript abundance of different
hyperhydricity related genes 116
4.1 Experimental model adopted in the pre-treatment study showing
various treatments 140
4.2 Moisture content at different periods of curing of vanilla beans after
various pre-treatments 142
4.3 Texture analysis of vanilla beans during different stages of curing
after various pre-treatments 143
4.4 Flexibility of vanilla beans cured for 10 days after pre-treatment
with NAA during blanching-pretreated beans 143
4.5 HPLC profiles of standard compounds and pre-treated vanilla beans 145
4.6 Total protein content of vanilla beans (at 25% moisture level) at
different incubation periods as estimated by Macro-Kjeldahl method 148
4.7 Electronic nose analysis pattern of vanilla pods from various sources 152
4.8 HPLC patterns showing profiles of vanilla flavour compounds
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formed after 10 days of curing at 38 oC in elicitor-treated vanilla
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beans as compared with standards and control beans 157
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4.9 Sensory profile of vanilla beans cured for 10 days with or without
elicitors and different pre-treatments
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LIST OF TABLES
Table Page
Title
No. No.
G1 World Vanilla Market 01
G2 Concentrations of major volatile compounds in cured vanilla bean 04
G3 Vanillin content in different plants 09
G4 Widely used markers and their applications 16
1.1 List of accessions of V. planifolia and their geographical origin
used in this study 31
1.2 List of selected RAPD and ISSR primers 33
1.3 List of selected primers used in RAPD analysis and number of
scorable bands 35
1.4 List of selected primers used in ISSR analysis and number of
scorable bands 36
2.1 List of selected primers used in RAPD analysis 53
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2.2 List of selected primers used in ISSR analysis 54
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2.3 Effect of different contact periods on growth and development of
vanilla shoot cultures 62
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2.4 Effect of different combinations of soil mixtures used for hardening
the plants under green house conditions 63
2.5 Field performance of Conventionally propagated and
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Micropropagated vanilla 63
3.1 List of anchor primers and arbitrary primers used for Differential
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Display 85
3.2 Gene specific primers and annealing temperatures used for RT-
PCR 94
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3.5 List of the isolated bands, their size and expression based on their
expression in PAGE gel electrophoresis 105
3.6 Homology search results for differentially expressed cDNAs during
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LIST OF ABBREVIATIONS
AFLP : Amplified Fragment Length Polymorphism
APS : Ammonium persulphate
BAP : Benzylaminopurine
BLAST : Basic Local Alignment Search Tool
bp : base pairs
BSA : Bovine Serum Albumin
BSR : Beet Seedling Root
ºC : Degree Centigrade
CAT : Catalase
cDNA : Complementary Deoxyribonucleic Acid
CES : Cellulase
CIS : Complete Immersion System
cm : Centimeter
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CMC : Carboxymethyl cellulose
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DAC : Days After Curing
DCP : Dry Cell Powder
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DD : Differential Display
DDRT-PCR : Differrential Display Reverse Transcription- PCR
DIG : Digoxigenin
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DMF : Dimethyl formamide
DNA : Deoxyribonucleic Acid
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dNTP : Deoxynucleotide triphosphate
DTT : Dithiothreitol
EDTA : Ethylene diamine tetra acetic acid
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IPTG : Isopropyl-Dthiogalactopyranoside
kb : Kilobase
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MAPK : Mitogen activated protein kinase
mg : milli gram
min : minute
mL : milli Liter
mM : milli Molar
mm : milli meter
MOPS : 4-Morpholinepropanesulfonic acid
mRNA : messenger RNA
MS : Murashige and Skoog
mS : milli Siemen
N : Normal
NAA : Naphthalene acetic acid
nm : nanometer
ng : Nano gram
nt : nucleotide
NCBI : National Centre for Biotechnology Information
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NBT : Nitroblue tetrazolium
OD : Optical density
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OPA : Operon A
PAs : Polyamines
PAGE : Polyacrylamide gel electrophoresis
PAL : Phenylalanine ammonium lyase
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PCA : Principal Component Analysis
PCR : Polymerase Chain Reaction
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PEG : Polyethylene glycol
pG1 : Polygalacturonase
POD : Peroxidase
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PP : Phenyl propanoid
QDA : Quantitative Descriptive Analysis
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RNase : Ribonuclease
rRNA : ribosomal RNA
rpm : revolution per minute
RT : Room Temperature
RT-PCR : Reverse Transcriptase Polymerase Chain Reaction
SA : Salicylic acid
SAGE : Serial Analysis of Gene Expression
SD : Standard Deviation
SDS : Sodium dodecyl sulphate
Sec : Second
SEM : Scanning Electron Microscopy
SOD : Superoxide dismutase
SS : Semi-solid
SSR : Simple Sequence Repeat
TAE : Tris-acetate-EDTA
Taq : Thermus aquaticus
TBE : Tris-Borate-EDTA
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TE : Tris-EDTA buffer
TIS : Temporary Immersion System
Tris : Tris (hydroxymethyl) amino methane
X-GAL : 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
U : Unit enzyme
UBC : University of British Columbia
UTM : Universal Texture Measurement
UV : Ultra Violet
V : Volt
v/v : Volume per volume
WAI : Week after inoculation
w/v : Weight per volume
α : Alpha
β : Beta
μg : Micro gram
μM : Micro molar
μL : Micro liter
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% : Percent
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General Introduction and Review of Literature
Vanilla is the most popular and widely used flavor by both monitory and tonnage
basis. Being the second most expensive spice traded in world market only after
saffron (Minoo et al. 2008), natural vanilla flavor is obtained as an extract from
cured vanilla beans and is universally used as aromatic flavoring in food,
pharmaceutical, beverage and cosmetic industries. The top category beans
classified as “gourmet” grade are above 15 cm or more in length and are selected
from A grade beans having over 1.75% vanillin (the chief flavouring compound)
and 25-30% moisture content. Approximately 20-25% of the total production
from well maintained farms often account for this grade. Natural vanilla
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consumption in India at present is only about 5% of a total consumption of 400-
500 tonnes per year (Indian Food Industry, 2008). Export of Indian vanilla has
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steeply gone up from 125 tonnes in 2006-2007 season to 200 tonnes in 2007-2008
season showing 133% increase from the target of 150 tonnes (Spice India, 2008).
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The major vanilla producing countries and their market sizes are presented
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in Table G1. Madagascar being the largest producer of vanilla accounts for 67%
of market share, followed by Papua New Guinea with 9% share. Both Uganda
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and Indonesia produce 7% each of world vanilla whereas India is 4th major
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General Introduction and Review of Literature
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by a cap or hood like structure called ‘rostellum’, which separates stamen from
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stigma and prevents natural pollination. The flower is so constructed that self-
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pollination is rather difficult unless hand-pollinated. Stingless bees of the genus
Melipona (Apoidea) and humming birds are known to pollinate some flowers in
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Mexico and Central America and elsewhere hand-pollination is practiced.
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A B C
Figure G1. A: Vanilla plant; B: Inflorescence; C: Beans
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General Introduction and Review of Literature
The syncarpous fruit of Vanilla planifolia develops from an inferior ovary and
splits open along the three lines at maturity becoming a capsule. There are two
principal regions in the fruit: The fruit wall or the ‘green’ region which includes
epidermis, ground and vascular tissues. The ‘white’ region composed of three
parietal placentae and the three bands of glandular hairs between them. The
glandular hairs play a role in the biosynthesis of the flavouring compounds. The
most important for the vanilla bean are the unusual glandular hairs that begin to
develop quickly in the regions between the placentae. Vanillin and related
intermediates of vanillin biosynthetic pathway accumulate in the inner white
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tissue of a developing vanilla pod, around placental hairs. This information may
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be important for understanding the rationale for the control of the curing process.
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The distinct aroma imparted by cured vanilla beans is due to a group of phenolic
aromatic compounds accounting for over 170 flavoring compounds, of which the
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major ones are vanillin, vanillic acid, para-hydroxybenzoic acid and para-
hydroxybenzaldehyde. The constituents responsible for the aroma and flavor are
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aromatic acids, phenols, phenol ethers, aliphatic alcohols, carbonyls, acids, esters
and lactones, aromatic hydrocarbons, terpenoids, aliphatic hydrocarbons and
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heterocyclics. The nonvolatile constituents are the tannins, polyphenols, resins and
free amino acids. All these constituents together produce the delicate, rich and
mellow aroma with spicy, woody and balsamic notes. The highest quantity of
about 2-2.5% of the dry matter of cured pod is due to vanillin, the cost of latter is
about a hundred-fold higher than the synthetic one. Concentrations of major
volatile compounds in cured vanilla bean are presented in Table G2. Earlier
studies have noted that vanillin or its glycoside do not accumulate in the interior
of cells may be to avert the reactivity and possible toxicity of the carbonyl group.
Therefore, vanillin biosynthesis in Vanilla species occurs in specialized cells
where vanillin is glycosylated and expelled from the cellular interiors. This
suggests that biological cells not equipped to deal with the cellular turnover of
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General Introduction and Review of Literature
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Aliphatic acids
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1 Linoleic acid 225.6±17.25
2 Hexadecanoic acid 126.6±5.94
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3 Acetic acid 124.3±11.1
4 Oleic acid 16.3±1.56
5 Nonanoic acid 15.7±1.73
Aromatic acids
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1 Cinnamic acid (isomer 2) 9.5±1.13
2 Benzene propanoic acid 3.9±0.28
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3 Cinnamic acid (isomer 1) 3.4±0.57
4 Benzoic acid 2.6±0.35
Alcohols
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1 2-Heptenal 2.1±0.28
2 2-Decenal 1.8±0.16
3 2,4-Decadienal 1.4±0.11
Esters
1 Ethyl linolenate 13.5±0.35
2 Anisyl formate 2.3±0.35
3 Methyl cinnamate 1.1±0.07
Hydrocarbons
1 Pentacosine 19.9±1.48
2 Tricosane 15.9±2.19
This brings to the fore the particular suitability of green vanilla beans for vanillin
production by direct enzymatic treatments. Proper agronomic practices and
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General Introduction and Review of Literature
selection of lines producing high levels of vanillin could result in high quality
vanilla pods since the present cultivation practices show that glucovanillin can
accumulate to levels of up to 20% of the dry weight (Havkin-Frenkel and
Belanger 2008). This being the case, with proper control of curing process, it
might be possible to obtain cured beans with 10% vanillin (Havkin-Frenkel and
Belanger 2008).
Based on qualitative variations in the aroma and flavor of beans of vanilla various
species and their geographical origins are as follows:
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Bourbon vanilla (V. planifolia Andrews) - It is the collective term used for the
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beans of Madagascar, Reunion, the Comoro islands and the Seychelles origin.
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Bourbon vanilla is characterized by its sweet, creamy, rich, full-bodied, tobacco-
like, woody and animal, and deep balsamic, sweet spicy flavor back notes.
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Mexican vanilla (V. planifolia Andrews) - The flavor notes in this are described
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as sharp, slightly pungent and sweet spicy notes lacking body compared to
Bourbon vanilla.
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Java vanilla (V. planifolia Andrews) - This is from Indonesian islands and is less
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sweet and creamy than Bourbon type. It lacks bouquet note and has a strong
woody and slightly smoky character with a freshly sharpened pencil note.
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Indian vanilla (V. planifolia Andrews) - The flavor of carefully cured Indian
vanilla is full-bodied but less sweet and creamy than Bourbon vanilla. It lacks
balsamic note but has slightly spicy and pungent sour notes.
Uganda vanilla (V. planifolia Andrews) - These beans almost have aroma and
flavor similar to Bourbon vanilla but less creamy and sweet.
Thaitian vanilla (V. tahitiensis) - This type has a distinctly perfumey and
flowery, fragrant, helitropine-like, with a rather shallow vanilla character.
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General Introduction and Review of Literature
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vanillic acid (nearly 0.1%), p-hydroxybenzoic acid (nearly 0.02%), p-hydroxy
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benzyl methyl ether (nearly 0.02%) and acetic acid (nearly 0.02%).
G2. Vanillin
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Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the most widely appreciated
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flavor compound with an odor threshold of 11.8 × 10-14 M for humans
(Buccellato 2005). It has the unique characteristic that even at a very high dose,
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It is found in traces in many plants. Essential oils like clove, cinnamon and mace
contain vanillin. Plants from the genus Vanilla have large amounts of vanillin.
Vanilla planifolia (syn. V. fragrans), V. tahitensis and V. pompona of the family
Orchidaceae are the commercially cultivated species for the production of natural
vanilla flavor where V. planifolia is the most preferred one.
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General Introduction and Review of Literature
Some early studies indicated vanillin secretion ‘in tissue around seeds’. A
comprehensive localization study in developing fruit using catechin-HCl which
binds to various phenolic compounds including vanillin as a staining agent
revealed that endocarp parenchymatic cells contained vanillin and intermediates in
the biosynthetic pathway. A descending staining gradient from endocarp in the
fruit cavity outwards was observed indicating the site of synthesis. The study also
showed that vanillin accumulation begins after 3 to 4 months of fruit
development. As vanillin is sparingly soluble in water, particularly in acidic plant
vacuoles, glycosylation of vanillin to glucovanillin is a likely mechanism for
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increasing the hydrophilicity of the compound, thus aiding in the sequestering and
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storage of the compound in aqueous extracellular regions. Special cells in the pod
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interior are thought to be dedicated to vanillin biosynthesis (Havkin-Frenkel and
Belanger 2008).
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G2.3 Biosynthesis of vanilla flavor compounds:
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Though extensive studies have been made on the biosynthesis of vanillin and
allied flavor compounds in plant and cell cultures, several questions remain
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ring. It is not yet clear whether vanillin is derived from the lignin precursors
having an alcohol function in the C3 part of phenylpropanoid, or from the
cinnamic acid type (acid group). Kanisaw et al. (1994) proposed that major
pathway would go via 4-coumaric acid glucoside, which is the precursor for para-
hydroxybenzaldehyde glucoside, the central intermediate for the biosynthesis of
the glucosides A and B as well as vanillin. Various experiments with vanilla cell
cultures give different results which might be due to the fact that different
biosynthetic pathways operate in the beans and cell cultures. Figure G2 shows
the formation of vanillin through different ways in a complex network of
compounds.
7
General Introduction and Review of Literature
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Figure G2. Shikimic acid pathway showing the formation of vanilla flavour
compounds
8
General Introduction and Review of Literature
Though several plant species produce vanillin in traces (Table G3), committed
biosynthetic route has been identified only in Vanilla species and the biosynthetic
pathways in others are still not clear. Various biotechnological approaches have
been explored to produce natural vanillin at a lower price through various
microorganisms, cell-free systems using enzymatic degradation and plant tissue
culture.
I
Unicorn plant (Proboscidae cuisianica) Roots and pods 0.01
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Potato (Solanum tuberosum) Tuber skin 0.01
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Clove (Syzygium aromaticum) Dry flower buds Traces
Narcissus (Triandrus narcissi, Tazetta arsissi) Roots and basal plate 0.01-0.60
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Hyacinth (Hyacinhus orientalis) Roots and basal plate 0.20-0.50
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Vanilla planifolia Pod (cured) 1.00-8.00
9
General Introduction and Review of Literature
I
food and beverages reacted predictably by searching for alternatives for natural
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vanilla. Both these activities took time and so the for the next three years, vanilla
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prices spiraled upward. By the end of 2003, worldwide consumption of
approximately 1100 metric tons was roughly half of what it had been in 1999 and
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the prices exceeded $500 per kg cured beans, nearly 15 times higher than they
were four years ago (Rick 2006).
@
growth and popularity and will probably take a decade to reach the level of
consumption which existed before the crisis. Like all who experience and survive
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natural vanilla has already started primarily in the retail (home) and food service
(restaurant) segments but yet to get initiated in the large food and beverage
manufacturers as they are adamant to reformulate their product to the use of
natural vanilla again. Ultimately, consumer will decide what to use and the
manufacturers need to rebound.
10
General Introduction and Review of Literature
The Vanilla genus contains more than 800 genera distributed in more than 25,000
species (Govaerts et al. 2006). The basic chromosome number of genus vanilla is
X=16 and V. planifolia is a diploid with 2n=32. The genus Vanilla belongs to the
Orchidaceae family (largest plant family) and V. planifolia is probably endemic
from eastern Mexico tropical forests and its natural habitat roughly follows a
straight line between the Oaxaca state towards Guatemala and Belize (Figure G3)
(Soto Arenas 1999a). Another Vanilla species, V. tahitensis J.W. Moore is
cultivated in several Pacific countries. Some other aromatic species grown locally
or harvested in the wild having no economical importance are V. pompona
I
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Schiede in the West Indies, V. chamissonis Klotzsch in Brazil, V. odorata C. Presl
in America, V. claviculata (W. Wright) Sw., V. griffithii Rchb. f. and V.
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abundiflora J.J.Sm. in the West Indies and in Asia (Soto Arenas 2003). The genus
Vanilla is widely distributed throughout tropical and subtropical regions around
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the world (Indonesia, South and Central America, Mexico and Africa), and this
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distribution supports the theory that it is very old genus. Three species have
economic value and are the Vanilla planifolia Andrews (earlier known as
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resistant to diseases. V. tahitensis yields a distinctly different flavor and the beans
are more expensive compared to the other two.
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11
General Introduction and Review of Literature
Itzco´atl (Aztec Emperor) in 1427, and citing vanilla as a remedy for fatigue in
Badianus manuscript in 1552 (Lubinsky 2004). Establishment of first vanilla
plantation was by the Totonac Indians (in the Veracruz region), particularly in the
Papantla and Misantla areas (Figure G3), from 1767 which marks the start of
vanilla cultivation. According to Ecott (2004), Soto Arenas considers that Totonac
Indians did not use manual pollination to produce vanilla pods. There is no
evidence of manual pollination before the 19th century. From 1841, the technique
of manual pollination discovered in Europe was transferred to Mexico and
Totonac Indians became the world most important producers, until the supremacy
of Madagascar in 1924 (Lucas 1990).
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12
General Introduction and Review of Literature
After C. Colombus discovered the New World in 1492, the first record of
dissemination of vanilla from Mexico is by Father Labat who imported three
V. planifolia vines into Martinique in 1697 and from there to Guadeloupe in 1701.
It was then introduced in Reunion Island in 1793. Early in the 19th century, one
major event is V. planifolia introduction by Marquis of Blandford into the
collection of C. Greville at Paddington where it flowered in 1807. Greville then
supposedly sent some cuttings to the botanical gardens of Antwerp (Belgium) and
from there to Paris (Correll 1953). From the Botanical Garden of Antwerp, it was
introduced into Buitenzorg in Java in 1819 by Marchal (Purseglove et al. 1981)
I
and in Reunion Island from the Jardin du Roi in Paris in 1822 by the ordinance
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officer of Bourbon, Marchant.
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Vanilla was introduced to Europe from Mexico, in about 1,500 numbers and its
reputation of being an aphrodisiac followed it to countries where it was
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introduced. An introduction event is documented in India in 1835 but the plant
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died after flowering (Correll 1953). Lack of natural pollinators in the areas of
introduction prevented sexual reproduction and pod production until the first half
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of the 19th century. The British introduced V. planifolia into India about 200 years
ago where five other species are native viz, V. pilifera Holtt., V. andamanica
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13
General Introduction and Review of Literature
I
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diversification and production, while developing a more sustainable agriculture.
Molecular markers have already played a major role in the genetic
FT
characterization and improvement of many crop species. They have also
contributed to and greatly expanded our abilities to assess biodiversity, reconstruct
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accurate phylogenetic relationships, and understand the structure, evolution and
interaction of plant and microbial populations. Molecular markers are now
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14
General Introduction and Review of Literature
polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) have been
widely used to survey genetic structure of populations. Among various molecular
markers, the RAPD technique is simple, rapid, and requires only a few nanograms
of DNA, has no requirement of prior information of the DNA sequence and has
feasibility of automation with higher frequency of polymorphism, which makes it
suitable for routine application for the analysis of genetic diversity (Babu et al.
2007). It is also proven to be quite efficient in detecting genetic variations, even in
closely related organisms like two near isogenic lines of tomato (Martin et al.
1991). For the use of ISSRs, primers are not proprietary as in Microsatellites or
Simple Sequence Repeats (SSRs) and can be synthesized by anyone and also
I
allow the production of a high number of reproducible polymorphic bands. ISSR
R
is found to be very simple, quick, cost-effective, highly discriminative and most
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reliable method which combines most of the advantages of SSRs and Amplified
Fragment Length Polymorphism (AFLP) to the universality of RAPD (Reddy et
al. 2002). ISSRs though considered mostly as dominant markers; they are shown
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to segregate co-dominantly in some cases (Sankar and Moore 2001) thus enabling
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distinction between homozygotes and heterozygotes. They are found to be more
useful and reproducible than isozymes, RAPD and RFLP (Fang et al. 1997) and
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are also known to give more polymorphism than any other assay procedure (Virk
et al. 2000). Detection of additional polymorphism could be done by the use of
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RAPD in combination with ISSRs (Joshi et al. 2000). Martins et al. (2004) suggest
the use of a combination of two types of markers that amplify different regions of
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the genome and hence a better analysis of genetic variation can be made.
15
General Introduction and Review of Literature
Markers Applications
PCR Based markers
Fingerprinting, mapping, F1, Varietal
AFLP: Amplified Fragment
identification, Gene tagging, Marker-assisted
Length Polymorphism (D)
selection, Map-based gene cloning
CAPS: Cleaved Amplified Framework mapping, Can be converted to allele-
Polymorphic Sequences (CD) specific probes, F1 identification, Gene tagging,
Bulk segregant analysis, Diversity studies,
SCAR: Sequence Marker-assisted selection, Map-based cloning
Characterized Amplified
Region (CD)
Fingerprinting, Varietal identification, Genetic
I
EST: Expressed Sequence Tag maps, F1 identification, Gene tagging and
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(D/CD) identification, Bulk segregant analysis, Diversity
studies, Marker-assisted selection, Novel allele
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STS: Sequence Tagged Site detection, High-resolution mapping, Map-based
(D/CD) cloning
IPCR: Inverse Polymerase
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Chain Reaction (CD) Fingerprinting, Varietal identification
IRAP: Inter-Retrotransposon F1 identification, Gene tagging
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Microsatellite Amplified
Polymorphism (CD)
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16
General Introduction and Review of Literature
The first molecular data on specimens from crops in northern Veracruz, Oaxaca
and other Mexican regions obtained using iso-enzymes show low levels of total
genetic variation. A molecular analysis using RAPD markers in Meso-America by
I
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Schlu¨ter (2002) differentiated V. planifolia from Costa Rica and Mexico. Among
Mexican V. planifolia, two main groups were revealed: individuals from Oaxaca,
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Chiapas and Quintana Roo on one hand, and individuals from Veracruz, Federal
District of Mexico, San Luis Potosi, Tabasco and Oaxaca on the other hand. The
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individuals from Oaxaca and Tabasco present in the second group most probably
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correspond to specimens that were collected from the Veracruz region at the time
of the establishment of new crops. Attempts to study some hypervariable regions,
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and the history of origin of cultivated vanilla suggests that the entire stock outside
Mexico may be from a single genetic source. A study on genetic diversity of
V. planifolia by Besse et al. (2004) using RAPD markers in vanilla cultivated
areas of Reunion Island and Polynesia reveled a very low level of diversity.
Vanilla cultivation was initiated in India, through the East India Company, nearly
250 years back in the spice garden at Kurtallam in Tamil Nadu (George 2005). Its
organized cultivation started in 2001-2002 in 1600 ha yielding 60 tonnes of cured
vanilla beans and steadily gained importance doubling its cultivation to 3427 ha in
2003-2004 resulting in the production of 131 tonnes
17
General Introduction and Review of Literature
I
vanilla is extremely essential to protect it from erosion due to epidemic diseases
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and pests apart from planning strategies for conservation of the genetic resources
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(secondary gene pools and cultivated resources).
G5. Micropropagation
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Vanilla is generally propagated by stem cutting which is a slow, time consuming
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and labour intensive process. Removal of cuttings may also cause injury to the
mother plant resulting in a set-back of growth and reduction in yield.
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as the market demand for the propagules is hardly met through these cuttings. In
vitro propagation of vanilla has been established by culturing axillary buds
(George and Ravishankar 1997; Geetha and Shetty 2000; Giridhar et al. 2001,
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Kalimuthu et al. 2006), aerial root tips (Philip and Nainar 1988), through callus
(Davidonis and Knorr 1991) and protocorms. Micropropagation using shoot tips
or nodal segments has been found as an appropriate technique for clonal
propagation of vanilla (George and Ravishankar 1997).
18
General Introduction and Review of Literature
propagules (Hvoslef-Eide and Melby 2000; Dey 2001). Low-cost culture vessels
and minimization of contamination are the other options available. Techniques at
present for the micropropagation need a large number of containers, gelled media
and aseptic conditions and a complicated and costly production technology. It
involves periodic transfer of plant material to fresh media, after subcultures, due
to exhaustion of the nutrients in the medium, continuous proliferation of the plant
material and limited size of the container. Though liquid culture systems are
considered as advantageous in term of uniform culture conditions, ease for change
of the medium and reduced manual labour requirement, it is usually associated
with hyperhydricity disorder. An intermittent exposure of the culture to liquid
I
medium rather than continuous was found to solve this problem. For this the
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bioreactors developed earlier do not suit as they are mainly adapted to bacterial
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culture and do not take in to consideration specific requirements of plant cultures
like the shear force, mechanical damages or foam formation in bubble aerated
bioreactors (Berthouly and Etienne 2005). High production cost limits the
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commercial use of micropropagation to markets with a high unit value, such as
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vanilla. It has been concluded for various species that extensive expansion of
micropropagation would only take place if new technologies became available to
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shoot cultures in a micropropagation industry. Initial trials for this are normally
conducted using shake-flask cultures, often known as complete immersion system
(CIS) as the shoots are continuously bathed in liquid medium. During cultivation
in vitro, the plantlets are exposed to a wide range of stress conditions caused by
high relative humidity, gas accumulation in the headspace, altered
nutrient/hormonal combinations and non-congenial osmoticity of the culture
medium. Although most plant cultures adapt to changes in environmental
conditions, some of them become abnormal with turgid, translucent, less green,
watery, hypo-lignified, wrinkled and brittle appearance. This phenomenon,
known as hyperhydricity syndrome (HHS), can lead to irreversible loss of
multiplication as well as regenerative potential. HHS has also been a generic
19
General Introduction and Review of Literature
I
The DDRT-PCR technique only requires small amounts of RNA and it allows the
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comparison of several RNA populations simultaneously (Bosch and Lohmann
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1997; Jorgensen et al. 1997). Nonetheless differential display has advantages over
other methods which include: the use of small amounts of total RNA, the
identification of mRNA species independent of prevalence (Wan et al. 1996), and
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the detection of rare and abundant transcripts of both known and novel genes
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(Matz and Lukyanov 1998). Though effective, the technique does have some
drawbacks. The major limitation is the high incidence of false positive. However
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the single greatest contributor to false positives is arbitrarily primed PCR using
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short oligos (Matz and Lukyanov 1998), which can be solved with the use of
longer arbitrary primers (Zhao et al. 1995). Classical differential display employs
the use of a degenerate single base or two-base- anchored oligo-dT primer [d(T)11
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20
General Introduction and Review of Literature
I
both up- regulated and down-regulated genes and the comparison of more than
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two RNA populations; in fact limitations to the number of comparisons that can
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be done are only imposed by the size of the gel.
Some of the notable advantages of differential display are:
4. Speed
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21
General Introduction and Review of Literature
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major ones after vanillin are vanillic acid, p-hydroxybenzoic acid and
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p-hydroxybenzaldehyde (Figure G4). In green vanilla beans, these phenolic
FT
aromatic compounds are present as their respective glucosides major being
glucovanillin (Figure G4) synthesized from phenylalanine of shikimic acid
pathway and curing process is meant to release the aglycones as the free aroma
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compounds. Curing also induces the formation of many other compounds that
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complement to the delicate aroma of natural vanilla flavour. In fact, it is the
presence of these minor compounds in large numbers that fetch high price for
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Botanical study of vanilla beans reveals that the flavour precursors are
found in the bean interior, i.e., placental region around the seeds, whereas the
hydrolytic and other degenerative enzymes that are known to catalyze the
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reactions for the release of flavour compounds are localized mostly in the outer
fruit wall (Havkin-Frenkel et al. 2005). The purpose of curing is to create contact
between the flavour precursors and the enzymes that catalyze the hydrolysis of
precursor compounds (Havkin-Frenkel et al. 2004).
22
General Introduction and Review of Literature
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Figure G4. Chemical structures of the major flavor compounds found in
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cured beans of Vanilla planifolia
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Despite the long time required for the curing process, the enzymatic
transformation of the glycosides to flavouring compounds is not very efficient.
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23
General Introduction and Review of Literature
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containers at 38 oC for 2-3 months resulting in pleasing aroma was also suggested
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(Broderick 1956). From then onwards, blanching in hot water has been an
essential step traditionally followed before curing of vanilla beans. This has been
a convention for several decades in various vanilla-growing countries of the
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world. For imparting the mild temperature treatment, sunning of the fruits has
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also been a regular practice, which invariably leads to losses at each exposure.
Thus, there is a need for a process, which is simple and effective.
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24
General Introduction and Review of Literature
With this background, present research work was conducted with the following
objectives
I
hyperhydric shoot cultures of Vanilla planifolia as depicted by Differential
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Display analysis
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To study the quality of green vanilla beans and their curing using various
elicitors for increasing flavor production as well as reduction in curing
period
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vanilla shoot cultures and novel improved methods for curing vanilla beans. The
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25
Genetic Diversity of Indian Vanilla
Summary
In the present study, use of isozymic markers to study genetic diversity was found
to be inappropriate due to variation in zymogram of peroxidase enzyme within the
plants. Therefore the usefulness of genetic markers such as RAPD and ISSR for
assessing the diversity among clones of V. planifolia cultivated in India was
evaluated. The genetic diversity among 25 accessions collected from 13 major
locations was studied. Forty random amplified polymorphic DNA (RAPD) and 11
inter-simple sequence repeats (ISSR) primers resulted in 326 scorable bands
ranging in size from 200 bp to 2800 bp and 83 scorable bands from 200 bp to
2500 bp, respectively. Banding pattern among the different samples collected
within accessions was similar indicating that the morphological difference
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observed within accession had no genetic background. On the other hand,
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molecular analysis among different accessions from different locations also
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yielded identical PCR band profiles in both RAPD and ISSR analysis. These
results clearly indicate that V. planifolia cultivated in India appears to share the
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same genetic background and therefore, the genetic diversity is extremely low.
Hence other biotechnological approaches may be considered to induce genetic
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Publication
Sreedhar RV, Venkatachalam L, Roohie K, Bhagyalakshmi N (2007) Molecular
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Analyses of Vanilla planifolia Cultivated in India using RAPD and ISSR Markers.
Orchid Science and Biotechnology 1(1): 29-33
26
Genetic Diversity of Indian Vanilla
1.1 Introduction
Vanilla planifolia (syn. V. fragrans), V. tahitensis and V. pompona of the family
Orchidaceae are the commercially cultivated species for the production of natural
vanilla flavour where V. planifolia is the most preferred one. Vanilla is cultivated
in an area of 37,000 ha with a production of 2230 tonnes of cured beans globally
(Gassenmeier et al. 2008). Natural vanilla flavour, extracted from cured vanilla
beans, is one of the most important and universally used aromatic flavours in
food, pharmaceutical, beverage and cosmetic industries. Vanilla was indigenous to
Mexico and was introduced to Europe by the Spanish Conquistadores in 1520
(Dignum et al. 2001). Vanilla cultivation was initiated in India, through the East
India Company, nearly 250 years back in the spice garden at Kurtallam in Tamil
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Nadu (George 2005). Its organized cultivation started in 2001-2002 in 1600 ha
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yielding 60 tonnes of cured vanilla beans and steadily gained importance doubling
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its cultivation to 3427 ha in 2003-2004 resulting in the production of 131 tonnes.
Indian vanilla occupies 5% of International vanilla market with a production
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volume of nearly 120 tonnes (Gassenmeier et al. 2008). In India, only a few
cultivars have been recognized from the species V. planifolia. In the countries
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where vanilla has been introduced, variability is likely to be highly limited as the
species is propagated only vegetatively. However, seed germination is also
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reported for vanilla (Havkin-Frenkel and Dorn 1997) indicating the possibility of
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bird dispersal leading to the chance for variations in the populations. Therefore, it
is necessary to analyze the extent of variations in V. planifolia plants collected
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27
Genetic Diversity of Indian Vanilla
as RAPD and AFLP have been used to study genetic diversity in various plants
like Wyethia (Ayres and Ryan 1999), sunflower inbreds (Popov et al. 2002) and
Lentil (Sultana and Ghafoor 2008). PCR-based random amplified polymorphic
DNA (RAPD) and inter-simple sequence repeats (ISSR) have been widely used to
survey genetic structure of populations. Among various molecular markers, the
RAPD technique is simple, rapid, and requires only a few nanograms of DNA, has
no requirement of prior information of the DNA sequence and has feasibility of
automation with higher frequency of polymorphism, which makes it suitable for
routine application for the analysis of genetic diversity (Babu et al. 2007). It is
also proven to be quite efficient in detecting genetic variations, even in closely
related organisms like two near isogenic lines of tomato (Martin et al. 1991). For
I
the use of ISSRs, primers are not proprietary as in Microsatellites or Simple
R
Sequence Repeats (SSRs) and can be synthesized by anyone and also allow the
FT
production of a high number of reproducible polymorphic bands. ISSR is found to
be very simple, quick, cost-effective, highly discriminative and most reliable
method which combines most of the advantages of SSRs and Amplified Fragment
C
Length Polymorphism (AFLP) to the universality of RAPD (Reddy et al. 2002).
@
ISSRs though considered mostly as dominant markers; they are shown to
segregate co-dominantly in some cases (Sankar and Moore 2001) thus enabling
ts
useful and reproducible than isozymes, RAPD and RFLP (Fang et al. 1997) and
are also known to give more polymorphism than any other assay procedure (Virk
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growing plant to maturity. Maize, tomato, Brassica, wheat, barley, soybean and
sugar-beet are the major crops where this technique has been routinely applied.
1.1.2 RAPD
RAPD technique uses any DNA segment that is amplified using short oligo-
deoxy-nucleotide primers of arbitrary nucleotide sequence (amplifiers) and
polymerase chain reaction procedures (PCR). Random amplified polymorphic
DNAs (RAPDs) are produced by PCR using genomic DNA and arbitrary primers.
PCR is typically carried out using two random oligonucleotide primers that flank
the DNA fragment to be amplified. These primers hybridise to complementary
strands of the target sequence and are oriented so that DNA synthesis by the
polymerase proceeds across the region between the primers. The result is an
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exponential accumulation of the specific target fragment by the de novo synthesis
of the region of DNA flanked by the two primers. Any variation in the DNA
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sequence representing the genetic variation among different individuals leads to
variation in the size or presence/absence of PCR product when amplified by
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RAPD primers. This is displayed as variation in the banding pattern when
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separated on agarose or acrylamide gel.
1.1.3 ISSR
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This technique is a variant of the PCR that uses simple sequence repeat primers
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(eg. [AC]n) to amplify regions between their target sequences. The technique
exploits the abundant and random distribution of SSRs in plant genomes by
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amplifying DNA sequences between closely linked SSRs. More complex banding
patterns can be achieved using 5’-anchored primers that incorporate the SSR
regions in their amplification. ISSR technique is nearly identical to RAPD
technique except that the primer sequences are designed from microsatellite or
SSR regions and the annealing temperatures used are higher than those used for
RAPD. Any variation variation in the DNA sequence is depicted as change in the
banding pattern after gel electrophoresis.
29
Genetic Diversity of Indian Vanilla
Mediterranean basin and the Caucasus, which were then considered to be the
major targets for conservation based on their results. They also showed that the
results of phylogenetic analyses and genetic data obtained with molecular tools
could offer an alternative measure of biodiversity that is not sensitive to
taxonomic inflation. Study of genetic diversity and phylogenetic relationships
among and within species of Cymbidiums using RAPD analysis showed full
agreement with the groups identified by morphological, physiological and
ecological characteristics. RAPD markers have also been successfully employed
to reveal relationships and classifications in Cymbidiums at cultivar levels (Obara-
Okeyo and Kako 1998; Ok et al. 2004). Chung et al. in 2006 successfully
differentiated Paphiopedilum and Phragmipedium using RAPD which were in
I
good agreement with morphologically-based classification. Schlu¨ter (2002) in
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Meso-America successfully used RAPD markers to differentiate V. planifolia
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from Costa Rica and Mexico. A study on genetic diversity of V. planifolia by
Besse et al. (2004) using RAPD markers in vanilla cultivated areas of Reunion
Island and Polynesia reveled that there exists a very low level of genetic diversity.
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The only study on genetic diversity of Indian vanilla using molecular markers is
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by Minoo et al. (2008). RAPD analysis of two indigenous collections of
V. planifolia made from vanilla cultivating regions of India reveled that there is
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very limited variation within the collections indicative of its narrow genetic base.
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In the present study, both the PCR based techniques, RAPD and ISSR,
were adopted for the evaluation of genetic variation in V. planifolia. Therefore,
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the objectives of the present study were to assess the usefulness of genetic
markers for assessing the diversity among clones of vanilla cultivated in India and
to create a database for the available germplasm.
30
Genetic Diversity of Indian Vanilla
Table 1.1 List of accessions of V. planifolia and their geographical origin used
in this study
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6 Udupi, Karnataka 1
7 Coimbatore, Tamil Nadu 1
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8 Kanyakumari, Tamil Nadu 1
9 Dindigul, Tamil Nadu 1
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10 Ooty, Tamil Nadu 1
11 Mallapuram, Kerala 1
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12 Kasargud, Kerala 1
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13 Pallakad, Kerala 1
* Refer to Figure 1.1 for location of accessions.
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Native-PAGE analysis
For analyzing the isozymes of POD from different clones of vanilla, standard
protocol was used. An initial experiment was conducted to check the variation in
zymogram pattern within same clone. For this, leaf material obtained from
different stages of leaves (unopened, first and fourth leaf pairs from shoot tip)
from the same plant was used for extraction of the proteins. Protein extract was
made in sodium phosphate buffer (pH 6.0) containing 1 mM Dithiothreitol and 0.1
mM Phenyl Methyl Sulfonyl Fluoride. Zymogram was prepared by
polyacrylamide gel electrophoresis (7.2% (w/v)) (PAGE) carried out at 120 V for
4 h using 12 × 14 × 0.5 cm gel without SDS. The gel was stained for POD activity
with a 100 ml solution of sodium phosphate buffer containing 10 ml of 0.25%
31
Genetic Diversity of Indian Vanilla
Approximately 100 mg of young leaf tissue was ground into fine powder in liquid
nitrogen and total genomic DNA was extracted using the GenEluteTM Plant
Genomic DNA Mini prep Kit (Sigma Aldrich, India). Quality and quantity of
DNA preparations were checked by standard spectrophotometry and the samples
were diluted to 25 ng µL-1 in TE buffer and stored at 4 °C.
1.2.4 Primer selection
Various RAPD and ISSR primers were selected, based on specific relevance to
family Orchidaceae to which vanilla belongs, from the studies of Besse et al.
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(2004), Tsai et al. (2002) and NCBI-database. Others were those for monocots
that were successfully used in our earlier study in banana (Venkatachalam et al.
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2007). Out of the 60 RAPD 10-mer primers and 20 ISSR primers, 40 RAPD and
11 ISSR primers were selected depending on their consistency in amplification
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(Table 1.2).
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32
Genetic Diversity of Indian Vanilla
KA
I
6
17
18
16
15
14
13
KA
11 24
10
TN 22
19
KE
I
9 21
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12 20
8
25
5 23
4
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7
3
1
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Figure 1.1 Map showing sampling locations (filled triangles) in India (I) and
Karnataka (K)
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RAPD primers
Kit OPA A-03; A-04; A-11; A-14; A-20
Kit OPC C-01; C-02; C-04; C-05; C-06; C-07; C-08; C-09; C-10; C-12
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33
Genetic Diversity of Indian Vanilla
were performed routinely using a PCR mixture (25 µL) which contained 25 ng of
genomic DNA as template, 1X PCR buffer (Fermentas GMBH, Germany), 200
µM dNTPs (Fermentas GMBH, Germany), 1 unit (U) of Taq DNA polymerase
(Bangalore Genei, India), 0.5 µM of each primer (Operon Technologies, Alameda,
California, USA) with varied concentration of MgCl2 (Fermentas GMBH,
Germany) depending on the primer (Table 1.3). PCR was performed at initial
denaturation at 93 °C for 4 min followed by 36 cycles of 1 min denaturation at 94
°C, 1 min annealing at 36 °C and 2 min extension at 72 °C with a final extension
of 72 °C for 10 min using a thermal cycler (Eppendorf thermal cycler 5332,
Germany).
For ISSR primers, optimal annealing temperature was found to vary
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according to the base compositions of the primers. PCR mixture (25 µL)
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contained 25 ng of genomic DNA as template, 1X PCR buffer, 200 µM dNTPs, 1
FT
unit (U) of Taq DNA polymerase, 0.5 µM of each primer with varied
concentration of MgCl2 depending on the primer (Table 1.4). PCR was performed
at initial denaturation at 94 °C for 4 min followed by 40 cycles of 1 min
C
denaturation at 94 °C, 1 min at 2 °C lower than the specified annealing
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temperature for each primer and 2 min extension at 72 °C with a final extension
also at 72 °C for 10 min using a thermal cycler.
ts
34
Genetic Diversity of Indian Vanilla
Table 1.3 List of selected primers used in RAPD analysis and number of
scorable bands
I
14 OPC 10 TCTCTGGGTG 0 3
R
15 OPC 12 TCTCATCCCC 1 7
16 OPD 04 TCTGGTGAGG 1 6
17 OPD 11 AGCGCCATTG 0 9
FT
18 OPD 16 AGGGCGTAAG 0 8
19 OPF 12 ACGGTACCAG 1 8
20 OPJ 07 CCTCTCGACA 0 9
21 OPJ 08 CATACCGTGG 2 4
C
22 OPJ 09 TGAGCCTCAC 1 7
23 OPJ 10 AAGCCCGAGG 2 8
@
24 OPJ 11 ACTCCTGCGA 1 5
25 OPJ 12 GTCCCGTGGT 0 7
26 OPJ 13 CCACACTACC 1 6
27 OPJ 15 TGTAGCAGGG 0 5
ts
28 OPJ 16 CTGCTTAGGG 1 8
29 OPJ 17 ACGCCAGTTC 2 11
30 OPJ 18 TGGTCGCAGA 2 6
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31 OPJ 19 GGACACCACT 1 10
32 OPM 16 GTAACCAGCC 0 15
33 OPM 18 CACCATCCGC 2 6
eP
34 OPM 20 AGGTCTTGGG 2 10
35 OPN 03 GGTACTCCCC 0 12
36 OPN 04 GACCGACCCA 1 10
37 OPN 06 GAGACGCACA 0 11
38 OPN 09 TGCCGGCTTG 2 11
39 OPN 10 ACAACTGGGG 1 14
40 OPN 14 TCGTGCGGGT 1 10
35
Genetic Diversity of Indian Vanilla
Table 1.4 List of selected primers used in ISSR analysis and number of
scorable bands
Sl. Primer name Primer sequence MgCl2 concentration No. of scorable bands
No. (5′–3′) (mM)
1 UBC 809 (AG)8G 0 4
2 UBC 810 (GA)8T 0 9
3 UBC 811 (GA)8C 0 10
4 UBC 813 (CT)8T 1 5
5 UBC 823 (TC)8C 0 4
6 UBC 824 (TC)8G 0 6
7 UBC 826 (AC)8C 1 15
8 UBC 834 (AG)8YT 0 8
9 UBC 836 (AG)8YA 0 5
10 UBC 840 (GA)8YT 1 11
11 UBC 848 (CA)8RG 0 6
I
Y : C or T
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1.3 Results
FT
A total of five isozymes of POD were observed in the zymogram out of which
only three were found in the protein extracts from unopened and first leaf pair.
C
Protein extract from the fourth leaf pair displayed all the five isozymes in the gel
@
(Figure 1.2). Standardization of conditions for PCR amplification such as
concentrations of magnesium chloride, Taq-polymerase, template DNA and
ts
effects. Forty RAPD primers (Table 1.2) were selected from 60 arbitrary primers
(having 60-70% GC content) based on their amplification products. Each RAPD
eP
primer generated a unique set of amplification products ranging in size from 200
bp to 2800 bp where the number of bands for each primer varied from 4 in OPJ 08
and 15 in OPM 16 (Table 1.3). The 40 primers used in this analysis yielded 326
scorable bands with an average of 8.15 bands per primer. Screening with the 20
ISSR primers generated 83 scorable bands in 11 primers (Table 1.2) ranging from
200 bp to 2500 bp. An average of 7.54 bands per ISSR primer was obtained
ranging from 4 to 15 (Table 1.4). Banding pattern among the different samples
collected within an accession was similar indicating that the morphological
difference observed within accessions had no genetic background. On the other
hand, molecular analysis among different accessions from different locations also
36
Genetic Diversity of Indian Vanilla
yielded an identical PCR band profile in both RAPD and ISSR analysis (Figures
1.3, 1.4).
I
R
FT
C
@
37
Genetic Diversity of Indian Vanilla
1.4 Discussion
Isozymic analysis of POD enzyme for its possible use in analysis of genetic
diversity among various clones of vanilla showed that there was variation in the
zymogram pattern within same clone between protein extracts of leaf material
from different stage of development. This difference within same plant/clone may
be due to variation in isozymes synthesized during various stages of growth and
development of the plant depending on the biochemical status of the plant tissue.
These observations suggested exclusion of isozymic analysis for diversity study in
vanilla clones collected which otherwise lead to erroneous conclusions. Of the
various biochemical and molecular techniques used to resolve genetic diversity in
lentil, isozymes and seed proteins gave low levels of genetic diversity. RAPD was
I
found to be the best option for determining inter- and intra-accession variation
R
(Sultana and Ghafoor 2008). A population genetic study of Goodyera procera
FT
with allozyme and RAPD markers supported that RAPD can detect higher levels
of genetic variation than allozyme (Wong and Sun 1999). In Brassica oleracea,
C
high variability in the banding pattern within and among cultivars was observed
(Arus et al. 1985). RAPD analysis proved to be more informative and effective
@
approach for estimation of genetic diversity in inbred sunflower lines (Popov et al.
2002). The use of isozymes in limited by the lower number of polymorphic
ts
The present study involved two types of efficient genetic markers involving
a large number of primers for marker-based genetic analyses of 25 accessions
eP
collected from different locations of India (Figure 1.1; Table 1.1). This study has
clearly showed the absence of genetic variation within and among V. planifolia
populations. A very low level of genetic diversity was detected in V. planifolia in
geographical areas such as Mexico (Soto and Arenas 1996; Cibrian 1999),
Reunion Island (Besse et al. 2004) and Polynesia (Pacific Ocean) (Besse et al.
2004), which is in accordance with the vegetative mode of dispersion as stem
cuttings and the history (introduced plant species) of recent introduction in these
regions. A thorough analysis of different species (such as V. planifolia, V.
tahitensis and V. pompona) and clones within the species of Vanilla cultivated in
Reunion and Central America (Besse et al. 2004) showed no variation in the
introduced locations.
38
Genetic Diversity of Indian Vanilla
I
abiotic factors (Li et al. 2006). The introduced plant need not accumulate enough
R
genetic variation to adapt to its newer environment in its spread phase if its
FT
phenotypic plasticity is stronger and could buffer against the selection pressure
(Weber and Schmid 1998). The degree to which introductions are accompanied by
genetic bottlenecks depends on the species breeding systems and is expected to be
C
lowest in highly selfing species or those that reproduce vegetatively (Nei et al.
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1975).
Vanilla, being an introduced crop in most countries where it has been
ts
propagation, which largely limits the genetic variability in the crop (Divakaran
2006b). Vanilla might have been introduced to India through a “stepping-stone”
eP
process resulting in all cultivars sharing the same genetic background. Vanilla
cultivation, having begun in India by the East India Company nearly 250 years
back in the spice garden at Kurtallam in Tamil Nadu indicates that a few vines
belonging to same mother plant or plantation might have been introduced and
further expansion to all other parts is from these plants, which are of a similar
genetic background. Reports indicate that plantations of Reunion, Mauritius,
Seychelles and Malagasy Republic can all be traced back to a single clone
(Madhusoodanan et al. 2003). Besse et al. (2004) while studying genetic diversity
of V. planifolia by using RAPD markers in vanilla cultivated areas of Reunion
Island and Polynesia reported a very low level of diversity. The only study on
genetic diversity of Indian vanilla using molecular markers is by Minoo et al.
(2008) by RAPD analysis of two indigenous collections of V. planifolia made
39
Genetic Diversity of Indian Vanilla
from vanilla cultivating regions of India reveled that there is very limited variation
within the collections indicative of its narrow genetic base.
The development of strong adaptability of a plant species to its current
environments is more important for its survival than the accumulation of rich
genetic diversity, which usually takes a long time to achieve (Xu et al. 2003).
Obviously, the shortcomings of low genetic diversity in a plant species can be
highly compensated by the development of its strong adaptability, at least for a
temporal period of time. However, it is difficult to predict the long-term effect
caused by the low genetic variation of the clonal species. Through rapid and
massive expansion, a few successful clones with favorable genotypes might be the
essential component in all individuals in its new colonies, although overall genetic
I
variability in these clones might appear to be low.
R
FT
1.5 Conclusion
This preliminary investigation has determined the absence of genetic variations in
C
introduced and then commercially cultivated V. planifolia in India indicating a
threat of extinction due to pest and environment vagaries. These observations
@
indicate the need to increase the number of introductions and broaden the gene
pool of cultivated vanilla in India to reduce its vulnerability to diseases and insect
ts
pests apart from its genetic improvement for other attributes. Genetic variability is
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40
Genetic Fidelity and Advanced Micropropagation Techniques
Summary
Occurrence of genetic variants during micropropagation is occasionally
encountered when the cultures are maintained in vitro for long period. Therefore,
the micropropagated multiple shoots of vanilla developed from axillary bud
explants that were established 10 years ago were used to determine somaclonal
variation using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple
Sequence Repeats markers (ISSR). One thousand micro-plants were established
in soil of which 95 plantlets (consisting of 4 phenotypes) along with the mother
plant were subjected to genetic analyses using RAPD and ISSR markers. Out of
the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers
showed 317 clear, distinct and reproducible band classes resulting in a total of
I
R
30,115 bands. However, no difference was observed in banding patterns of all the
samples for a particular primer indicating the absence of variation among the
FT
micropropagated plants. The results suggest that the micropropagation protocol
used for in vitro proliferation of vanilla plantlets for the last 10 years might be
C
applicable for the production of clonal plants over a considerable period of time.
@
To study the possibility of up-scaling the protocol, vanilla shoot
multiplication in semi-solid (SS), complete immersion system (CIS) and partial
ts
immersion system (PIS) were evaluated keeping track of kinetics of growth and
nutrient uptake. Significant reduction in osmolarity due to high uptake of sucrose
rin
was higher in CIS than in PIS with no difference in the conductivity pattern,
indicating that the mineral uptake was probably similar in both. The rate of shoot
eP
multiplication was although marginally higher in SS than in CIS and PIS, by the
end of five-weeks culture period, the biomass production and shoot elongation
were significantly higher in PIS than in SS and CIS. Shoot cultivation in CIS was
associated with hyperhydric shoots (>80%) having poor ability to establish in
fresh medium or soil. GrowtekTM bioreactor, the cheaper version of bioreactor,
functioning on the principle of PIS enabling constant supply of the nutrients and
aeration to the plants was found to perform better than SS and CIS and can be an
efficient liquid culture system for shoot cultivation of vanilla. To obtain
preliminary data on the conditions required for the shoot multiplication in
bioreactor where the shoots are intermittently bathed with liquid medium,
different medium contact periods were provided to shoot cultures in CIS where in
41
Genetic Fidelity and Advanced Micropropagation Techniques
the 30 min contact three times a day appeared most congenial for best shoot
multiplication and elongation. An equi-proportion of red soil: sand:
vermicompost was found to be the best hardening soil medium for vanilla. The
shoots sub-cultured in PIS were robust with good elongation producing both
geotropic and aerial roots resulting in highest survival (90.5%) on transfer to this
soil mixture. When a comparison was made on the growth and yield performance
of plants derived from micropropagation and stem cuttings, plants form
micropropagation flowered early and were higher yielders than those from the
stem cuttings.
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Publication
Sreedhar RV, Venkatachalam L, Bhagyalakshmi N (2007) Genetic fidelity of long -
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term micropropagated shoot cultures of Vanilla (Vanilla planifolia Andrews) as
assessed by molecular markers. Biotechnology Journal 2: 1007-1013
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42
Genetic Fidelity and Advanced Micropropagation Techniques
2.1 Introduction
Genus Vanilla Swartz belongs to the family Orchidaceae which consists of 110
species, of which 15 are known to yield aromatic pods. The seeds of vanilla do
not generally germinate and hence the plants are propagated by vegetative means
through stem cuttings that result in slow rate of multiplication and non-uniformity
in planting material. Removal of cuttings may also cause injury to the mother
plant resulting in a set-back of growth and reduction in yield. Market demand for
the propagules is hardly met through these cuttings; therefore, micropropagated
plantlets have gained importance in the vanilla cultivating areas. In vitro
propagation of vanilla by culturing axillary buds (George and Ravishankar 1997;
Giridhar et al. 2001), aerial root tips (Phillip and Nainar 1988), through callus
I
R
(Davidonis and Knorr 1991) and protocorms has been reported earlier.
Micropropagation using shoot tips or nodal segments has been found to be an
FT
appropriate technique wherein about one lakh plantlets may be obtained in fifteen
sub cultures (George and Ravishankar 1997).
C
True-to-type clonal fidelity is important for utilizing the advantages of
@
micropropagation. A major problem encountered with in vitro culture is the
occurrence of somaclonal variation amongst sub-clones of one parental line,
ts
buds and shoot tips have been reported to maintain clonal fidelity as organized
meristems are more resistant to genetic changes compared to unorganized callus
eP
under in vitro culture (Ostry et al. 1994). However, the possibility of occurrence
of somaclonal variants even in such cultures cannot be ruled out (Devarumath et
al. 2002). The exact causes for such variations are still unknown, although it is
believed to be induced by alterations in the supply of nutrients, auxin-cytokinin
concentrations and their ratio, in vitro stress due to unnatural conditions and
disturbed diurnal rhythm. Cultured plant tissues are also known to undergo high
levels of oxidative stress and are exposed to reactive oxygen species (ROS), the
latter being known to cause DNA damage including microsatellite instability
(Jackson et al. 1998). Thus, the array of variations is often heritable and
undesirable, challenging the very “clonal” nature of micropropagated plants.
Reliable assays to assess the genetic stability of a genotype throughout in vitro
43
Genetic Fidelity and Advanced Micropropagation Techniques
I
R
(SSRs) consist of short tandem repeats of 2 to 5 base pair motifs, distributed
throughout eukaryotic genomes and hence are highly informative. However, for
FT
their efficient use, flanking regions must be known so that polymerase chain
reaction (PCR) primers may be generated. On the other hand, Random Amplified
C
Polymorphic DNA (RAPD) is simpler and has proven to be quite efficient in
detecting genetic variations, even in closely related organisms (Martin et al.
@
1991). With Inter-Simple Sequence Repeats (ISSRs), primers are not proprietary
as in SSRs and can be synthesized by anyone and also allow production of a high
ts
effective, highly discriminative and reliable method which combines most of the
advantages of Simple Sequence Repeats and Amplified Fragment Length
eP
Polymorphism with the universality of RAPD (Reddy et al. 2002). They are
found to be more useful and reproducible than isozymes and RAPD; less
cumbersome and cost-effective for routine application than RFLP (Fang et al.
1997). In addition, the ISSRs are also found to give more polymorphism than any
other assay procedure (Virk et al. 2000). Thus, detection of additional
polymorphism could be done by the use of RAPD in combination with ISSRs. At
present, RAPD and ISSR markers have been widely utilized to detect the genetic
similarities and dissimilarities in micropropagated material in various plants
(Devarumath et al. 2002; Martins et al. 2004; Venkatachalam et al. 2007).
Mass propagation of plants by tissue culture is a costly and labour
intensive technology. The gelling agents used are not inert medium components
44
Genetic Fidelity and Advanced Micropropagation Techniques
and do not enable easy automation for commercial mass propagation. Agar, the
most commonly used non-nutrient gelling agent is one of the costliest ingredients
of the culture medium. High production costs limit the commercial use of
micropropagation to markets with a high unit value for crops such as vanilla. It
has been concluded for various species that extensive expansion of
micropropagation would only take place if improved technologies became
available for automation and acclimatization (Kitto 1997). Liquid culturing has
been considered as an ideal technique for mass propagation as it reduces manual
labor and facilitates better control over medium manipulation apart from rendering
automation opportunities of the entire process. Moreover, plant tissues from
numerous species are known to perform better when cultured in liquid medium
I
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rather than on agar medium. It is also considered to be an ideal solution for
reducing plantlet production costs and the system provides more uniform culturing
FT
conditions. Use of liquid medium allows scale-up in bioreactors minimizing the
number of operations with the advantage of medium manipulation, thereby
C
reducing the cost of production of micro-propagules (Hvoslef-Eide and Melby
2000; Dey 2001). The available bioreactors are basically the modified microbial
@
bioreactors and are un-suitable for higher plants due to their high sensitivities to
shear force leading to mechanical damages. Alternatively, the aerated type exhibit
ts
2005).
Vanilla shoots when grown in liquid medium undergo hyperhydricity or
eP
45
Genetic Fidelity and Advanced Micropropagation Techniques
I
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been well documented (Dey 2005).
Reports on in vitro propagation of vanilla indicate the usefulness of
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culturing axillary buds (George and Ravishankar 1997; Geetha and Shetty 2000;
Giridhar et al. 2001, Kalimuthu et al. 2006) and aerial root tips (Phillip and Nainar
C
1988). Reports on the plantlet formation through callus cultures (Davidonis and
Knorr 1991) and protocorms are sporadic. Micropropagation using shoot tips or
@
George and Ravishankar 1997 has been adopted as an initial step for
establishment and maintenance of shoot cultures in the present study. While
eP
46
Genetic Fidelity and Advanced Micropropagation Techniques
I
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maintained for about 10 years at 252 oC under 16 h photoperiod having
illumination of 37.5µE m-2 s-1 provided by fluorescent lamps. Over 1000 plantlets
FT
from such cultures were established as rooted plantlets, of which four groups of
plants were selected where group one had 26 normal-looking and the other three
C
groups consisted of 23 plantlets each showing unusual phenotypes such as pale
@
green leaves, hyper-hydric, multiple-apiced and stunted growth (Figure 2.1). The
plantlets from these four groups were used for genetic analysis along with the leaf
ts
material from the mother plant (maintained in the departmental garden). Thus a
total number of 96 samples (including mother plant) were subjected for genetic
rin
marker analyses.
eP
47
Genetic Fidelity and Advanced Micropropagation Techniques
2.2.2 Micropropagation
2.2.2.1 Plant material
The plant material, establishment and maintenance of shoot cultures were as in
section 2.2.1.1. Shoot bud clusters (Figure 2.6A) were trimmed to have an
average of five buds per cluster and were used as initial inoculum for all the
studies. Each cluster weighed approximately 1 gm and each bud was 0.5-1 cm in
length.
I
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2.2.2.2 Culturing conditions
The medium used for all the studies was modified MS medium (supplemented
FT
with 30 g of sucrose, 8.87 µM BAP and 2.69 µM NAA). Medium gelled with 7.2
g L-1 of agar (HiMedia, India) served as SS system. As a CIS, Erlenmeyer’s flasks
C
(150 mL) with 40 mL of medium (Figure 2.6B) were used. For PIS, GrowtekTM
bioreactors (100×150 mm (×h), Tarsons Products, India) (Figure 2.2) filled with
@
200 mL of liquid medium having unique features like floating, rotating, non-
absorbing explant holder with perforated explant support matrix; side-tube with
ts
silicon rubber septum for changing media and online monitoring of medium
rin
medium was used. The bubble column bioreactor was made of a glass column
(Corning glass, height 22 cm and diameter 14 cm) of 3 liters capacity with a
working volume of 1.75 liter. The upper lid had provision for air inlet/outlet for
sparger and ports for inoculation and sampling. Air was supplied through a glass
sparger of 45 cm height and 7 cm diameter, molded into a circular shape at the
bottom having pores of size 1 mm. An autoclavable basket made of stainless steel
wire mesh of 10.5 cm height and 8.5 cm diameter was used to provide anchorage
for the biomass. This anchorage had pores of 0.5 cm diameter at the bottom as
well as sides and was placed at a height of 7 cm from the bottom of the bioreactor
vessel supported by a stainless steel stand. The distance between sparger and the
48
Genetic Fidelity and Advanced Micropropagation Techniques
Figure 2.2 Growtek™ bioreactor used in the present study. A: The entire
I
setup; B: Container with lid and side-tube with silicon rubber septum, C:
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Explant holder with perforated explant support matrix
Perforated
basket for 22 cm
anchorage
eP
14 cm
49
Genetic Fidelity and Advanced Micropropagation Techniques
I
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pumping liquid medium in and out to provide required period and number of
contacts by the medium. Effect of different contact periods on the shoot
FT
multiplication, elongation and morphology were studied for a total period of five
weeks.
C
Clusters of shoot buds (shoot bud length 0.5-1 cm) were used as initial
inoculums. Erlenmeyer’s flasks and GrowtekTM bioreactors were maintained at
@
50
Genetic Fidelity and Advanced Micropropagation Techniques
Gonotech, GmbH, Germany). The calibration of the instrument was done using
triple distilled water. Osmolarity was expressed as Osmol kg-1.
I
2.2.2.5 Rooting, hardening and green house cultivation
R
Rooting of the elongated shoots from GrowtekTM bioreactor was achieved on MS
FT
medium with 1/2 strength NH4NO3 supplemented with 5.37 µM NAA and
15 g L-1 sucrose which was found to be most suitable by earlier observations.
C
Well rooted plantlets of 8-10 cm length were then planted in soil mixture
containing varied proportion of red soil, sand and vermicompost and observations
@
on percent survival and growth response were made four weeks after planting.
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51
Genetic Fidelity and Advanced Micropropagation Techniques
I
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presented as an average of mean±error bars (p≤0.05).
2.3 Results
2.3.1 Genetic fidelity FT
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Table 2.1 lists the RAPD primers used in the present study, their sequence, the
@
additional levels of MgCl2 needed for proper amplification, the number of
scorable bands formed for each primer and the range of fragment sizes. A
ts
comparison of RAPD and ISSR profiles of all the DNA samples extracted was
done for analyzing the variations, if any. Out of the 45 arbitrary primers (having
rin
60-70% GC content) tested for RAPD, 30 primers were selected based on their
amplification products. Each RAPD primer generated a unique set of
eP
amplification products ranging in size from 200 bp to 2,800 bp where the number
of bands for each primer varied from 4 in OPJ 08 and 15 in OPM 16. The 30
primers used in this analysis yielded 258 scorable bands with an average of 8.60
bands per primer. Out of 20 ISSR primers, 7 were selected and they produced 59
scorable bands which ranged from 200 bp to 2,500 bp (Table 2.2). An average of
8.43 bands per ISSR primer was obtained which ranged from 4 to 15. A total of
30,115 bands (no. of plantlets analysed × no. of band classes with all primers)
were generated by the RAPD and ISSR techniques giving rise to monomorphic
band classes for all the 95 DNA samples of micropropagated plants analyzed.
Both micropropagated plants and the mother plant showed identical banding
52
Genetic Fidelity and Advanced Micropropagation Techniques
Table 2.1 List of selected primers used in RAPD analysis, the respective
additional levels of MgCl2, the number of scorable bands obtained and the
range of fragment size for each primer
I
06 OPC 01 TTCGAGCCAG 0 7 450-1300
R
07 OPC 02 GTGAGGGCTC 2 6 300-1200
08 OPC 04 CCGCATCTAC 1 8 450-1500
FT
09 OPC 05 GATGACCGCC 1 8 300-1700
10 OPC 06 GAACGGACTC 0 6 900-2500
11 OPC 07 GTCCCGACGA 0 8 400-2800
12 OPC 09 CTCACCGTCC 1 9 400-2500
13 OPC 08 TGGACCGGTG 0 7 300-1500
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14 OPD 04 TCTGGTGAGG 0 6 900-2300
15 OPD 11 AGCGCCATTG 1 9 300-2800
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16 OPD 16 AGGGCGTAAG 1 8 400-1500
17 OPF 12 ACGGTACCAG 0 8 300-2000
18 OPJ 07 CCTCTCGACA 0 9 300-2000
19 OPJ 08 CATACCGTGG 1 4 400-1000
ts
53
Genetic Fidelity and Advanced Micropropagation Techniques
Table 2.2 List of selected primers used in ISSR analysis, the respective
additional levels of MgCl2, the number of scorable bands obtained and the
range of fragment size for each primer
I
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FT
C
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ts
rin
eP
54
Genetic Fidelity and Advanced Micropropagation Techniques
M P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
3000
1031
500
M P 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
3000
1031
500
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FT
M P 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
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3000
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1031
500
ts
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M P 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
eP
3000
1031
500
55
Genetic Fidelity and Advanced Micropropagation Techniques
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FT
C
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rin
eP
56
Genetic Fidelity and Advanced Micropropagation Techniques
2.3.2 Micropropagation
I
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2 weeks after inoculation (WAI). Hence, the cultivation was terminated and
GrowtekTM bioreactor (Figure 2.6C, D) which showed a good response was
FT
considered as PIS for further studies. In order to evaluate the effectiveness of
PIS, it was compared with semi-solid and CIS for various parameters like pH,
C
conductivity, osmolarity and their effects on the rate of biomass accumulation,
@
shoot multiplication and elongation.
The pH of the medium which had been adjusted to 5.8 during preparation,
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dropped to 5.7 after autoclaving, with a steep drop further to 4.3 after inoculation.
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It later stabilized at around 4.7 throughout the culture period from 1 WAI. The
pH changes were almost similar for both PIS and CIS (Figure 2.7). The medium
conductivity depends on the electrolyte concentration and ignores the changes in
eP
sugars which are present in higher concentration (Madhusudhan et al. 1995). The
trend of uptake was almost similar in both CIS and PIS with a slight higher rate of
uptake in CIS 4 WAI. A decrease in conductivity from an initial value of 6.4 mS
to 4.2 mS in PIS and 4.05 mS in CIS was notable (Figure 2.7). The Osmolarity
takes into account the number of moles of all solutes present in the medium.
Initial osmolarity of the medium before inoculation was 0.225 Osmol kg-1 which
gradually decreased to 0.086 Osmol kg-1 in PIS and 0.069 Osmol kg-1 in CIS
indicating all solutes in the media are probably exhausted by 5 weeks of culture
(Figure 2.7). Though there was a similar trend of uptake of solutes in both PIS
and CIS in the first week, a higher uptake of nutrients in case of CIS between 1st
and 4th week was apparent from the osmolarity data.
57
Genetic Fidelity and Advanced Micropropagation Techniques
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FT
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58
Genetic Fidelity and Advanced Micropropagation Techniques
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FT
Figure 2.7 pH, osmolarity and conductivity of the medium in Erlenmeyer’s
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flasks (CIS) and GrowtekTM bioreactor (PIS) used at various stages of
culturing shoot cultures. WAI: weeks after inoculation. All treatments had at
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least fifteen replicates and the data are presented as an average of
mean±error bar (p≤0.05) of replicates of two separate experiments.
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The rate of shoot multiplication was 2.71 in SS and 2.46 in PIS 5WAI
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which did not differ significantly (Figure 2.8). However, there was a significant
difference in shoot multiplication rate between SS, CIS and PIS from 2 WAI to 4
eP
59
Genetic Fidelity and Advanced Micropropagation Techniques
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FT
C
Figure 2.8 Shoot multiplication, shoot elongation and biomass production in
semi-solid medium (SS), Erlenmeyer’s flasks (CIS) and GrowtekTM
@
bioreactor (PIS) at various stages of culturing shoot cultures. WAI: weeks
after inoculation. All treatments had at least fifteen replicates and the data
are presented as an average of mean±error bar (p≤0.05) of replicates of two
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separate experiments.
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There was no significant difference in shoot length during first and second
week of culturing. Highest biomass accumulation was found in PIS 5 WAI which
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was 23.45-fold (Figure 2.8). It was significantly higher compared to CIS (11.89-
fold) and SS (7.78-fold). A significantly higher increase in biomass production
was found in PIS from 3 WAI. No significant variation in biomass accumulation
was found between SS, CIS and PIS during the first two weeks of culturing.
However shoot cultivation in CIS was associated with hyperhydricity syndrome
wherein more than 80% of shoots appeared hyperhydric (Figure 2.6B).
The shoots derived from PIS (GrowtekTM bioreactor) appeared better and
well- grown than those from CIS or SS (Figure 2.6D). Development of root
initials was a notable feature in the GrowtekTM bioreactor grown shoot clusters.
Each shoot in the cluster developed 2-3 root initials 5 WAI. All the shoots were
then separated and were inoculated into rooting medium. Uniform rooting (3-4
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Genetic Fidelity and Advanced Micropropagation Techniques
roots per plantlet) in MS medium with 1/2 strength NH4NO3 supplemented with
5.37 µM NAA and 15 gm L-1 of sucrose was observed (Figure 2.6F).
In case of Temporary Immersion System, a contact period of 30 min three
times a day was found to be the best compared to the other treatments for
automation of vanilla shoot cultivation. Shoot multiplication rate was 2.42 which
was significantly higher and 1.25-fold higher than the 15 min treatment (Table
2.3). Although the treatment with 45 min contact period showed significantly
higher number of shoots with 1.19-fold higher than 30 min treatment, the shoots
were stunted and associated with hyperhydricity syndrome which started by 3rd
week of inoculation. Highest shoot elongation was observed in 30 min contact
treatment which was 9.45 cm by the end of 5 week cultivation (Table 2.3). It was
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1.43-fold higher than the 15 min treatment and 2-fold higher compared to 45 min
contact treatment. Total biomass accumulation was found to be highest in 30 min
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contact period treatment which was around 654.1 g and was 3.04-fold higher than
the 45 min contact treatment and 1.53-fold higher than 15 min treatment (Table
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2.3). The shoot cultures which got a 15 min contact period three times a day
appeared dried and less vigorous whereas the ones which received 30 min contact
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period treatment were more vigorous and dark green. Shoots which received a 45
min contact treatment were pale and translucent probably due to hyperhydricity
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2.9) with a medium contact period of 30 min three times a day. This resulted in a
biomass accumulation of more than 663.5 g with shoot multiplication rate of
nearly 2.55 fold and shoot elongation of around 9.55 cm five weeks after
inoculation.
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Genetic Fidelity and Advanced Micropropagation Techniques
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Genetic Fidelity and Advanced Micropropagation Techniques
Table 2.4 Effect of different combinations of soil mixtures used for hardening
the plants under green house conditions (Average of 25 plantlets)
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compared to 15-20 days taken by those in micropropagation. Shoots of stem
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cuttings reached a height of 13-15 cm in 16-18 weeks which was comparatively
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lesser in the case of micropropagated ones which took just 12-14 weeks in the
greenhouse conditions (Table 2.5). After a six month period, the conventionally
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propagated plants reached a height of 30-35 cm which was significantly higher in
micropropagated ones which reached a height of more than 50 cm.
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3 years after planting Flowering and fruit set Flowering and fruit set (higher)
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Genetic Fidelity and Advanced Micropropagation Techniques
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Genetic Fidelity and Advanced Micropropagation Techniques
A B C
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D E F
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trained to a bamboo support. D: One year old plant grown under shade net
condition. E: Profuse flowering in vertically trained branch of vanilla of two
year old tissue culture derived plant. F: Fruiting in plant of tissue culture
origin.
2.4 Discussion
2.4.1 Genetic fidelity
The present study involved two types of efficient genetic markers involving a
large number of primers for marker-based genetic analyses of 95 micropropagated
plants. This study has clearly shown the absence of genetic variation among the
plantlets of V. planifolia. By using a combination of two types of markers which
amplify different regions of the genome a better analysis of genetic stability of
plantlets can be made (Martins et al. 2004). It is (Palombi and Damiano 2002)
65
Genetic Fidelity and Advanced Micropropagation Techniques
suggested that the use of more than one DNA amplification technique proves to be
advantageous in evaluating somaclonal variation while screening the
micropropagated plants of kiwi fruit for any genetic variation. Hence, in the
present study, two PCR based techniques, RAPD and ISSR were adopted for the
evaluation of clonal fidelity of micropropagated V. planifolia plantlets.
The large number of bands produced by RAPD and ISSR primers indicates
that several sites were effectively amplified by these primers and hence they
appear appropriate for genetic evaluation of Vanilla planifolia. Absence of
genetic variation using RAPD has been reported in several cases like in
micropropagated shoots of Pinus thunbergii Pral. (Goto et al. 1998), axillary bud
proliferations of chestnut root stock hybrids (Carvalho et al. 2004) and almond
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plantlets (Martins et al. 2004). In contrast, somaclonal variations were reported in
micropropagated plants of Populus tremuloides (Rahmann and Rajora 2001),
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cauliflower (Leroy et al. 2001) and Actinidia deliciosa (Palombi and Damiano
2002). In the present study, no variation in the banding pattern was observed
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among the micropropagated plants in any of the RAPD or ISSR profiles,
indicating the absence of variation in DNA sequence and therefore the absence of
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somaclonal variation.
The vanilla shoots cultured in vitro for several subcultures appeared to
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different phenotypes during prolonged in vitro culturing was also observed while
working on micropropagated phenotypes of Pinus thunbergii Pral. (Goto et al.
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1998) and P. radiate (Ishii et al. 1987). In these studies, the use of cytokinins,
especially the exposure to BAP (6-Benzylaminopurine), has been noted to induce
hyper-hydricity. The latter phenomenon has been prevalent in xerophytic plants.
Since vanilla plants are also partially xerophytic in nature one can expect high
morphological variability and concomitant genetic changes, especially when shoot
cultures are exposed to cytokinins for a long-period. Morphological changes such
as stunted growth of shoots and unusual shapes of leaves observed in the present
study have also been commonly known to occur during micropropagation in many
plants either due to direct effect of plant growth regulators or due to re-
juvenilization during long-term culture (George 1996). Formation of callusy
shoot apices in some of the cultures during prolonged multiplication could be due
66
Genetic Fidelity and Advanced Micropropagation Techniques
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somaclonal variation (George 1996). Though a high concentration of BAP and
NAA have been continuously used in the medium for sub culturing over a period
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of 10 years, in the present study, no somaclonal variation could be detected within
the micropropagated vanilla shoots. The morphological changes that occurred
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seem to be purely due to varied physiological and/or developmental states with no
effect on genetic composition of the plantlets. Almond plantlets multiplied
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through axillary branching maintained their genetic integrity even after 4 and 6
years of in vitro multiplication (Martins et al. 2004). Similar was the case in long-
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term (70 months) tissue cultured silver birch (Ryynanen and Aronen 2005).
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(Bennici et al. 2004) which appears applicable even to vanilla since no genetic
variations were observed. Somaclonal variation has often been linked to the
source of explant and the method of culture. In the present study, axillary buds
have been used for micropropagation of vanilla and were found to be appropriate
explants for initiation of in vitro shoot cultures. Such explants are considered to
be a low-risk material, as organized meristems are proven to be more resistant to
genetic changes than un-organized callus (Ostry et al. 1994).
From the results obtained in the present study using RAPD and ISSR
markers it is apparent that there exists genetic homogeneity of vanilla plantlets
and therefore the present micropropagation system for vanilla appears practically
feasible and can be carried out for a considerable length of time (for at least 10
67
Genetic Fidelity and Advanced Micropropagation Techniques
years) without any risk of genetic instability. The present study of evaluating
different primers (RAPD and ISSR) for amplification of genomic DNA in vanilla
can also form a basis for the use of these primers for other genetic studies like
DNA fingerprinting.
2.4.2 Micropropagation
The culture medium kinetics with reference to pH shows that the pH drops down
after inoculation and stabilizes after first week and remains constant throughout.
The narrow change in pH due to autoclaving the medium is known and the change
in presence of explant (Escalona et al. 1999). Although vanilla shoots were
obtained from previous cultures, the trimming of unwanted tissues creating cut
ends probably results in leaching of sap. The initial sudden drop in pH may be
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due to the inoculation with acidic plant material. This would lead to a sudden
drop in the pH of the medium to 4.3, and its improvement and stabilization later at
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4.7 suggests that the ionic status is maintained and hence there could not be a
catastrophic effect of medium pH on the development of hyperhydricity syndrome
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(HHS). The later lower pH can be attributed to the preferential uptake of
ammonium ions in the medium in exchange to protons. In the medium containing
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both NO3ˉand NH4+ ions, preferential uptake of NH4+ ions leads to drop in pH
during early growth stage which increases NO3ˉ utilization and a gradual increase
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A rapid fall of the osmolarity of the medium noted between the 1st and 3rd
week of culture indicates high uptake of minerals as well as sugars and the steady
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Genetic Fidelity and Advanced Micropropagation Techniques
nutrient uptake is limited to the explant basal region only. CIS works the other
way where in the entire explant is well covered with liquid medium providing
more surface for nutrient uptake and less area for gas exchange and hence is
usually associated with hyperhydricity. PIS which provide enough exposure of
the explant to the nutrient medium along with good exposure to air for better O2
exchange invariably resulting in good elongation of shoots promoting pronounced
growth of aerial roots.
Vanilla micropropagation using nodal explants requires a period of 5-6
months to obtain plantlets in good numbers. Use of an intervening liquid medium
also requires at least 5 months for moderate rate of shoot multiplication (George
and Ravishankar 1997). Use of a single culture medium for initiation,
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multiplication, elongation and rooting was proposed by Kalimuthu et al. (2006)
and the entire protocol takes more than 15 weeks to obtain plantlets of 8-9 cms.
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Culturing of shoot tip and nodal explants for 10-12 weeks in MS medium
supplemented with BAP and coconut water yielded 9-10 shoots and 2-3
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vigorously growing shoots which elongated with expanded leaves and root
initials. Further culturing of such shoots in fresh medium with similar
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needs 4-5 weeks for shoot bud proliferation after initiation of shoot cultures from
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nodal explants and another 5 weeks for elongation of the shoots to a length of 9-
10 cms. Rooting could be achieved in 2-3 weeks and the total period required is
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Genetic Fidelity and Advanced Micropropagation Techniques
of liquid and semi-solid medium in the same culture vessel enhanced axillary bud
and shoot production in many plants (Molnar 1987). Use of successive semi-
solid and liquid media for establishing propagation systems have been reported for
Lilium hybrids (Simmonds and Cumming 1976). Shoots of vanilla cultivated in
CIS started developing HHS by the end of 2 WAI and were completely
hyperhydric by the end of 3 weeks. A similar observation was noted wherein
stunted growth and hyperhydricity of shoots occured in cultures liquid medium as
early as within 21 days after sub-culture (George and Ravishankar, 1997).
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normal growth and morphogenesis in CIS. On the other hand, in GrowtekTM
bioreactor, circular floating explant holder provides unique advantage to use it in
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agitated mode apart from providing support for the shoot culture for partial
immersion which allows better gaseous exchange compared to CIS. Perforations
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in the explant holder permits free access to nutrient medium. It was observed that
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plant material propagated by temporary immersion can perform better during the
acclimatization phase than those grown on semi-solid or in liquid media (Etienne
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and Berthouly 2002). There are other similar reports on the advantages of
GrowtekTM bioreactor (Dey 2005). Lesser physical stress seems to improve
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70
Genetic Fidelity and Advanced Micropropagation Techniques
(Krueger 1991). Shoots of radiate pine derived from temporary immersion system
were longer and of better quality than those obtained on agar medium (Aitken-
Christie and Jones 1987). Krueger et al. in 1991 reported that cultures that grew
in intermittent contact with the culture medium gave higher values for the shoot
multiplication rate, shoot weight and shoot length. Work on sugarcane with twin
flask system immersion system showed that it clearly stimulates shoot formation
and length (Lorenzo et al. 1998). Hyperhydricity in outer leaf sheaths of stem
portion immersed continuously in liquid medium with continuous bubble-aeration
in banana was reported by Alvard et al. 1993 which was successfully prevented
when cultured in temporary immersion system.
Immersion or continuous/intermittent contact period was reported to have
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a major influence on the quality of shoots. Longer contact periods and complete
immersion resulted in hyperhydricity development in coffee and rubber. Several
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immersions for shorter time (1 min) were found to be more appropriate in case of
coffee somatic embryos whereas several immersions with an immersion time of
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15 min resulted in glassy embryos (Berthouly and Etienne 2005). In case of
rubber, excessive time of immersion (15 min every 6 hours) induced
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serviceberry shoots but more frequent immersion of 5 min every 30 min resulted
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in hyperhydricity with highly translucent shoots with curled and thickened leaves
(Krueger et al. 1991). A similar observation has been made in the present study
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wherein immersion of the shoot cultures for lengthier period of time (45 min per
day) resulted in hyperhydricity problem which initiated in a period of 3 weeks
after inoculation. An immersion of microcuttings of Coffee arabica for a period
of 15 min every 6 hours showed no symptoms of hyperhydricity whereas the same
was not true for Coffee canephora which reveled hyperhydricity symptoms for the
same treatment. The sensitivity to the duration and frequency of immersion in
liquid medium may vary with species of same genera (Berthouly et al. 1995).
Immersion or contact period of 30 min for 3 times per day was found to be most
appropriate for vanilla shoot cultures which yielded dark green vigorous shoots
with better shoot elongation and biomass accumulation than the other treatments.
This can form basis for development of automated large scale micropropagation
71
Genetic Fidelity and Advanced Micropropagation Techniques
technique for vanilla. Based on the above results, the bioreactor used earlier
(Figure 2.9) was re-considered for a study with intermittent bathing of shoot
cultures. Immersion or contact period of 30 min for 3 times per day which was
found to be most congenial was provided and the shoot cultures were successfully
propagated with lesser hyperhydricity and excellent shoot multiplication and
elongation. Thus, an automated micropropagation system for large scale
multiplication of vanilla shoot cultures was achieved by developing a modified
bioreactor (Figure 2.3) for intermittent submergence of the shoot cultures. Apart
from the positive effects of TIS on multiplication and plant material quality, it also
provides an opportunity to reduce cost of production, thereby making the whole
process of micropropagation simpler.
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2.4.3 Greenhouse hardening and field performance studies
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An equi-proportion of red soil: sand: vermicompost was found to be the best
hardening soil medium for vanilla by the present study. Vanilla is reported to
thrive well in the soil with good amount of humus and sand and needs soil which
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provides good drainage for the excess water to drain down. The plants are
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sensitive to water logged conditions. A combination of red soil, sand and
vermicompost in equal proportion in perforated poly-bags provides an excellent
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growing substrate for the vanilla plantlets and aids maximum survival (90.5%)
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micropropagation were found to perform better than those from the stem cuttings.
They were early and high yielders. A comparative study using morphological and
biochemical tests of field established micropropagated and conventionally cutting-
derived plants of mulberry genotypes conducted reveled that the micropropagated
mulberry plants showed significantly better vigour than plants raised through
cuttings (Zaman et al. 1997). A similar observation was made even in this study
wherein the micropropagation derived plants were found to be more vigorous than
the stem cutting derived ones. In a study on field performance of blueberries
derived from softwood cuttings and from micropropagation, propagation methods
were found to exert significant influence on nursery and field performance.
Cutting-derived plants grew more slowly, produced significantly less and shorter
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Genetic Fidelity and Advanced Micropropagation Techniques
shoots and were more variable than micropropagated plants. However, the
majority of cutting-derived plants developed flowers one year earlier, flowered
more abundantly, bore significantly larger berries than tissue cultured plants.
Plants obtained through in vitro culture were more uniform than cutting-derived
plants for the number of inflorescences per plant. On the contrary, better yielding
of tissue cultured plants without deterioration of berry quality was reported for
half-highbush blueberry (El-Shiekh et al., 1996), and lingonberry (Gustavsson
1999). Thus such differences may be attributed to different effects of
micropropagation protocol on different plant species (Litwin´czuk et al. 2005).
The conventionally vegetatively propagated plants are known to carry latent
systemic infections, leading to low yields. Higher plant size with more
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pseudostem height and higher uniformity in fruit characteristics were observed in
banana plants derived from micropropagation compared to those from
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conventional method. Bunches from plants derived from micropropagation could
be harvested earlier by nearly a month than the conventionally cultivated ones
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(Msogoya et al. 2006). In the present study, micropropagation derived plants
started yielding earlier than the cutting derived ones which might be due to
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micropropagated plants may also be due to the well developed roots and leaves in
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2.5 Conclusion
The results from this study clearly indicated that the micropropagation protocol
followed for establishment and maintenance of shoot cultures is appropriate and
does not induce any genetic instability during long-term cultivation in vitro.
Through the use of partial immersion system in the form of GrowtekTM bioreactor
for micropropagation, plantlets for greenhouse hardening could be obtained within
a period of 12-14 weeks compared to 20-24 weeks required in conventional
methods with the advantage of cutting the cost of laborious sub-culturing in solid
medium. Moreover the plants derived from this micropropagation system were
found to outperform those from stem cuttings in both growth and yield
parameters. Operating conditions for a bioreactor with net support and temporary
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Genetic Fidelity and Advanced Micropropagation Techniques
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Hyperhydricity Syndrome of Vanilla
Summary
Shoot cultures of Vanilla (Vanilla planifolia) when transferred to liquid medium
from gelled solid medium showed a progressive increase in their watery
appearance resulting in hyperhydricity syndrome (HHS) which caused necrosis of
shoot buds. HHS in in vitro cultures is a major constraint and a generic problem in
cultured shoots. HHS hinders automation and large-scale production of plantlets.
HHS was also associated with severe damages at cellular and sub-cellular levels,
increase in free polyamines (PAs) and accumulation of water, and decrease in
quantities of chlorophyll, protein and drastic changes in reducing and non-
reducing sugars. Spermine was far the major polyamine in all the analyzed
cultures. The onset and progression towards HHS showed higher activities of
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antioxidant enzymes, indicative of shoots’ defensive efforts against oxidative
stress. The specific enzyme activities of normal and H2 stages respectively were
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342.6 and 350.35 U mg -1 protein for peroxidase (POD, EC 1.11.1.11), 38.4 and
30.38 U mg -1 protein for superoxide dismutase (SOD, EC 1.15.1.1) and 71.3 and
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-1
82.75 U mg protein for catalase (CAT, EC 1.11.1.6). The kinetic parameters of
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culture medium, suggested that nutrient utilization being normal in HHS, the
severe biochemical alterations and cellular damages mainly occur due to oxidative
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Polymerase Chain Reaction was employed to have information about the up/down
regulated genes during this disorder. Of the 114 HH-associated cDNAs identified,
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31 were cloned in T/A cloning vector and sequenced using M13 forward and
reverse primers. Electronic homology searches using BLASTX analysis resulted
in identification of 23 cDNA clones showing homology with various stress related
proteins like Zinc-finger like protein, gag-pol polyprotein, apoptosis related MAP
kinase activating protein, DNA replication related GANP protein, DNA repair
related DNA-binding SAP zinc finger. Endopolygalacturonase, pG1 protein,
Triglycerol lipase and Enolase responsible for carbohydrate breakdown, Biotin-
carboxylase having a role in fatty acid biosynthesis were also found to be
differentially expressed during HHS. BLASTN analysis yielded 18 fragments
having homology with different stress linked cDNA clones whilst the remainder
did not show any significant homology to known sequences. Quantitative reverse
75
Hyperhydricity Syndrome of Vanilla
transcriptase polymerase chain reaction analysis of selected genes indicated that there
was a more than 20-fold increase in the relative expression levels of genes having
homology to Zinc finger family protein, Enolase and MAP kinase activating
protein during the course of HHS. The relative transcript abundance level of clone
having homology with putative GANP protein showed a drastic reduction to a
0.06-fold in hyperhydric shoots compared to normal shoots indicating down-
regulation of this particular gene during HHS. A partially characterized
transcriptome of hyperhydric condition in Vanilla planifolia has been developed
which paves the way for a better insight into gene expression during this common
physiological disorder.
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Publication
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Sreedhar RV, Venkatachalam L, Bhagyalakshmi N (2009) Hyperhydricity-related
morphological and biochemical changes in Vanilla (Vanilla planifolia). Journal of
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Plant Growth Regulation DOI 10.1007/s00344-008-9073-4
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Hyperhydricity Syndrome of Vanilla
3.1 Introduction
Switching over to submerged cultivation is the first step towards automation of
shoot cultures production in a micropropagation industry. Initial trials for this are
normally conducted using shake-flask cultures, often known as complete
immersion system (CIS) as the shoots are continuously bathed in liquid medium.
During cultivation in vitro, the plantlets are exposed to a wide range of stress
conditions caused by high relative humidity, gas accumulation in the headspace,
altered nutrient/hormonal combinations and non-congenial osmoticity of the
culture medium. Although most plant cultures adapt to changes in environmental
conditions, some of them become abnormal with turgid, translucent, less green,
watery, hypo-lignified, wrinkled and brittle appearance. This phenomenon,
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known as hyperhydricity syndrome (HHS), can lead to irreversible loss of
multiplication as well as regenerative potential. HHS has also been a generic
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problem in continuous cultivation or scale-up of plant organs in vitro. Such a shift
towards hyperhydricity has been linked to various metabolic disorders, changed
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array of proteins and altered stress responsive pathways.
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Under stressful conditions cells undergo a surge of reactive oxygen species
(ROS) such as superoxide anion (O2-) and hydrogen peroxide (H2O2). The
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generation of ROS has been associated with oxidases in plasma membrane and the
electron transport of chloroplast and mitochondrion (Laloi et al. 2004, Ye et al.
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2006) and known to affect photosynthetic pigments, membranes and cell ultra-
structure (Xu et al. 2008). A higher concentration of intracellular ROS has been
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Hyperhydricity Syndrome of Vanilla
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the cells. This can be achieved by applying different techniques, such as RT-PCR
differential display, serial analysis of gene expression (SAGE), subtractive
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hybridization, and cDNA microarray. RT-PCR differential display (Liang and
Pardee, 1992, 1995) has been widely used to isolate genes whose expression
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profiles have been altered under different abiotic or biotic cues because of its
technical simplicity and lack of requirement for previous genomic information of
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the species of interest (Kuno et al. 2000; Carginale et al. 2004; Basse 2005; Lang
et al. 2005).
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rare enzymes that were previously difficult to purify. These results suggest that
differential display is a powerful tool used to investigate the rare genes involved
in the plant life cycle without using information from proteins. The stress response
genes affected by environmental factors such as ultraviolet (UV) light exposure,
extreme temperatures, oxygen, salt and desiccation was isolated using by DD and
characterized (Yamazaki and Saitom 2002).
Transcriptome analyses in apricot (Grimplet et al. 2005) and tomato (Alba
et al. 2005) have also been performed in an effort to get an insight into the vast
array of genes that may be involved in ripening. These studies have been
successful in identifying several novel genes related to fruit ripening whose action
in the ripening process was not obvious earlier. The isolation and characterization
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Hyperhydricity Syndrome of Vanilla
of the promoters of these novel genes could provide important cis-elements useful
for delayed ripening through targeted repression of ripening related genes in fruit
or for the expression of target proteins for value addition.
Vanilla planifolia is a member of Orchidaceae, and vanilla is popular for
its natural flavour prepared from the extract of carefully cured vanilla beans.
Although vanilla plants are propagated by cuttings, there is a large demand for
elite planting material produced via micropropagation technique. While
developing a protocol for micropropagation of vanilla, George and Ravishankar
(1997) studied the effects of various growth regulators as well as different culture
conditions, where a maximum number of shoots was formed in liquid medium,
and a higher number of shoots than in solid medium occurred in two-phase culture
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system. Long-term culturing of shoots in vitro and the use of genetic markers for
testing the clonal nature of plantlets thus produced have also been evaluated
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(Sreedhar et al. 2007a). Further for scale-up of shoot cultures in bioreactor, initial
trials in fully submerged cultivation of vanilla shoots in air-lift bioreactor (2-L
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capacity) resulted in rapid death and leaching of white exudates into the medium.
Therefore, a thorough investigation was considered aiming at acclimatizing the
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shoot cultures to liquid medium. The present study focuses on unraveling the
major structural, biochemical and molecular changes occurring in vanilla shoots
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grown in liquid medium. The study also records the changes in kinetics of
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nutrient utilization from the medium, measured in terms of pH, osmolarity and
conductance. To have a better insight on the molecular regulation of
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Hyperhydricity Syndrome of Vanilla
culture medium supplemented with 8.87 µM BAP and 2.69 µM NAA and
maintained at 252 oC under 16-h photoperiod having illumination of 37.5 µmol
photons m-2s-1 provided by fluorescent lamps.
Transferring to liquid medium involved the culturing of shoots in
Erlenmeyer flasks (150 ml) containing 40 ml of MS based medium supplemented
with 8.87 µM BAP and 2.69 µM NAA. Approximately 2.5 g of the plant material
was used as initial inoculum, which included 1 to 2 cluster(s) having 5 to 6 shoot
buds (of length 0.5-1 cm) each. The culture vessels were maintained at 252 oC
under 16-h photoperiod, having illumination of 37.5 µmol photons m-2s-1 on a
gyratory shaker set at 90 rpm throughout the culturing period of 5 weeks. The
cultures thus established were continuously bathed in liquid medium and termed
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as continuous immersion system. The shoots cultured on a solid medium having
composition as above but gelled with 7.2 g L-1 of agar (HiMedia, India) served as
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control. Observations in terms of morphological and biochemical changes were
made at various stages of HHS for 5 weeks.
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3.2.1.2 Morphological changes
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While the shoot multiplication was better in complete immersion system (CIS),
compared to solid system (Figure 3.1), the shoots in CIS displayed HHS towards
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the end of the culture period of 5 weeks and the four stages towards HHS are as
shown in Figure 3.2. The other drastic morphological changes occurring
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morphology and changes at tissue level. For latter purpose freehand sections of
about 0.5 to 1 mm thickness were made using new stainless steel razor blade.
The sections were mounted on a slide and observed under an inverted light
microscope (Leitz, LABOVERT, Ernst Leitz GmbH, Wetzlar, Germany) at 320×
magnification and the responses were documented through photomicrographs.
For SEM, the normal and hyperhydric cultures were processed according to
Fowke et al. (1994). The samples were fixed in 2% (v/v) glutaraldehyde in 0.2 M
phosphate buffer (pH 6.8)
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Hyperhydricity Syndrome of Vanilla
1 2
Figure 3.1 Shoot cultures of Vanilla planifolia cultured on (1) solid and (2)
complete immersion system (CIS) (liquid media) after 5 weeks of culture
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showing hyperhydricity and bud death
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1 2 3 4
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Figure 3.2 Shoots cultivated in normal and liquid medium showing various
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(H3 stage)
for 6 h, dried in alcohol series up to 100% (v/v), sputter coated with gold and
examined in a LEO Scanning Electron Microscope 435 VP (Leo Electron
Microscopy Ltd., Cambridge, UK).
3.2.1.3 Measurement of pH, conductance and osmolarity
The pH of the medium was adjusted to 5.8 prior to autoclaving and the changes in
the pH after autoclaving and after the placement of shoot bud inoculum were
monitored using digital pH meter (Control Dynamics, India) pre-calibrated with
buffer standards.
Osmolarity was measured in order to determine the level of total solutes,
both charged and neutral compounds in the medium, by using an automatic
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Hyperhydricity Syndrome of Vanilla
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(Lichtenthaler 1987) after measuring the absorbance of the acetone extracts at 645
and 661.5 nm.
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3.2.1.5 Carbohydrate content
Samples were repeatedly crushed in absolute ethanol and the alcohol solubles and
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insolubles were separated by filtration and vacuum evaporation of alcohol. Total
carbohydrate in each fraction was estimated by phenol sulphuric acid method
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values from the total sugar values. The alcohol insoluble material was dried and
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after recording the weight, a known weight was hydrolyzed using 72% (v/v)
H2SO4 keeping samples in ice bath. The mixture was appropriately diluted and
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Hyperhydricity Syndrome of Vanilla
of 1.0 ml min-1. The benzoyl-polyamines were eluted through a C18 column (300 ×
4.6 mm i.d., with pore size of 5 μM), an SLC-6A system controller, and a CR4A
data processor was used. Detection of eluted compounds was done by a UV
detector SPD-AV set at a sensitivity of 0.04 AUFC and absorbance at 254 nm. A
relative calibration procedure was used to determine the polyamines in the
samples, using data of standards – Put, Spd and Spm by comparing peak areas and
retention times. Results were expressed as nanomoles per gram of fresh weight.
Extractions from three different samples were made independently and each
extract was quantified in triplicate.
3.2.1.7 Protein content and antioxidant enzyme activity
Protein content of the plant material was estimated by Lowry’s method (Lowry et
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al. 1951). The plant material (1g each) for enzyme assay was extracted by
crushing using pestle and mortar with 10 ml of respective buffer and after
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homogenization, the homogenate was centrifuged at 12,000 rpm twice and the
supernatant was used.
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POD enzyme was estimated following the method explained earlier
(Sreedhar et al. 2007b). The material was extracted in sodium phosphate buffer
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described by Murthy et al. (2002). Units of SOD activity were expressed as the
amount of enzyme required to inhibit the reduction of NBT to 50%. The specific
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activity was expressed in terms of units mg-1 protein. CAT activity was
determined by adding sample extract in a 50 mM phosphate buffer (pH 7)
containing 18 mM H2O2 in a total volume of 3 mL. The consumption of H2O2 by
CAT was measured by the decrease in absorbance at 240 nm (ε = 39.4 mM cm-1)
at 25 oC (Beers and Sizer 1952).
Chlorophyll, carbohydrates, free polyamines and protein contents along
with enzyme activities were recorded initially, 1st, 3rd and 5th week after
inoculation in both normal(solid) and liquid cultures at respective moisture
contents.
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Hyperhydricity Syndrome of Vanilla
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of 0.25% (w/v) orthodianisidine dihydrochloride and 10 ml of 1% (v/v) hydrogen
peroxide and immediately photographed (Thimmaraju et al. 2007).
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3.2.2 Molecular Changes
3.2.2.1 RNA isolation
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Total RNA was extracted from shoot cultures of vanilla at four stages of HHS
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(Figure 3.3) for 5 weeks using RNAqueous® kit (Ambion, Austin, TX, USA)
according to the manufacturer’s protocol.
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The experimental model adopted for this study is given in Figure 3.3. The mRNA
Differential Display was performed using the RNAimage kit® (GenHunter
Corporation, Nashville, TN, USA) according to the manufacturer’s protocol
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provided. The analysis was conducted in four RNA samples using 0.2 µg of total
RNA from each independent sample. Briefly, poly(A)+–RNA (0.2 µg) was heated
at 65 C for 10 min and chilled on ice immediately. First-strand cDNA synthesis
was performed in a reaction mixture containing 50 mM Tris–HCl (pH 8.5), 50
mM KCl, 4 mM MgCl2, 10 mM DTT, 1mM each dNTP, 40 units of RiboLock
Ribonuclease inhibitor (MBI (Fermentas GmbH, St. Leon-Rot) and 40 units of H-
minus M-MuLV Reverse Transcriptase (MBI Fermentas) with 50 mM of different
anchor primer (Table 3.1) for 1 h at 42 C. The reaction was stopped by heating
the mixture at 70 C for 10 minutes and chilling immediately. Enzymes were then
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Hyperhydricity Syndrome of Vanilla
heat-denatured at 95 C for 5 min. Product thus obtained was used for PCR
amplification of the cDNAs.
Table 3.1 List of anchor primers and arbitrary primers used for Differential
Display
Anchor primers Arbitrary primers
Sl. No. Name of the Sequence
primer (5’-3’) Operon Primers:
1 DD1 (T)11A
OPA-03, OPC-08, OPD-11, OPJ-09,
2 DD2 (T)11G
OPM-16, OPN-04, OPN-06, OPN-10
3 DD3 (T)11C
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3.2.2.2.1 PCR amplification
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primers and each arbitrary primer in all possible combinations (Table 3.1). PCR
was performed using a thermal cycler (MWG peqlab, Germany) and the reaction
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parameters were as follows:
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a. Initial Denaturation : 94 °C for 4 min
b. Denaturation : 94 °C for 60 sec
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Hyperhydricity Syndrome of Vanilla
and immediately cooled on ice. Samples (12 µL) were loaded on pre-warmed
denaturing 6% polyacrylamide standard sequencing gel of 28:2 ratio
acrylamide:bisacrylamide, 7.5 M urea, 1X TBE buffer (89 mM Tris, 89 mM boric
acid, 2 mM EDTA; pH 8.0), then electrophoresed in 1X TBE buffer at 50W
constant power for about 2 h and 30 min; until the loading dye reached the bottom
of the gel. The gel was fixed by incubating the gel slab in fixation solution (2%
ethanol and 0.1% acetic acid) for 10 min with gentle shaking. Then the gel was
rinsed with distilled water for twice for 5 min each and incubated in staining
solution (chilled 0.2% AgNO3 prepared in fixation solution) for 20 min followed
by brief wash in double distilled water for 10 sec. The gel was developed with
developing solution (0.6% NaOH and 0.2% of 37% formaldehyde). The
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developing solution was discarded as soon as it turned yellow and was replaced
with a fresh portion. When a sufficient degree of staining has been obtained,
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developing solution was replaced with 5% acetic acid and the gel was washed
with distilled water.
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3.2.2.3 Elution of differentially expressed amplicons
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The gel slice containing differential DNA bands was excised using an aseptic
surgical sharp scalpel blade (no.11) fitted to handle. DNA was extracted using
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3.2.2.4 Re-amplification
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A-tailing: The PCR product was heated at 95 ○C for 20 min and 1µL of 2 mM
dATP per 10 µL of PCR product was added. This was incubated at 70 ○C for 15
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Hyperhydricity Syndrome of Vanilla
min. The mixture was purified using HiPura PCR clean-up kit (HiMedia, India)
before ligation.
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10X Ligase Buffer : 1.0 L
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T4 DNA Ligase, 5 U L-1 : 0.5 L
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Deionized water : vol. made up to 10.0 L
The reaction components were mixed by pipetting or a brief spin and the mixture
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was incubated at 16 °C for 4 hours. The enzyme was then inactivated by heating
the reaction mixture to 65 ○C for 10 min.
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Reagents
Luria-Bertani broth (LB) (components used per litre)
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Bacto-tryptone : 10 g
Bacto-yeast extract : 5g
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Sodium chloride : 10 g
The pH was adjusted to 7.0 with 2 N NaOH and the total volume was made up to 1
litre with deionized water.
SOB (components used per litre)
Bacto-tryptone : 20.0 g
Bacto-yeast extract : 5.0 g
Sodium chloride : 0.6 g
Potassium chloride : 0.19 g
Magnesium sulphate : 10.0 mM (added from 1.0 M stock)
Magnesium chloride : 10.0 mM (added from 1.0 M stock)
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Hyperhydricity Syndrome of Vanilla
The first four components and the magnesium salts were autoclaved separately and
mixed to constitute the SOB medium.
SOC (per 100 mL): To 1.0 mL of SOB, 7 L of filter-sterilized (Millipore, 0.4 μm)
glucose solution (50% w/v) was added to prepare SOC medium.
0.1 M CaCl2 stock solution: 1.47 g of CaCl2 was dissolved in 100 mL of deionized
water. The solution was sterilized by filtration and stored as 20 mL aliquots at -20
○
C.
Ampicillin stock solution: 100 mg of Ampicillin (Ranbaxy, India) was dissolved in
1.0 mL of deionized water and the solution was sterilized by filtration. It was stored
at 4 ○C and used at a working concentration of 100 g mL-1.
0.1 M IPTG stock solution: 0.12 g of IPTG was dissolved in 5 mL of deionized
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water and the solution was filter-sterilized and stored as aliquots at -20 ○C.
X-Gal stock solution: 100 mg of X-Gal was dissolved in 2 mL of N, N'-
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dimethylformamide (DMF) and the solution was stored in micro-centrifuge tube
wrapped in aluminium foil at -20○C.
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Hyperhydricity Syndrome of Vanilla
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Figure 3.3 The experimental model adopted for the differential display study
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Hyperhydricity Syndrome of Vanilla
A single colony of E. coli (DH5 strain) from freshly grown culture (at 37 ○C for
16-20 h) was picked and transferred into 50 mL of LB broth in a 250 mL conical
flask. The culture was incubated at 37 ○C with rigorous shaking. The OD600 of the
culture was determined periodically to monitor cell growth. When the OD600
reached 0.4-0.5, the cells were transferred aseptically to 50 mL sterile
polypropylene tube. The culture was cooled by storing the tube on ice for 10 min
and the cells were recovered by centrifugation at 4000 rpm for 8 min at 4 ○C. The
medium was decanted and the pellet was re-suspended in 10 mL of ice-cold 0.1 M
CaCl2 and was stored on ice. The cells were recovered by centrifugation at 4000
rpm for 10 min at 4 ○C. The fluid from the cell pellet was decanted and the tubes
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were kept in an inverted position for 1 min to allow the last traces of fluid to drain
away. The cell pellet was re-suspended in 10 mL of ice-cold 0.1 M CaCl2 and cells
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were stored at 4 ○C for 4-8 hours.
3.2.2.5.5 Transformation of competent cells
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About 200 L of the suspension of competent cells was added to sterile micro-
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centrifuge tubes along with the Plasmid DNA (~50 ng) or 4 L of ligation mixture.
The contents of the tubes were mixed by swirling gently and the tubes were stored
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on ice for 30 min. The samples represented (a) competent cells that received
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standard supercoiled plasmid DNA and (b) competent cells that received no plasmid
DNA. The tubes were transferred to water-bath set at 42 ○C for 90 sec to subject the
cells to heat shock. The tubes were then rapidly transferred to ice and the cells were
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allowed to chill for 45 sec. An 800 L of SOC medium was added to each tube and
the cultures were incubated at 37 ○C for 45 min.
About 100 L of transformation mix was plated onto LB agar plates containing 100
g mL-1 ampicillin, 0.5 mM IPTG and 80 g mL-1 X-Gal. The plates were
incubated overnight at 37 ○C for the colonies to grow.
Colonies harbouring the recombinant plasmid have a disrupted lacZ gene
and appeared white while non-recombinant vectors were blue. White colonies
were selected from the LB plate and screened by PCR for the presence of insert
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Hyperhydricity Syndrome of Vanilla
using the T3 and T7 primers flanking the cloning site. PCR amplifications were
performed using PCR mixture (250 μL) containing 25 μL of 10X PCR buffer, 5
μL of 10 mM dNTPs, 10 U of Taq DNA polymerase and 5 μM of each primer (T7
and T3 primers) where volume was made up to 250 μL using nuclease free water.
Transformed cells from white colonies were picked by sterile tips and were mixed
to the PCR mixture. PCR was performed at initial denaturation at 94 °C for 5
min, 30 cycles of: 30 sec at 94 °C; 30 sec 42 °C; 50 sec at 72 °C, and final
elongation for 10 min at 72 °C using a thermal cycler according to the
manufacturers protocol for pKRXT vector cloning and detection of transformants
(SBS Genotech, Beijing, China). The PCR products obtained were separated on
1.8% agarose gel, stained with ethidium bromide (0.001%), and documented. The
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size of the amplification products was estimated from the 100-bp DNA ladder
(Fermentas GmbH).
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3.2.2.5.7 Isolation of plasmid DNA from the transformed colonies
Plasmids from recombinant clones were isolated with the Qiagen mini prep
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Plasmid Mini Kit according to the manufacturers’ protocol.
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3.2.2.6 Sequencing of the clones
DNA sequencing was carried out by dideoxy chain termination method (Sanger et
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al. 1977). The reaction was carried out in an automatic DNA sequencer (ABI
prism, Applied Biosystems, USA) with fluorescent dideoxy chain terminators at
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Hyperhydricity Syndrome of Vanilla
and then precipitated in ethanol. One µL of total RNA (RNA from the stock 1µg
µL -1) was denatured with 2 volumes of denaturing buffer (1X MOPS containing
formamide and formaldehyde) by boiling for 5 minutes and immediately chilled
on ice and spotted to a Hybond-N nylon membranes (Ambion Inc., Austin, TX).
The RNA was fixed to the membrane by using a 302 nm ultraviolet cross linker
for 45 min. Then the membranes were pre-hybridized for more than 1 h in
ULTRAHyb buffer (Ambion Inc., Austin, TX) and hybridization was then
performed overnight with the same buffer containing the gene specific DIG-
labeled probe at 42 °C. Two selected clones VP2 (zinc finger family protein) and
VP9 (Putative GANP protein) one representing up-regulation and the other down-
regulation were assessed for regulation by RNA blots using total RNA isolated
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from four stages of hyperhydricity. The clone 18S (18S ribosomal RNA gene of
Vanilla planifolia) was used as a control. The hybridization, post-hybridization
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treatments and detection were done as follows:
Pre-hybridization
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The membranes with RNA blots were placed in a polythene bag containing 15 mL
pre-warmed (65 ○C) hybridization buffer and were sealed and incubated at 42 ○C
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Hybridization
A 5 L aliquot of probe for respective gene was heat-denatured by incubating in
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boiling water for 5 min, followed by snap cooling on ice and 2 L aliquot of
denatured probe was added to 5 mL of pre-warmed (65 ○C) hybridization buffer.
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Hybridization buffer containing the denatured probe was added to the polythene bag
containing the membrane and sealed and it was incubated at 42 ○C for 6 h with mild
agitation in a water bath.
Post-hybridization washes
The hybridization buffer was discarded and the membrane was washed twice in 50
mL of post hybridization washing buffer-I for 5 min at room temperature under
mild agitation. The membrane was again washed twice in 50 mL of post
hybridization washing buffer-II for 15 min at 65 ○C under mild agitation.
Detection
1. The membrane was rinsed briefly at room temperature in maleic acid buffer and
incubated in 50 mL 1X blocking solution for 30 min.
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Hyperhydricity Syndrome of Vanilla
2. The blocking solution was discarded and the membrane was incubated in 10 mL
of antibody solution (1:5000 Anti-DIG-AP conjugate in 1X blocking solution)
at room temperature for 45 min under mild agitation.
3. The membrane was then washed in 50 mL maleic acid buffer twice for 15 min
and incubated in 20 mL detection buffer for 5 min.
4. The detection buffer was discarded and the membrane was incubated overnight
in 10 mL freshly prepared color solution.
5. The membrane was kept in dark for color development.
6. The reaction was stopped by washing the membrane for 5 min with 50 mL of
deionized water. The results were documented by photography of the wet
membrane and the membrane was stored at 4 °C.
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3.2.2.8 Quantitative (relative) reverse transcriptase polymerase chain
reaction (qRT-PCR)
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Quantitative relative RT-PCR was used to confirm the differential expression of
DNA fragments isolated from normal and hyperhydric shoot cultures. The sample
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selection and RNA isolation was done as mentioned earlier. Specific
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oligonucleotide primers were designed for each DD product for RT-PCR (Table
3.2) and synthesized (Bioserve Biotechnologies Pvt. Ltd., Hyderabad, India). For
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amplification of the 18S rRNA sequence, primers from banana were used with
success as there was non-availability of 18S rRNA sequence in the GenBank
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Hyperhydricity Syndrome of Vanilla
products obtained were separated on 1.8% agarose gel, stained with ethidium
bromide (0.001%), and documented. The size of the amplification products was
estimated from the 100-bp DNA ladder (Fermentas GmbH). The band intensity of
each gel was checked using the Herolab E.A.S.Y Win 32 software. The transcript
levels of each gene in control shoots were taken for comparison in calculating the
transcript abundance of respective genes during hyperhydricity. Five genes
(Table 3.2) were selected based on their vital role played during various stress
responses for the qRT-PCR study.
Table 3.2 Gene specific primers and annealing temperatures used for RT-
PCR
Annealing
Primers Primer sequence (5’-3’)
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temperature (°C)
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VP2 Forward GACCGTCGAGTCGCTAAGAG
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VP2 Reverse GGTCGGTGCCTGTGTGTAT
VP9 Forward AGCCACCTCCTCCAGGTACT
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VP9 Reverse CAATGTGATCGCAAGGTGAG
VP11 Forward CGTTTAAGGAGGCCATGAAG
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61
VP11 Reverse TCCAATTACAACCTTGCCAGT
VP12 Forward CGGGAATTCGATTAGTCAGC
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60
VP12 Reverse ATCATTCCGGATAACGCTTG
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Hyperhydricity Syndrome of Vanilla
3.3 Results
3.3.1 Morphological and Biochemical changes
3.3.1.1 Morphological and ultra structural changes
Normal and HHS shoots of vanilla displayed significant differences, when leaves
and stem portions were observed by optical and scanning electron microscopy
(SEM). Normal vanilla shoots cultured on solid medium displayed xerophytic
morphology, with succulent waxy leaves. The moisture content was 88.8% in
normal shoot cultures, 90.3% in H1 stage cultures, 93.7% in H2 stage and 96.4%
in H3 stage cultures. Light microscopic observations of stem sections of H2
cultures revealed distinct degradation of vascular tissues and lesser degradation of
cortical tissues, reduction in size of the cortical cells and the hypertrophy of
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tissues inside the vascular cylinder. Degradation of the endodermal cells was
noted in the hyperhydric shoots (Figure 3.4), whereas the leaf sections showed
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higher degradation of vascular bundles, loss of compactness of palisade
parenchyma with abnormal enlargement and more intercellular space (Figure
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3.5). The scanning electron microscopy of stem surface revealed uniform
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cuticular ridges in normal cultures and scattering in hyperhydric cultures (Figure
3.6A). Vascular tissue was severely collapsed in hyperhydric shoot cultures
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(Figure 3.6B & 3.6C). Stomata showed regular structure in normal leaf and
lacked closure mechanism in hyperhydric leaf (Figure 3.6D).
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The pattern of variation in key media characteristics like pH, osmolarity and
conductance during the course of HHS development is presented in Figure 3.7. A
sudden fall in the pH of the medium from 5.7 to 4.3 was observed just after
inoculation (JAI) of the shoot cultures, which stabilized within a week attaining an
equilibrium pH of 4.7 throughout the culturing period of five weeks (Figure 3.7).
Omolarity of the medium, which was initially 0.225 Osmol kg-1, showed a steady
decline to a level of 0.069 Osmol kg-1 by the end of culturing period with a steep
fall between 2nd and 3rd week (Figure 3.7). The medium conductance, which
reflects the electrolyte concentration, showed an initial value of 6.4 mS. There
was a steady decline to a level of 4.05 mS by the end of culturing period
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Hyperhydricity Syndrome of Vanilla
indicating a slow and continuous uptake of the nutrients by the shoot cultures
(Figure 3.7).
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Normal Hyperhydric
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Figure 3.4 Cross-sections of stem in normal and hyperhydric (stage H2) shoot
cultures of vanilla. Vascular tissue (white arrows) and endodermis (black
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arrows) of stem C
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Normal
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Hyperhydric
Figure 3.5 Cross-sections of leaf in normal and hyperhydric (stage H2) shoot
cultures of vanilla. Vascular tissue (white arrows) and palisade parenchyma
(black arrows) of leaf
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Hyperhydricity Syndrome of Vanilla
Normal Hyperhydric
A
10 µm 10 µm
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C FT
100 µm 100 µm
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10 µm 10 µm
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Hyperhydricity Syndrome of Vanilla
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Figure 3.7 Kinetic parameters of the medium used for culture of vanilla
shoots. pH, osmolarity, and conductance in complete immersion system
(CIS). BI: before inoculation; JAI: just after inoculation; WAI: weeks after
inoculation. Data presented as mean of five replicates. Means with different
letters are significantly different at p0.05, according to Duncan’s multiple-
range test
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Hyperhydricity Syndrome of Vanilla
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was highest in normal shoots whereas the total insoluble sugar was highest in both
normal and stage H1 cultures. Hyperhydric shoot cultures had significantly lower
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concentrations of soluble reducing sugars compared to the normal shoots. Total
soluble and soluble reducing sugars were very low in stage H3 cultures. Highest
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amount of reducing sugars was found in the insoluble fraction in H1 stage.
However, normal cultures showed highest content of non-reducing sugars in the
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significantly sharp increase in the concentration of all the three PAs was noted in
stage H2 cultures. A sudden increase in the concentration of Put was found in
cultures of stage H2 from base level in normal and H1 stage cultures. However, it
showed a pitfall in H3 stage cultures.
Almost a 1.8-fold increase in Spd content was observed in H2 compared to
normal and H1 stage, which dropped in H3 stage. Spm was high in the normal
cultures, with a significant decline at H1 and steeply increased at H2 stage. The
increase in Spm concentration was to an extent of 1.6-fold in H2 stage cultures
compared to H1 stage shoots. It again declined to nearly half of its concentration
from H2 to H3 stage.
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Hyperhydricity Syndrome of Vanilla
Table 3.3 Changes in the levels of chlorophyll and carbohydrates in normal (N) (shoots cultivated on solid-medium)
and hyperhydric shoot cultures of vanilla
Chlorophyll (% FW) Soluble sugars (mg g-1 FW) Insoluble sugars (mg g-1 FW)
Stage Total Chlorophyll-a Chlorophyll-b Total Reducing Non- Total Reducing Non-
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sugars reducing sugars reducing
sugars sugars
N† 0.0057±0.0005a 0.0040±0.0003a 0.0017±0.0002a 13.45±1.24a 8.86±0.34a 4.58±0.13a 4.55±0.2a 2.94±0.18b 1.6±0.04a
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H1 0.0051±0.0005a 0.0034±0.0003b 0.0018±0.0002a 10.04±0.5b 6.01±0.22b 4.04±0.3a 4.84±0.21a 4.11±0.2a 0.73±0.07b
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H3 0.0003±0c 0.0002±0d 0.0001±0c 2.78±0.21d 2.15±0.15d 0.63±0.06c 1.46±0.08c 1.1±0.1c 0.37±0.02c
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Hyperhydric shoots: 1 week after inoculation (H1), 3 weeks after inoculation (H2) and 5 weeks after inoculation (H3)
†
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No significant differences in the chlorophyll and carbohydrate contents were noticed among solid-medium cultivated shoot
cultures of initial, 1st, 3rd and 5th weeks of culturing.
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Data presented as mean±SD of five replicates. Means with different letters are significantly different at p≤0.05,
according to Duncan’s Multiple Range Test
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Hyperhydricity Syndrome of Vanilla
Table 3.4 Levels of free polyamines (Putrescine (Put), Spermidine (Spd) and Spermine (Spm)) in solid-medium and
CIS cultured shoots
Put (nmol g-1 FW) Spd (nmol g-1 FW) Spm (nmol g-1 FW)
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Solid CIS Solid CIS Solid CIS
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2. 16.9±1.91a 9.05±0.91b 17.93±0.99a 18.64±1.76b 1985.4±89.12a 1291.77±122.34b
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4. 14.68±1.17a 64.5±6.22b 20.1±1.99a 8.48±0.88c 1947.36±34.88a 1087.63±104.7b
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Solid-medium cultured shoots: (1) 0 weeks after inoculation (N), (2) 1 week after inoculation, (3) 3 weeks after inoculation and (4) 5
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weeks after inoculation; CIS cultured shoots: (1) 0 weeks after inoculation (N), (2) 1 week after inoculation (H1 stage), (3) 3 weeks
after inoculation (H2 stage) and (4) 5 weeks after inoculation (H3 stage).
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Data presented as mean±SD of triplicates. Means with different letters are significantly different at p≤0.05, according to Duncan’s
Multiple Range Test
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Hyperhydricity Syndrome of Vanilla
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showed three isoforms in both normal and HH shoot cultures with a significant
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difference between the two, i.e., the bands of H2 stage cultures showed high intensity
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indicative of high activity whereas those of normal shoots (N) showed faint activity.
SOD: The activity of SOD was highest in normal and H1 stage cultures with a
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significant loss in H2 stage cultures. A substantial drop in the SOD activity was
noted (nearly 6-fold) (6.01 U mg-1 protein) in H3 stage cultures compared to normal
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82.75 U mg-1 protein in H2 stage cultures with more than 1.4-fold increase in the
activity compared to H1 stage (58.04 U mg-1 protein). However, there was a steep fall
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(more than 2-fold) in its activity in H3 stage cultures (38.48 U mg-1 protein)
compared to H2 stage cultures (Figure 3.8).
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Hyperhydricity Syndrome of Vanilla
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Figure 3.8 Protein content and activity of antioxidant enzymes in normal (shoots
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cultured in solid medium) and hyperhydric shoot cultures: shoots from CIS 1
week after inoculation (H1 stage), shoots from CIS 3 weeks after inoculation (H2
stage), and shoots from CIS 5 weeks after inoculation (H3 stage). Data presented
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as mean of five replicates. Means with different letters are significantly different
at p0.05, according to Duncan’s multiple-range test. † No significant differences
in the protein content and activity of antioxidant enzymes were noticed among
solid-medium cultivated shoot cultures of initial, first, third, and fifth week of
culturing.
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Hyperhydricity Syndrome of Vanilla
N N H2 H2
P1 P1
P2 P2
P3 P3
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normal shoots and H2: of hyperhydric (stage H2, shoots from complete
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immersion system 3 weeks after inoculation) shoot cultures. P1, P2, and P3
represent various isoforms of PODs
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In the present study, mRNA Differential Display technique was employed by the
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means of RNAimage kit® (GenHunter Corporation, Nashville, TN, USA) as a general
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strategy to obtain fragments of genes differentially expressed under hyperhydric
stress condition. This technique amplifies cDNA sequences from subsets of mRNAs
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by reverse transcription and PCR. Total RNA was isolated from four different stages
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of shoot cultures and was used for cDNA synthesis. The use of 27 primer
combinations (Table 3.1) in the four RNA populations resulted in 114 distinct
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scorable fragments in the silver-stained polyacrylamide gels within the scorable area
of 100 to 1000 bp. Thirty one fragments were selected based on differential display
pattern of either present/absent or of higher/lower intensity (Figure 3.10). These
were eluted from the gel and were re-amplified using the same primer combinations
as used earlier for amplification of respective cDNA fragment after reverse
transcription. Most of the candidate bands generated single target PCR product which
corresponded to the size observed in the original DD gel. The remaining candidate
bands produced more than one PCR product from which the target PCR product
appeared more intense than the non target one. These single bands and the intense
bands were selected for cloning and sequence analysis. Initially, the up-regulation
and down-regulation (differential expression) of the genes was screened and
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Hyperhydricity Syndrome of Vanilla
documented based on the variation in intensity levels of the selected candidate bands,
which were eluted from the DD gel (Table 3.5). Later Northern dot-blot analysis was
performed for selected clones to confirm the differential expression which was
followed by analysis of the expression levels of important genes by qRT-PCR.
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Figure 3.10 Differentially expressed bands visualized on polyacrylamide gel
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(M: Protein marker; 1, 2, 3, 4: PCR amplified products of different cDNAs)
Table 3.5 List of the isolated bands, their size and expression based on their
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Hyperhydricity Syndrome of Vanilla
3.3.2.1 Cloning
All the selected re-amplified amplicons were cloned into pKRX-T cloning system
which takes advantage of the ability of Taq polymerase to add a terminal
3’deoxyadenosine during the PCR. These vectors contain a 3’T overhang at the
cloning site, allowing sticky end ligation which is more successful than blunt end
ligation. However, the resolution of the re-amplified PCR products on a 1.5% agarose
gel was insufficient to detect 5-20 bp differences and multiple products were not
observed until cloned fragments were amplified using the universal sequencing T3
and T7 primers.
3.3.2.2 PCR analysis to confirm cloning
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Both the T3 and T7 primers anneal to sites in the pKRXT vector 60 bp upstream or
downstream respectively from the cloning site. Therefore, colonies that contained
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plasmids with inserts gave a band of 160 plus the size of the insert. Based on the PCR
results, clones with the correct insert size were selected. All the 31 cloning and
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transformation reactions yielded recombinant clones from which the plasmid DNA
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was isolated for sequencing.
3.3.2.3 Sequence analyses of cDNA fragments
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Clones from all the 31 cDNA fragments were sequenced using both T3 forward as
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close to the estimated size from the original display from which these fragments were
obtained. All the cDNA fragments were amplified with their respective arbitrary
primers on both the 5’ and 3’ ends as determined by sequencing. Nucelotide
sequences of these cloned cDNA fragments are presented in the Appendix.
3.3.2.4 BLASTN analysis
Analysis of the 31 sequences of the cloned products for homology with the EST
sequences available in the genetic data base resulted in four groups on their functional
similarity (Table 3.6). They are as follows:
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Hyperhydricity Syndrome of Vanilla
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and other biotic and abiotic stress.
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Group II. Biotic Stress
FT
The five cloned sequences of this group were detected to share homologies with the
genes expressed during various biotic stresses. While three of them had sequence
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similarity with the cDNA library of the wild leaf response of Zingiber species to
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Phythium aphanidermatum (VP14, VP18 and VP19), the other two had homology
with the cDNA library of the soybean disease resistance (VP13) and Citrus sinensis
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The third group included seven clones homolog to different cDNA libraries. Two of
them (VP9 and VP10) were found related to genes expressed during tillering stage of
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Oryza sativa. Another two sequences (VP16 and VP30) showed homology with
cDNA library associated with callus induction in oil palm and Citrus sinensis. The
clone VP3 showed significant homology with genes encoding non-stressed condition
in Musa while the clone VP7 showed homology with cDNA induced in banana by
ethylene treatment. Clone VP27 had homology with EST sequence of developing
shoot buds of Actinidia deliciosa.
Group IV. Novel sequences
This group is constituted by the sequences which were novel and had no homology
with the genetic database available. A total of six clones were found in this category.
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Hyperhydricity Syndrome of Vanilla
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Group II. Metabolism
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Eight sequences were found to make this group in which three clones (VP5, VP12
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and VP26) showed homology with pG1 protein having endopolygalacturonase
activity. The clone VP11 had homology with gene coding for enoloase which has a
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major role in the glycolytic pathway. Clone VP15 showed homology to gene
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encoding triacylglycerol lipase enzyme involved in carbohydrate breakdown. The
clone VP17 showed similarity to alpha-gliadin protein encoding gene while VP22 had
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sequence similarity for Golgi complex component Cog3 gene. VP28 displayed
sequence homology with biotin-carboxylase encoding gene having a role in fatty acid
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biosynthesis.
Group III. DNA replication and repair
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The third group constitutes of sequences which showed homology with the genes
coding for proteins and enzymes having a major role in DNA replication and repair.
While the first two (VP9 and VP10) sequences shared similarity with putative GNAP
protein encoding genes involved in DNA replication, the next two (VP18 and VP19)
had homology with the genes coding for transposase which is involved in DNA
transposition. The other clone (VP21) displayed similarity with genes coding DNA
binding SAP related to chromosomal organization and DNA repair. The clone VP24
was closely related to spliceosome-associated factor gene which splices hnRNA and
clone VP29 had good homology with transcriptional regulator coding gene.
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Hyperhydricity Syndrome of Vanilla
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functions are yet to be arrived at.
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Hyperhydricity Syndrome of Vanilla
Table 3.6 Homology search results for differentially expressed cDNAs during hyperhydricity syndrome of vanilla:
BLASTN analysis
Fragment Accession Size Homologue with highest homology in the genetic Significance
ID No. (bp) database E score; identities (%)
EST-cDNA library Acc. No.
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Group I. Abiotic Stress
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VP1 GE746280 250 Emiliania huxleyi grown on phosphate GE205735.1 0.006; 75%
starved media
VP2 GE746281 392 Ethiolated Panicum virgatum seedlings FL901957.1 3e-16; 68%
FT
VP5 GE746284 254 Tamarix hispida leaves at different stress EH055866.1 6e-117; 97%
times
VP6 GE746285 147 Light stressed Haematococcus pluvialis GE649945.1 5.6; 100%
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VP8 GE746287 358 Wheat during abiotic stress CK163419.1 0.37; 86%
VP11 GE746290 264 Salt stressed Fragaria vesca EX663966.1 8e-64; 85%
VP12 GE746291 345 Salt stressed Oryza sativa EX451515.1 1e-121; 97%
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VP21 GE746300 684 Abiotic and biotic stressed Panicum GD019023.1 2e-13; 83%
virgatum
VP22 GE746301 615 Nitrogen deprived Neurospora crassa GH152236.1 8.2; 93%
VP24
VP25
GE746303
GE746304
729
420
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Glucose deprived Neurospora crassa
Nutrient deprived roots of Tomato
Salt stressed Fragaria vesca
GH122179.1
BF096278.1
EX657176
8.2; 93%
9.8; 84%
0; 96%
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VP26 GE746305 506 Heat stressed Fragaria vesca EX673998.1 0; 96%
VP31 GE746310 376 Water stressed Glycine max CX705303.1 5e-06; 66%
Group II. Biotic Stress
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VP13 GE746292 216 Soybean disease resistance cDNA library CV998036.1 6e-08; 97%
VP14 GE746293 309 Zingiber species wild leaf response to ES560490.1 5e-17; 98%
Phythium aphanidermatum
VP18 GE746297 218 Zingiber species wild leaf response to ES560490.1 7e-19; 98%
Phythium aphanidermatum
VP19 GE746298 361 Zingiber species wild leaf response to ES560490.1 6e-17; 98%
Phythium aphanidermatum
VP28 GE746307 537 Citrus sinensis leaf affected by Xylella EY665891.1 1e-09; 76%
fastidiosa
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Hyperhydricity Syndrome of Vanilla
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VP30 GE746309 416 Callus of Citrus sinensis CV717662.1 4e-40; 90%
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Group III. Novel sequences
VP4 GE746283 285 NA
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VP15 GE746294 175 NA
VP17 GE746296 241 NA
VP20 GE746299 771 NA
VP23 GE746302 506 NA
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VP29 GE746308 533 NA
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Hyperhydricity Syndrome of Vanilla
Table 3.7 Homology search results for differentially expressed cDNAs during hyperhydricity syndrome of vanilla:
BLASTX analysis
Fragment ID Accession Size Homologue with highest homology in the genetic database Significance
No. (bp) E score;
identities (%)
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Protein homologue Putative Acc. No.
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function
Group I. Stress
VP2 GE746281 392 Zinc finger family Oxidative stress NP850020.1 6e-19; 50%
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protein response
VP14 GE746293 309 Truncated disease Disease CAP66362.1 3.4; 100%
resistance protein of resistance
Musa
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VP16 GE746295 324 Gag-pol polyprotein Stress resistance AAR13317.1 4e-35; 62%
Group II. Metabolism
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VP5 GE746284 254 pG1 protein Endopolygalactu AA066461.1 3e-14; 61%
-ronase
VP11 GE746290 264 Enolase Glycolytic AAQ77241.1 8e-37; 91%
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Hyperhydricity Syndrome of Vanilla
DNA segmants
VP21 GE746300 684 DNA binding SAP Chromosomal ABD33066.1 3e-08; 34%
organization,
DNA repair
VP24 GE746303 729 Spliceosome- Splice hnRNA XP954790.1 0.1; 28%
associated factor
VP29 GE746308 533 Transcriptional Transcription ZP02620321.1 5e-04; 22%
regulator regulation
Group IV. Ungrouped sequences
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VP1 GE746280 250 phi-1 like protein Phosphorylation, AAM08535.1 8e-05; 56%
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cell division
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VP20 GE746299 771 Oxidoreductase domain Oxidation- YP821867.1 9e-81; 57%
protein reduction
VP30 GE746309 416 MAP kinase activating Apoptosis BAD61807.1 1e-19; 45%
protein
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Group IV. Novel sequences
VP3 GE746282 239 NA
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VP4 GE746283 285 NA
VP6 GE746285 147 NA
VP7 GE746286 115 NA
VP8 GE746287 358 NA
VP13
VP23
GE746292
GE746302 ts
216
506
NA
NA
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VP25 GE746304 420 NA
VP27 GE746306 557 NA
VP31 GE746310 376 NA
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Hyperhydricity Syndrome of Vanilla
VP2
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VP9
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18S
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Each band was normalized against the intensity obtained with the same cDNA using
the internal 18S primers of Vanilla planifolia. All the comparisons of the abundance
for the transcripts were made with the abundance in normal shoot cultures. There
was a more that 20-fold increase in the relative transcript abundance level of VP2
clone corresponding to zinc finger family protein in the H2 stage of HHS and a
relatively higher level of 19-fold was maintained in the H3 stage compared to the
level in normal shoots (Figure 3.12A). The relative transcript abundance level of
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Hyperhydricity Syndrome of Vanilla
VP9 (putative GANP protein) was highest to an extent of 1.46-fold in the H1 stage
which showed a drastic reduction to a 0.06-fold compared to normal shoots indicating
down-regulation of this particular gene during HHS (Figure 3.12B). A slight but
significant up-regulation of clone VP12 (pG1 protein) was observed as HHS
progressed (Figure 3.12C). A near 20-fold increase in the relative expression level
of VP11 (Enolase) in the H2 and H3 stage cultures was seen compared to normal
shoots (Figure 3.12D). Clone VP30 (MAP kinase activating protein) showed an
increase of nearly 22-fold in H2 stage and 17.5-fold in H3 stage cultures (Figure
3.12E).
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Hyperhydricity Syndrome of Vanilla
25
A 20
15
10
0
N H1 H2 H3
1.8
B 1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
N H1 H2 H3
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2
C 1.8
1.6
1.4
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1.2
1
0.8
0.6
0.4
0.2
0
N
CH1 H2 H3
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25
D 20
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15
10
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0
N H1 H2 H3
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25
E 20
15
10
0
N H1 H2 H3
18S
rRNA
Figure 3.12 Expression and relative transcript abundance of different hyperhydricity
related genes. A: Zinc finger family protein (VP2); B: Putative GANP protein (VP9);
C: pG1 protein (VP12); D: Enolase (VP11); E: MAP kinase activating protein (VP30)
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Hyperhydricity Syndrome of Vanilla
3.4 Discussion
3.4.1 Morphological and Biochemical changes
Vanilla planifolia normally displays xerophytic characteristics and therefore, one can
expect the difficulty in growing this plant in submerged (hydrophytic) conditions.
However, as in most of the other shoot cultures, which easily adapt to agitated
medium, the vanilla shoots were also expected to grow and multiply better in CIS.
Although shoot number increased in CIS as expected (Figure 3.1), the shoots
underwent severe hyperhydricity conditions leading to HHS (Figure 3.2), where
progressive accumulation of water was evident. In other plants where hyperhydricity
conditions are reported, anatomically there was hypertrophy of the cortex and pith,
I
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and parenchyma displayed large intercellular spaces (Hazarika 2006). Even in case
of vanilla with HHS, there was a general loss of cellular integrity and pronounced
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degradation of vascular tissues. Severe structural damage was observed at the surface
of the leaves. The endodermis, palisade and vascular tissues were found severely
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collapsed (Figure 3.4 & 3.5). The conducting vessels, particularly xylem and
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tracheids in normal plants displayed beautiful architecture of secondary walls in
xylary tissues whereas the vasculatures of HHS appeared decomposed (Figure 3.4,
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3.5 & 3.6). Vascular bundles lacked the typical arrangement as found in normal
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in vitro plantlets do not have a closure mechanism, a cause linked to water loss and
death of plantlets during acclimatization under low relative humidity (Hazarika
2006). Contrarily, vanilla plants in vitro displayed structurally normal stomata
(Figure 3.6D). Regarding the induction of HHS stage, the significant changes in the
carbohydrates indicate the structural degradation of cell walls.
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Hyperhydricity Syndrome of Vanilla
stabilization later at 4.7 suggests that the ionic status is maintained and hence there
could not be a catastrophic effect of medium pH on the development of HHS. A
rapid fall of the osmolarity of the medium noted between the 1st and 3rd week of
culture indicates high uptake of minerals as well as sugars and the steady decline in
conductance reflects the progressive uptake of ions (minerals). These trends
inversely correlate with the increase in shoot growth and accumulation of biomass
(data not shown). The lesser rate of change in osmolarity and conductance from 3rd
week onwards indicates poor utilization of nutrients. Another possibility could be
saturation of the water/nutrient uptake system.
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are known to play important role in various cellular processes (Bais and Ravishankar
2002). PAs have been implicated in direct scavenging of free radicals, thereby
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reducing oxidative effects. They may also act indirectly by elevating the levels of
antioxidants. Put is known to suppress the level of superoxide and H2O2 in leaf of
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stressed plants. The chilling induced H2O2 production was found inhibited in
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cucumber seedlings after Spd pretreatment (Shen et al. 2000). Spm (a tetramine), Spd
(a triamine) and their precursor Put (a diamine) are known to play a major role in
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assisting the plants and their cells/tissues to adapt to stressful conditions through
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although both are derived from Put showing a particular dynamics of free PAs. Such
stress-dependant increase in the levels of Spm has also been observed in other studies
(Kumar and Rajam 2004; Silveira et al. 2006). A similar trend was observed even in
the case of banana cultures with progressive subcultures (Venkatachalam and
Bhagyalakshmi 2008). The control vanilla shoot cultures (in solid medium) did not
show significant variations in the contents of PAs during the culture of 5 weeks;
contrarily our study with banana cultures showed a slow accumulation of free PAs
during 12 weeks period (Venkatachalam and Bhagyalakshmi 2008). In the present
study, the cultures in CIS displayed the accumulation of free PAs, Spm, Put and Spd
increasing substantially during HHS progression. The H3 stage where shoots had
attained HHS, the levels of Spd and Put decreased and most of the proteins and
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Hyperhydricity Syndrome of Vanilla
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oxidative injury to stressed tissue could be reduced by exogenous application of
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respective PAs leading to concomitant increase in the endogenous levels of respective
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PAs, suggesting their direct or indirect role as antioxidants.
Reduction in the chlorophyll content was one of the earliest symptoms
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observable with the onset of HHS (Figure 3.2), which probably serve as an early
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marker for HHS. The changes in the levels of reducing and non-reducing sugars are
expected to occur due to hydrolysis of polysaccharides as well as inter-conversions of
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soluble sugars as happens in ripening fruits with higher water activity. The decrease
in soluble sugars at H3 stage may be due to their further degradation forming
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uronides. However, since there is no drastic change either in the pH of the medium,
its conductance or osmolarity, the degradative products may be held within the
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Hyperhydricity Syndrome of Vanilla
HHS is indicative of oxidative stress in vanilla shoot cultures. Among the three
enzymes, SOD appears the foremost one to defend against the injury caused by ROS,
catalyzing the dismutation of O2– to H2O2 and molecular oxygen. The H2O2 produced
is then scavenged by several classes of PODs, the activity of which is also high at H1
stage. PODs are homoproteins present as multiple isozymes in plant tissues and are
distributed throughout the cell carrying the function of catalyzing the reduction of
H2O2 to H2O. They belong to a large family of enzymes, having the ability to
oxidize/reduce different substrates in the presence of H2O2 and thus maintain redox
status of the cell. While the activity of POD was highest in vanilla cultures, the
simultaneous increase in the activity of this enzyme and of CAT in the hyperhydric
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cultures at H3 stage indicates their good orchestrated effects in scavenging H2O2 as
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well as other free radicals. It is also known that PODs exist as a large group of
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isoenzymes with an extreme range of isoelectric points, serving a multitude of
functions. Each group is thought to have a different function in the cell. Acidic
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(anodic) isoenzymes of POD are known for their involvement in growth and
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differentiation of cells, the basic forms are assumed to provide H2O2 for other PODs
(Gulen and Eris 2004). In the present study the zymograms of POD from normal and
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Earlier studies have indicated both physical and chemical methods for the
reversal and control of hyperhydricity. Among the physical methods, measures like
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flushing out ethylene, CO2 and other volatile components accumulated in the
headspaces of culture vessels appear essential to contain HHS (Lai et al. 2005).
Avoiding the complete submergence of shoots has also been suggested for which, the
use of temporary immersion systems providing intermittent contact between the plant
material and the liquid culture medium have been proposed (Etienne and Berthouly
2002). In case of vanilla, our earlier studies have shown that despite 10 years of
maintenance in solid medium (Sreedhar et al. 2007a) or any increase or decrease of
cytokinin concentration in solid medium (George and Ravishankar 1997), the
induction of HHS did not occur. Our present study shows that liquid medium in
particular induces HHS indicating that water potential plays an important role in the
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Hyperhydricity Syndrome of Vanilla
induction of HHS. However, in case of Vitis vinifera (cv. Albarino) although the
increase in cytokinin level enhanced HHS condition, the addition of liquid phase to
solid medium further increased HHS (Couselo et al. 2006). It is worth noting that our
present study does not involve any change in growth regulator, where the level of
cytokinin is constant both in liquid and solid medium. Agar at a concentration of
0.8% was found to be the best gelling agent to avoid hyperhydricity as lower
concentrations clearly induced hyperhydricity. A double-phase culture (using liquid
and gelling agent solidified culture system) was also proposed to obtain higher
biomass and shoot number compared to solid medium in Japanese pear (Kadota et al.
2001). However, as mentioned above, this aggravated HHS condition in Vitis vinifera
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(cv. Albarino) (Couselo et al. 2006). Among the chemicals, activated charcoal,
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phloridzin and paclobutrazol have been used for specific shoot cultures (George
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1996).
3.4.2 Molecular Changes
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To obtain comprehensive information on gene expression pattern a more effective
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and straightforward strategy would be the genomic approach where isolation of
numerous genes and their characterization on the basis of homologies and similarities
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Hyperhydricity Syndrome of Vanilla
database. The abiotic stress seems to have developed as a result of higher availability
of various salts and other nutrients in the liquid culture medium during cultivation of
the shoot cultures as depicted by variation in the patterns of expression of genes
involved in salt and nutrient stresses (Table 3.6). In view of the over expression of
carbohydrate hydrolases, one may expect influx of salts causing ionic imbalance and
expression of salt-stress-responsive genes. The development of callus in the
hyperhydric shoot cultures was also observable which was complemented by an
increase in expression levels of the genes related to growth response (VP27 and
VP30, Table 3.5 & 3.6), especially re-induced type.
BLASTX analysis reveled that majority of cDNAs (10) did not share protein
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homology with any of the entries in the GenBank database with only three of them
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sharing homology with proteins associated with stress response.
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Group I. Stress
Zinc-finger proteins have been reported to be involved in regulation of gene
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expression and RNA metabolism by direct associations with respective cognate target
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promoters. Some zinc-finger proteins have a role in defence or senescence regulation
while many of other identified proteins have no function associated to them. A zinc-
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finger transcription factor is known to increase during tomato ripening together with
other transcriptional factors, including MADS box genes, homebox genes and
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polycomb genes (Bartley and Ishida 2002). In pepper (Capsicum annum L.), a zinc-
finger protein gene was found to express in red fruit but was undetectable in the green
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ones (Kim et al. 2004). This gene was also proposed to function as an early defense
gene against pathogen (Kim et al. 2004). A zinc-finger identified by Luis and
Cristina (2007) had high homology to proteins having putative role in responses to a
variety of abiotic stresses.
The gag-pol poly protein which is a major protein imparting stress resistance
in plants was found to be up-regulated during the course of development of
hyperhydricity in vanilla. A similar observation was made by Maqbool et al. (2008)
while studying drought stress responsive transcripts by differential display in cotton
where two of the transcripts having homology with the gag-pol protein studied
showed a significant increase in their expression levels. But in another study
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Hyperhydricity Syndrome of Vanilla
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pectin of cell wall, causing loss of cell adhesion in the middle lamella is well
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established (Monic et al. 2007). Endopolygalacturonases have long been proposed to
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play an important role in fungal pathogenicity to plants by depolymerizing
homogalacturonan, a major component of the plant cell wall. Besides acting as
C
virulence factors, endoPGs may also function as avirulence determinants through
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release of oligogalacturonide inducers of plant defense and interaction with plant
proteins that modulate PG activity (PGIPs) (Cervone et al. 1989; Antonio and Isabel
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Hyperhydricity Syndrome of Vanilla
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regulators abscisic acid and 6-benzylaminopurine. Development of anaerobic
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environment around the shoot cultures of vanilla during their submerged cultivation
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in liquid medium might play an important role in up-regulation of the gene
controlling enolase (Figure 3.12D) and may play a vital role in HHS of vanilla. In
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roots of Mesembryanthemum crystallinum, enolase transcripts increased in abundance
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in response to salt, low and high temperature, and anaerobic stresses. Surprisingly, no
increase in enolase protein levels was observed despite the increased levels of mRNA
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and enzyme activity during salt stress. The stress-induced increase in enolase activity
was therefore found to be due to post-translational regulation of steady-state enzyme
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Hyperhydricity Syndrome of Vanilla
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stress. Down regulation of protein factors that are involved in replication,
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transcription and repair of DNA was an observable feature in the hyperhydric
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cultures.
Group IV. Ungrouped sequences
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Plants respond to a variety of biotic and abiotic signals that influence growth and
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development. Although the responses of plants to these signals have been extensively
studied at the physiological and the biochemical levels, the perception and the
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ABA. Extreme temperatures, drought, and salt stress induce a partially overlapping
set of genes in different organisms. Incubation of tobacco leaf pieces in high salt
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medium induced a protein kinase with very similar properties to a MAP kinase. MAP
kinases have been demonstrated to be activated upon injury such as cutting of leaves
(Seo et al. 1995, Usami et al. 1995) and biotic stress such as exposure of cells to
fungal elicitor (Suzuki and Shinshi 1995). Increased transcript levels of genes
encoding a MAP kinase module have been taken as a key evidence for the
involvement of a MAP kinase pathway in signaling response to touch, cold, salt, and
water stress. It is quite evident that environmental stresses are mediated by post-
translational activation of a specific MAP kinase in alfalfa. The MAP kinase pathway
appears to mediate only specific forms of stress, because cold and drought, but not
high temperature and osmotic stress, induce the activation of this pathway (Jonak et
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Hyperhydricity Syndrome of Vanilla
al. 1996). Many MAPKs are activated by osmotic stress, cold, salt, drought, and
wounding. All of these conditions are known to disturb the redox balance of plants.
A role of the Arabidopsis MAPK module MEKK1-MKK2-MPK4/MPK6 was
reported for cold and salt stress (Pitzschke and Hirt 2006).
3.5 Conclusion
The present study has given an insight into morphological and biochemical changes
occurring during the progression of HHS in vanilla shoots. One of the main
consequences of HHS appears to be the oxidative stress, which is evident from the
increase in the levels of activities of antioxidant enzymes. The rapid loss of
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chlorophyll with concurrent increase in the content of free PAs serve as useful
biochemical indicator of HHS in V. planifolia. On the basis of homology with known
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genes, the sequences which have been identified as preferentially expressed in vanilla
shoot cultures during hyperhydricity would be involved in a wide range of basic
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metabolic functions and hence the data generated here would be highly valuable for
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understanding the regulatory cascades of hyperhydricity syndrome.
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Biotechnological Approaches for Curing of Vanilla Beans
Summary
For developing an efficient rapid curing method, vanilla beans derived from the
micropropagated plants as well as the commercial beans were subjected for curing
following different biotechnological approaches. Aiming at reducing the curing
period, effects of pre-treatments on the flavour formation in vanilla beans during
accelerated curing at 38 oC for 40 days were studied. Moisture loss, change in
texture, levels of flavouring compounds and activities of relevant enzymes were
compared among various pre-treatments as well as the commercial sample. Use
of naphthalene acetic acid (5 mg L-1) or ethrel (1%) with blanching pre-treatment
resulted in 3-fold higher vanillin on 10th day. Other flavouring compounds -
vanillic acid, p-hydroxybenzoic acid and p-hydroxybenzaldehyde fluctuated
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highly showing no correlation with the pre-treatments. Scarification of beans
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resulted in nearly 4-fold and 3.6-fold higher vanillin formation on 10th day in
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NAA and ethrel-treated beans respectively as compared to control with a
significant change in texture. When activities of major relevant enzymes were
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followed, addition of NAA or ethrel helped to retain higher levels of cellulase
throughout the curing period and higher levels of β-glucosidase on 20th day that
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may find importance for realizing higher flavour formation in a shorter period
since the major quality parameters were found comparable to commercial sample.
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Biotechnological Approaches for Curing of Vanilla Beans
Publications
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128
Biotechnological Approaches for Curing of Vanilla Beans
4.1 Introduction
Natural vanilla flavour, obtained from cured beans of Vanilla planifolia forms the
highest-priced flavour ingredient in food (60%), cosmetics (33%) and
aromatherapy (7%) (Priefert et al. 2001). The major compound vanillin is the most
preferred flavouring compound among the universally used aromas and has a
great market potential in food, beverage, cosmetic and pharmaceutical industries.
Sixty five volatiles and 26 odour-active compounds are identified in the extract of
cured vanilla beans (Perez-Silva et al. 2006); the major ones after vanillin are
vanillic acid, p-hydroxybenzoic acid and p-hydroxybenzaldehyde. Characteristic
vanilla flavour in beans is formed only during a careful curing process resulting in
2% vanillin (on dry weight basis) and over 170 other compounds with delicate
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sweet fragrance. In green vanilla beans, these phenolic aromatic compounds are
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present as their respective glucosides major being glucovanillin synthesized from
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phenylalanine of shikimic acid pathway and curing process is meant to release the
aglycones as the free aroma compounds. Curing also induces the formation of
C
many other compounds that complement to the delicate aroma of natural vanilla
flavour. In fact, it is the presence of these minor compounds in large numbers that
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found in the bean interior, i.e., placental region around the seeds, whereas the
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hydrolytic and other degenerative enzymes that are known to catalyze the
reactions for the release of flavour compounds are localized mostly in the outer
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fruit wall (Havkin-Frenkel et al. 2005). The purpose of curing is to create contact
between the flavour precursors and the enzymes that catalyze the hydrolysis of
precursor compounds (Havkin-Frenkel et al. 2004). Curing of vanilla beans is a
traditionally well-established process which is laborious and takes 3 to 6 months
depending on different curing procedures adopted in different vanilla-producing
regions (Havkin-Frenkel et al. 2004). Despite the long time required for the
curing process, the enzymatic transformation of the glycosides to flavouring
compounds is not very efficient. Only a fraction of the vanillin is produced by
systematic curing of green beans of which a part may also be lost during exposure
to sun as well as during extraction (Frenkel and Havkin-Frenkel 2006). Finally,
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Biotechnological Approaches for Curing of Vanilla Beans
the total flavour yield depends upon the quality of starting material where the best
quality beans range in length from 15-20 cm.
The conventional curing depends on weather conditions as it involves
intermittent exposure to sun and sweating followed by conditioning (Dignum et al.
2001). Therefore, it is lengthy and cumbersome involving several months, may
often fail to completely hydrolyze glucosides resulting in only fractions of flavour
compounds (Frenkel and Havkin-Frenkel 2006). The curing process has been
conventionally developed over a few centuries in vanilla-growing countries, as an
art rather than a science. Thus curing is the most crucial and laborious step in the
entire process of natural vanilla production.
To overcome the above problems, earlier workers used methods such as
I
curing of the cut beans, covering beans in plastic sheets and heating them at 60 °C
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along with high humidity, freeze curing, treatment of green beans with various
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enzymes like -glucosidase, pectinase and hemi-cellulase, hot-air drying and solar
drying (Dignum et al. 2001; Ruiz-Teran et al. 2001). The development of vanilla
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flavour during these treatments is partly due to the hydrolysis of glycosylated
precursors occurring in the green bean (Arana 1943). The most important step in
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water of 63 oC for 2-3 minutes, followed by cutting the beans into pieces and
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(Broderick 1956). From then onwards, blanching in hot water has been an
essential step traditionally followed before curing of vanilla beans. This has been
a convention for several decades in various vanilla-growing countries of the
world. For imparting the mild temperature treatment, sunning of the fruits has
also been a regular practice, which invariably leads to losses at each exposure. In
the present study, vanilla beans derived from both micropropagated plants and
those bought from the market were cured following different biotechnological
approaches for development of an efficient curing technique.
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Biotechnological Approaches for Curing of Vanilla Beans
I
membranes enhancing the permeability. Such effects in the present context are
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expected to create appropriate physico-chemical conditions for the enzymes to
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come in contact with the substrates and act to release the flavouring compounds.
A preliminary screening was done and among the auxins, naphthalene acetic acid
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(NAA) was selected due to its better efficacy over others in supporting the
biosynthesis of extractable phenolics (Funk and Brodelius 1990a, 1990b). An
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earlier report on cell cultures of Vanilla planifolia showed that NAA increased the
secondary metabolism, resulting in high phenylalanine ammonia lyase (PAL)
ts
131
Biotechnological Approaches for Curing of Vanilla Beans
measure the change in voltage due to the presence of odorous volatile molecules.
Sensor responses are then analyzed by the software built-in with the equipment to
get an olfactive picture of the product. E-nose has been successfully used for
analysing coffee aroma (Gretsch et al. 1998) tea quality (Lucas et al. 1998). It has
been used by Ravi et al. (2007) for charactrization of coriander aroma. Evaluation
of flavour quality of pepper was carrriedout with the help of E-nose by Mamatha
et al. in 2008.
4.1.2 Use of elicitors
A large number of handlings for sunning and sweating generally result in a low
quality product. Due to these reasons, there are various attempts to modernize the
curing process, which involves solar drying, oven drying and enzyme treatment
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(Dignum et al. 2001). However, so far there has been no attempt to apply elicitors
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for curing vanilla beans. Elicitors are compounds that trigger the increased
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production of pigments, flavones, phytoalexins and other defence related
compounds. Elicitation has been found as an effective strategy for the induction
and enhancement of secondary metabolites at a commercial scale. For example,
C
synthesis of shikonin and its derivatives by suspension cultures of Lithospermum
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erythrorhizon was by the use of elicitors – agaro-pectins (Tabata and Fujita 1985).
In genetically transformed root cultures of red beet a significantly high
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productivity of 5-fold betalain was observed when pullulan was used and 4-fold
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higher pigment accumulated when cultures were treated with dry cell powder of
Penicillium notatum (Savitha et al. 2006). Peroxidase is one of the key enzymes in
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I
vanillin (Odoux et al. 2003). Peroxidase (POD) activity in vanilla beans is found
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to be quite high even during curing (Sreedhar et al. 2007b) and hence may be
FT
implicated in oxidation/reduction of flavour-forming compounds. For vanilla
flavour development one may also envisage the involvement of cell wall
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degrading enzymes like cellulase, hemicellulase and pectinase by way of breaking
down the cell walls and making the flavour substrates available for the enzyme to
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act (Ranadive 1992). Accordingly, the treatment of vanilla beans with additional
enzymes resulted in enhanced flavour formation. For example, successive
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Biotechnological Approaches for Curing of Vanilla Beans
I
obtained from the growers in “Western ghats” region of Karnataka, India during
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the second phase of harvesting season (early December) and transported within 24
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h to the laboratory. In addition, the mature beans of similar lengths were
harvested from the plants derived via micropropagation cultivated in the institute
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and mixed with the samples collected from the growers. For comparison with the
commercial samples, the conventionally cured beans from market where the
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curing is done by intermittent sun-drying and sweating process for 3-6 months
(Dignum et al. 2001) were used.
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Beans from each treatment were separately subjected for drying in an oven at 60
°C and the loss of water was recorded by gravimetric method throughout the
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Texture of the beans at various stages of curing was measured using Instron 4301-
UTM (Universal Texture Measuring system) by WB Shear at a speed of 100 mm
min-1 and a load of 100 kg. Ten beans were removed from each treatment at 10
days intervals for a total curing period of 40 days and were used for the analysis.
All the results were expressed as force in Newton (N).
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Biotechnological Approaches for Curing of Vanilla Beans
Vanillin (1.2 g), vanillic acid (0.08 g), p-hydroxybenzoic acid (0.02 g) and
p-hydroxybenzaldehyde (0.06 g) were separately weighed into a 100 mL
volumetric flask and diluted to 100 mL with 95% pre-distilled ethanol. From
these, 10 mL aliquot was further diluted to 100 mL with 40% ethanol separately
and was used as standard.
The extraction of flavour components from vanilla beans was done by the method
as described earlier (Ranadive 1992). Briefly, triplicate of 10 g cured vanilla
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beans were finely crushed in liquid nitrogen. The extraction was done with 75 mL
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of 44% aqueous ethanol for 48 h at 45 oC in stoppered conical flasks. The mixture
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was stirred occasionally, filtered and washed with 36% ethanol until the total
volume of filtrate, along with washings, was 100 mL. An aliquot of the filtrate
was taken in a syringe and passed through a membrane filter (Millipore, 0.45µm)
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to remove coarse particles and clear aliquot was used for HPLC analyses.
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(Waters Corp., Miford, MA) C18 Column (300 x 4.6 mm i.d., with pore size of 5
micron) a SLC-6A system controller and CR4A data processor was used. For
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135
Biotechnological Approaches for Curing of Vanilla Beans
Total protein was extracted from the beans and estimated by Macro-Kjeldahl
method (AOAC, Official Methods of Analysis No. 984.13) since the phenolic
compounds hindered the estimation by Lowry’s method.
Earlier reports indicated the involvement of three major enzymes during curing of
vanilla beans; the involvement of -glucosidase (-GLUC) for catalyzing the
conversion of glucovanillin and other glycosides to vanillin and respective flavour
compounds, cellulase (CSE) for cell-wall degradation assisting the
permeabilization of -GLUC from surface of the beans to the centre and
I
peroxidase (POD) in various bio-conversions of phenyl-propanoid compounds
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(Dignum et al. 2001). Therefore, the activities of these three enzymes were
FT
followed throughout the curing period after different treatments.
5000g for 15 min. and the supernatant was used as the enzyme source.
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4.2.7.1.2 -glucosidase
The buffer used was 0.1 M sodium citrate (pH 5). The activity of -GLUC was
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136
Biotechnological Approaches for Curing of Vanilla Beans
4.2.7.1.3 Cellulase
CSE activity was determined by measuring the reducing groups released from
carboxymethyl cellulose (CMC, Sigma) by following the method explained
elsewhere (Priya-Sethu et al. 1996). The reaction mixture contained 0.25 mL of
crude enzyme, 0.5 mL of 0.1% (w/v) CMC and 0.25 mL of sodium citrate buffer
(pH 5), incubated at 37 °C for 1 h. One unit is defined as amount of enzyme that
catalyzed the formation of one micro molar reducing group per minute.
4.2.7.1.4 Peroxidase
POD was extracted in sodium phosphate buffer (pH 6) at 4 °C and the activity was
determined following the procedure explained elsewhere (Agostini et al. 1997).
Briefly, 1 mL assay mixture was prepared which consisted of 100 L of 1% H2O2,
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100 L of 0.25% ortho-dianisidine dihydrochloride, 10 L of enzyme extract and
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790 L of sodium phosphate buffer (pH 6). The change in absorbance at 460nm
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per minute (dA min-1) at 27 °C was recorded using kinetic program in UV- visible
spectrophotometer (Shimadzu UV-160A). Activity was quantified on the basis of
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standard curve, using the same substrate, of horseradish POD enzyme obtained
from ICN-biochemicals. One unit of enzyme activity refers to the rate of change
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Activities in the fresh beans were also checked by tissue printing method where
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cross sections of beans were obtained from distal end (away from the petiole),
middle region and proximal end (near the petiole) and immediately and carefully
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placed on nitrocellulose paper supported on sterile blotter sheets. After the sap
from the sections imbibed into nitrocellulose, the latter was processed at 4 oC in
buffer solutions containing substrates for the enzyme in question. For peroxidase,
100 mL of (at 4 oC) sodium phosphate buffer (pH 6.0) containing 10 mL of 0.25%
o-dianisidine dihydrochloride and 10 mL of 1% H2O2 was used. For β-
glucosidase, 100 mL of reaction solution containing per ml concentration of
100µL of 0.1 M sodium citrate buffer (pH 5) and 100 L of 0.0055 M p-
nitrophenol β -D-glucopyranoside (Spagna et al. 2002) was used. For control, the
prints of bean slices were processed in respective buffers without substrate. After
10 min, each nitrocellulose sheet was kept on multilayered sterile blotters and the
colour developed was photographed. Localization of enzymes in bean tissues was
137
Biotechnological Approaches for Curing of Vanilla Beans
re-checked by keeping the thin sections on a glass slide and directly adding the
respective buffer solutions with or without substrate. The enzymatic reactions
were immediately photographed.
4.2.9 Sensorial properties
Sensory evaluations were carried out in seven separate booths maintained at a
temperature of 22±2 oC with 45±5% of relative humidity with fluorescent lights,
which was equivalent to day light illumination. The Quantitative Descriptive
Analysis (QDA) method (Stone and Sidel 1998) used for profiling sensory
attributes consisted of 15 cm line scale wherein 1.25 cm was anchored as low and
13.75 cm as high.
4.2.9.1 Odour profile analysis of vanilla pods
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One gram of vanilla pod was taken in 250 mL conical flask with stopper. Panelists
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were trained to sniff the headspace and mark the intensity of odour notes in the
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scorecard. The scorecards were decoded and mean values of the attributes were
calculated from three separate analyses. The profiles were generated as spider web
diagram by plotting attributes versus mean scores.
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4.2.9.2 Panel training
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A group of 12-15 panelists were trained over three sessions for descriptive sensory
analysis. The members of the staff were familiar with sensory analysis techniques
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used in plantation products and flavour technology and related fields. The training
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The common descriptors selected by at least one-third of the panel and few
important descriptors cited in the literature were utilized in the development of the
scorecard. In order to assist panelists in the selection of descriptors, dominant
flavour notes of vanilla and appropriately diluted reference compounds
corresponding to the flavour notes (Hariom et al. 2006) were provided. The
panelist evaluated the vanilla extracts in a group and recorded the perceived
attributes individually. Following this, an open discussion was held to reach to an
agreement on appropriate descriptors and the threshold levels were decided for
plotting the graph.
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Biotechnological Approaches for Curing of Vanilla Beans
An E-nose (Alpha Fox 3000, Alpha M.O.S. SA, Toulouse, France) equipped with
six doped and six undoped metal-oxide semiconducting sensors was used in the
present study. The samples that were subjected for the analysis were commercial
sample (1); green beans (2); blanched and cured beans (3) and beans cured after
scarification and pre-treatment with 5 mg L-1 NAA (4). Two grams of the vanilla
pods from each group were placed separately in the sample vials and the volatiles
were allowed to accumulate in the headspace by holding the vials at 25 oC for
120 sec. Then, the volatiles were carried by a stream of zero air (flow rate 150 ml
min-1) to the sensor chamber. Injection and acquisition times were 60 and 120 sec,
respectively.
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4.2.10 Statistical analyses
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Each treatment had at least 150 beans and thirty beans were randomly picked, at a
known point of time, for physical, chemical and sensorial analyses. The entire
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experiment was repeated in the subsequent year. Student ‘t’ test has been used to
compare the mean values and the tests were considered statistically significant at
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p<0.05. The data was analyzed by one-way analysis of variance (ANOVA) using
Microsoft Excel XP (Microsoft Corporation, Washington), and post-hoc mean
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1960).
4.2.11 Treatments
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Biotechnological Approaches for Curing of Vanilla Beans
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appropriately. For the entire experiment, tap-water washed untreated beans
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(without blanching/scarification and without NAA/ethrel-treatments) served as
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general control; thus all the treatments had respective controls (Figure 4.1).
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All the samples with different treatments as well as general control were
separately wrapped in double-layered wax paper and gently tied with cotton
thread. Incubation of the beans was carried out at 38 °C for a period of 40 days.
140
Biotechnological Approaches for Curing of Vanilla Beans
Since the moisture loss was very high initially, the wax wrappers were replaced
with fresh ones on 3rd, 6th and 10th days. For each treatment, 150 beans were
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used. After every 10 day thirty randomly picked beans from each treatment were
used for chemical and physical analyses. After different curing periods, the beans
were re-bundled in fresh butter paper and allowed to condition at room
temperature (28 to 30 °C) for 15 days, followed by storing in thermocole boxes at
RT in self-sealable polythene bags.
I
beans were scarified lengthwise with a brush having fine stainless steel bristles
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(automation for blanching and scarification is also possible). Different sets, each
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having 20 scarified beans (×6), were kept on two-layered butter paper sheet and
sprinkled with different elicitor powders at the rate of 5 mg DCP per bean. The
treated beans (20 in each bundle) were wrapped and incubated in an oven at
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38±1 oC as reported (Sreedhar et al. 2007b).
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4.3 Results
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o
C is shown in Figure 4.2. The initial water content of over 70% was drastically
reduced, in most of the treatments, to around 10% (w/w of the beans) by the 40th
day except in control. The water loss was higher in treated beans than control,
with more so in scarification treatments. The conventionally cured market
samples were also subjected for this test and found to contain 25% moisture. The
analysis of texture (Figure 4.3) showed progressively low values indicating
increase in softness till the end of the curing period in most of the treatments.
However, at the end of the curing period the control and scarified samples showed
higher resistance due to more hardness than in other treatments. The
conventionally cured commercial sample showed texture comparable to the 10
days cured samples of the present study indicating that 10th day is the right time to
141
Biotechnological Approaches for Curing of Vanilla Beans
terminate the incubation at 38 oC. The NAA-pretreated beans cured for 10 days
and conditioned at RT retained their flexibility even after one year (Figure 4.4).
Thus both texture and moisture levels of 10 day-cured beans and conditioned at
RT are comparable to the commercial sample in almost all the treated beans
except for un-blanched control.
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FT
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replicates±SD.
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Biotechnological Approaches for Curing of Vanilla Beans
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Figure 4.3 Texture analysis of vanilla beans during different stages of curing
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after various pre-treatments. The values presented are averages of 10
replicates±SD. C
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Figure 4.4 Flexibility of vanilla bean cured for 10 days after pre-treatment
with NAA during blanching-pretreated beans. The figure shows the
flexibility of the bean stored for one year at room temperature in air-tight
pouches.
enhanced vanillin content by nearly 40% on 10th day, which decreased further
during the entire curing period of 40 days showing 15-25% increase over the
respective control, i.e., un-scarified beans (Table 4.1). Blanching, the
conventional pre-treatment, showed an increase of nearly 1.5-fold (150%
increase) in vanillin content when compared to un-blanched control after 10 days
of curing. A very significant increase of vanillin content by nearly 2-fold was
evinced when blanching was combined with scarification treatment compared to
blanching alone. A similar 2-fold increase in vanillin was observed when NAA (5
mg L-1) treatment was followed after blanching treatment when compared to the
respective control i.e., blanching alone. Though all the treatments with NAA
resulted in significant increase in the turn-over of vanillin and other flavouring
I
compounds, the best concentration was 5 mg L-1.
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FT
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144
Biotechnological Approaches for Curing of Vanilla Beans
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C
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Figure 4.5 HPLC profiles of standard compounds (1), green bean (2),
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with 5 mg L-1 of NAA) (5) and commercial bean sample (6) showing elution
time for major vanilla flavour compounds detected at 254 nm. The HPLC
profiles of (3), (4) and (5) are of beans incubated for 10 days at 38 oC.
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145
Biotechnological Approaches for Curing of Vanilla Beans
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When blanching and scarification was combined with NAA treatment (5 mg L-1),
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there was a very high increase in vanillin formation, accounting to nearly 4-fold
FT
increase as compared to untreated control and 2.6-fold higher than blanched
control on 10th day of curing. In this treatment, the increase was to the tune of 4-
C
fold over the control on 10th and 20th day and 40th compared to control (untreated).
Other concentrations of NAA, i.e., 3 and 7 mg L-1, were lesser efficient than 5 mg
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L-1. Between the two ethrel treatments, use of 1% ethrel after blanching and
scarification showed significantly higher vanillin content on 10th day than the
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146
Biotechnological Approaches for Curing of Vanilla Beans
Table 4.2 Vanillic acid content in pre-treated beans (% g-1 bean weight*)
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FT
Table 4.3 p-hydroxybenzoic acid content in pre-treated beans (% g-1 bean
weight*) C
Sl No. Treatments 10 DAC 20 DAC 30 DAC 40 DAC
1 Control 0.003 a 0.006 a 0.003 b 0.001 b
0.002 b 0.002 c 0e 0.002 a
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2 Scarified
3 Blanched 0.001 c 0.003 bc 0.002 c 0.002 a
4 Blanched + Scarified 0.001c 0.001 d 0.001 d 0.002 a
5 Blanched + NAA (3 mg L-1) 0d 0e 0.001 d 0.001 b
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147
Biotechnological Approaches for Curing of Vanilla Beans
I
* at 25% moisture level; DAC: Days After Curing
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4.3.1.3 Protein content
FT
Total protein content estimated on the basis of nitrogen analysis showed the
presence of 5.64% protein on 25% moisture basis. This value remained constant
C
without any significant change throughout the curing process (Figure 4.6).
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Figure 4.6 Total protein content of vanilla beans (at 25% moisture level) at
different incubation periods as estimated by Macro-Kjeldahl method
148
Biotechnological Approaches for Curing of Vanilla Beans
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Sl Treatments Zero Day
No. Mean±SD 10 DAC 20 DAC 30 DAC 40 DAC
FT
1 Control 172.3±9.3 188.094 bc 311.306 bc 141.133 cd 119.846 cd
2 Scarified 187.013 bc 315.898 bc 208.593 bc 204.085 bc
3 Blanched 257.263 a 243.352 cd 126.931 d 201.480 bc
4 Blanched + Scarified 213.74 b 358.14 bc 318.660 a 246.639 b
C
5 Blanched + NAA (3 mg L-1) 105.90 d 174.9 d 101.21 d 108.55 d
6 Blanched + NAA (5 mg L-1) 211.876 b 349.875 bc 134.032 cd 217.112 bc
7 Blanched + NAA (7 mg L-1) 110.2 d 190.2 d 120.4 d 126.2 cd
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8 Blanched + Scarified + NAA
(3 mg L-1) 102.69 de 243.35 cd 100.24 d 106.22 d
9 Blanched + Scarified + NAA
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149
Biotechnological Approaches for Curing of Vanilla Beans
4.3.1.4.2 Cellulase
All the pre-treatments including blanching showed a significant increase in the
CSE activity compared to initial activity (0.74 U g-1 tissue at 25% moisture level)
in green beans. Though there was a higher activity on 20th day in all the
treatments, a sudden fall was noticed upon further incubation (Table 4.6).
I
6 Blanched + NAA (5 mg L-1) 2.695 ab 3.363 a 0.608 d 0.929 ab
R
7 Blanched + NAA (7 mg L-1) 1.462 c 1.841 c 0.421 de 0.562 cd
8 Blanched + Scarified + NAA
FT
(3 mg L-1) 1.324 c 1.415 cd 0.308 e 0.446 d
9 Blanched + Scarified + NAA
(5 mg L-1) 2.648 ab 2.83 b 0.617 d 0.892 ab
10 Blanched + Scarified + NAA
(7 mg L-1) 1.562 c 1.678 c 0.546 d 0.729 bc
C
11 Blanched + Ethrel (1%) 2.96 a 3.272 ab 0.665 d 0.819 b
12 Blanched + Ethrel (2%) 2.746 ab 2.944 ab 0.428 de 0.662 c
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13 Blanched + Scarified + Ethrel
(1%) 2.81 a 3.484 a 0.592 d 0.94 a
14 Blanched + Scarified + Ethrel
2.442 ab 2.991 ab 0.322 e 0.774 b
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(2%)
* at 25% moisture level; DAC: Days After Curing
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4.3.1.4.3 Peroxidase
An initial POD activity of 1125.3 U g-1 tissue (at 25% moisture level) was found
eP
in the green beans which increased upon scarification treatment on 10th day and
increased further on 20th day. Blanching generally retarded the activity compared
to the un- blanched control, though uniformity in the activity was found in most of
the treatments throughout the curing period (Table 4.7).
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Biotechnological Approaches for Curing of Vanilla Beans
I
12 Blanched + Ethrel (2%) 906.21 cd 801.46 d 524.67 d 502.03 d
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13 Blanched + Scarified + Ethrel
(1%) 1404.53 b 1277.64 c 816.46 c 779.69 cd
14 Blanched + Scarified + Ethrel
FT
(2%) 936.35 cd 851.76 d 544.3 d 519.79 d
The Electronic nose analysis of the vanilla pods is shown in the Figure 4.7 as a
two dimensional representation of Principal Component Analysis (PCA) with
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respect to axes. As seen in the graph, the data analysis was done based on the
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The results are shown as a two dimensional representation of PCA with respect to
two axes. Principal Component 1 (PC1) accounted for major differences (92.6%)
in variance and Principal Component 2 (PC2) accounted for very minor
differences of 6.7%. Beans cured by blanching pre-treatment (3) was placed in
one quadrant while the green beans cured without any pre-treatment (2) was
placed in a separate quadrant away from the others. Both the NAA pre-treated
beans (4) and beans from commercial sample (1) were placed in a similar
quadrant indicating that both have a similar odour profile.
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Biotechnological Approaches for Curing of Vanilla Beans
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Figure 4.7 Electronic nose analysis pattern of vanilla pods from various
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sources. 1: commercial sample, 2: green beans, 3: blanched beans and 4:
NAA pre-treated beans
content of 5.7% was found and the cured beans showed 5.55% protein indicating
an insignificant loss during the treatment period of 40 days (data not shown
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separately).
4.3.2.2 The enzymes
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The levels of activities of the three enzymes followed in the present study are
summarized in Table 4.8. POD was dominant in all the elicitor powders, with a
high level of activity (98,500±260 U g-1 DW) in acetone-dried powder of red beet
seedling root (BSR) than in the lyophilized counterpart (92,800±280 U g mg L-1
DW). The other elicitors, A. niger and S. cerevisiae, showed considerably high
activity of POD (over 1000 U g-1 DW), however, the values were significantly
lower than those in BSR elicitors. The activity of -GLUC was not traceable in
BSR whereas some activities of CSE and POD were found in all the elicitors.
Even in case of vanilla beans, the fresh ones showed high POD activity (1125±29
U g-1 tissue) distributed throughout the fruit, although the core and the distal
region showed higher activity of POD than the surface and the proximal (petiolar)
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Biotechnological Approaches for Curing of Vanilla Beans
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1125±29 U g-1 tissue at initial level during curing with a narrow increase on the
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20th day and a significant drop on the 30th day following a slight increase on 40th
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day (Table 4.9). The traces of cellulose activity present in the beans on 10th day
of curing steadily declined towards the end. Upon elicitor treatment, there was a
C
steady increase in the activity of β-GLUC, especially in the acetone dried A.
niger-treated beans, with a highest activity of 513 U g-1 tissue on the 40th day of
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153
Biotechnological Approaches for Curing of Vanilla Beans
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a
at 25% moisture level
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FT
C
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Biotechnological Approaches for Curing of Vanilla Beans
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Controlc 213.74 c 358.14 ab 318.66 bc 246.63 cd
A. niger-A 237.12 b 396.39 a 463.63 a 513.31 a
FT
A. niger-L 219.33 bc 288.99 bc 381.63 b 316.59 bc
BSR-A 249.33 a 198.31 d 222.43 cd 234.30 cd
195.43 b 241.36 cd 210.28 cd 207.16 cd
C
BSR-L
Yeast-A 228.31 b 264.33 c 240.39 cd 180.01 d
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Yeast-L 224.4 bc 259.21 c 238.81 cd 209.34 cd
a
Data are the mean value of three replicates
b
At 25% moisture level
c
Experiment-specific control where the beans were blanched and scarified
DAC, days after curing; A, Acetone-dried; L, Lyophilized; BSR, Beet Seedling
Root
Data followed by different letters within each column are significantly different
according to Duncan’s multiple-range test at p≤0.05
155
Biotechnological Approaches for Curing of Vanilla Beans
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day and much higher (3.23-fold) than the conventionally cured sample (0.82%)
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(Sreedhar et al. 2007b) and nearly 7-fold compared to zero day (0.38% in green
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bean). Nearly similar effects were observed in case of treatments with A. niger
and BSR-L elicitors, all producing nearly 2% vanillin with 2-fold increase in
vanillic acid and slight fluctuations in the levels of p-hydroxybenzaldehyde and
C
p-hydroxybenzoic acid. In the control sample itself, there were significant
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fluctuations in the quantities of flavour compounds formed during the entire
curing process, with a decline in vanillin after 20 days with a steady increase up to
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40 days.
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Biotechnological Approaches for Curing of Vanilla Beans
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Biotechnological Approaches for Curing of Vanilla Beans
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BSR-L 1.89 ab 0.008 a
0.006 a 0.004 b
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Yeast-A 1.58 c 0.006 b
0.002 c 0.00 cd
Yeast-L 1.58 c 0.005 bc 0.005 ab 0.006 a
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30 DAC
Controlc 1.59 c 0.004 bc 0.003 c 0.001 c
A. niger-A 1.80 ab 0.004 bc
0.004 c 0.000 c
A. niger-L 1.45 d 0.006 ab 0.002 d 0.007 a
C
BSR-A 1.87 a 0.006 ab
0.007 b 0.005 bc
BSR-L 1.70 bc 0.008 a
0.010 a 0.006 ab
Yeast-A 1.62 c 0.00 c 0.005 bc 0.006 ab
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Yeast-L 1.85 ab 0.007 ab
0.008 ab 0.008 a
40 DAC
Controlc 2.00 ab 0.009 b 0.005 cd 0.002 d
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b
At 25% moisture level
c
Experiment-specific control where the beans were blanched and scarified
DAC, days after curing; A, Acetone-dried; L, Lyophilized; BSR, Beet Seedling
Root
Data followed by different letters within each column are significantly different
according to Duncan’s multiple-range test at p≤0.05
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Biotechnological Approaches for Curing of Vanilla Beans
profile to the beans with high notes of vanilla, sweet and floral odour and low
intensity of woody, beany and smoky notes.
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Figure 4.9 Sensory profile of vanilla beans cured for 10 days with or without
elicitors and different pre-treatments. The data represents average of atleast
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12 panelists in three separate analyses.
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Biotechnological Approaches for Curing of Vanilla Beans
4.4 Discussion
4.4.1 Pre-treatments experiment
Traditionally the appearance, the flexibility and size characteristics have been of
importance since there is a fairly close relationship between any two factors and
the aroma/flavour quality. The moisture content is one of the several parameters,
which is important for bean quality. It is therefore, very important to realize that
moisture content is inter-dependent on other quality parameters and cannot be
considered, by itself, as an index of quality. The presence of appropriate water
content ranging from 25 to 30% has been noted by earlier workers, to result in the
desirable texture. The present study has clearly shown that the use of NAA or
ethrel coupled with bean scarification more quickly reduced the moisture content
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than with similar set of conditions with blanching treatment (Figure 4.2). Beans
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used for extraction have low moisture content while gourmet beans contain higher
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moisture content (Havkin-Frenkel et al. 2005). The moisture content is a major
factor in the preservation of cured vanilla beans since low moisture content is
essential to prevent microbial growth. Also, the water content of properly cured
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beans must be sufficiently low to prevent growth and activity of microorganisms,
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since low water, in combination with high phenolic content offer protection
against spoilage in cured beans. Our observation of NAA/ethrel-treated 10-day
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cured beans, stored at RT for one year did not show spoilage due to any
fungal/microbial infestation (Figure 4.4).
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160
Biotechnological Approaches for Curing of Vanilla Beans
I
respective glucosides, as compared to untreated beans, despite the well-
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documented fact that the specific β-GLUC gets arrested after blanching and
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missing during curing (Dignum et al. 2002). An extensive study of the thermal
sensitivity of β-GLUC showed that the activity of the enzyme was lost within 24h
after blanching (Dignum et al. 2002). However, in the present study, a much
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higher activity than reported elsewhere was observed throughout the curing period
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where most of the treatments significantly enhanced the enzyme activity on 20th
day. Although direct correlation could not be made between the enzyme levels
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and the quantity of flavours formed at a given period of treatment, one can reason
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pathway of vanillin and other flavour related compounds is shown in Figure G2.
The formation of vanillin precursor, the glucovanillin originates from glucosides
of ferulic acid or protocatechuic aldehyde, both the compounds being derived
from coumaric acid glucoside of phenyl-propanoid pathway (Figure G2). In
studying the biosynthesis of vanillin in vanilla beans, it was found that ferulic acid
was incorporated to a greater extent into vanillin than vanillic acid indicating that
ferulic acid is β-oxidized to vanillyl-Co-A, which either can be reduced to vanillin
or de-acetylated to vanillic acid (Zenk 1965). Thus the native β-glucosidase plays
a crucial role in the conversion of not only the glucovanillin into vanillin but also
the other precursors to glucovanillin. This probably could be the reason for
fluctuations in the levels of flavouring compounds during the curing period and
hence not strictly corroborative with the enzyme levels. The limiting factor in
161
Biotechnological Approaches for Curing of Vanilla Beans
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to enhance the flavour formation via up-regulation of the -GLUC and CSE.
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The E-nose analysis of vanilla beans from different sources complimented
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well with the hplc quantification of major flavour components. Sample from green
beans which consists of least amount of vanillin and allied flavouring compounds
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was placed in a separate quadrant away from the other samples derived from
cured beans. The PCA mapping clearly distinguished the samples based on the
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dominant volatiles from the beans. A similar trend was observed in a study on
coriander oil samples aroma analysis (Ravi et al. 2007).
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catalyzing lignification of plant cells. Since the natural vanilla flavour from cured
beans of V. planifolia comprises over a hundred flavour molecules derived from
the PP pathway, the effects of elicitors were studied, assuming that they might
hasten the curing process by catalyzing relevant enzyme activities.
The data presented in Table 4.8 show significant differences in the
activities of enzymes, indicating that the method used for elicitor preparation
possibly influenced their characteristics. Peroxidase showed lesser sensitivity to
processing conditions than other enzymes. Enzyme activity change due to
processing conditions is widely known. The major enzyme present in both vanilla
beans and elicitors was the POD. The formation of very high levels of vanillin
was also noted in the treatment with high levels of POD, which is indicative of the
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Biotechnological Approaches for Curing of Vanilla Beans
major role played by this enzyme in catalyzing the flavour pathway. Earlier
studies have established that peroxidases (of Class III type) are widely distributed
in higher plant cells. Plant PODs and their various isoenzymes have been proven
responsible for a plethora of physiological functions where they preferentially use
phenolics as electron donors resulting in the formation of oxidized phenolic
compounds of brown colour (Hanum 1997). Since their isoenzymes are diversely
regulated (Welinder and Gajhede 1993), one can expect wide variations as well as
an array of the end product. Whereas the fungal peroxidases (Class II type) are
known to act at extra-cellular level and are mainly involved in lignin degradation;
often contributing for the re-formation of vanillin (Hanum 1997; Priefert et al.
2001). Ferulic acid (an iso-lignin) occurs abundantly in most of the plant cells
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including vanilla beans. Earlier labeling studies have established its preferential
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incorporation into vanillin over other closely related substrates leading to the
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higher turnover of vanillin (Zenk 1965). Thus, there is a good relationship
between the levels of POD (in situ + that of elicitor) and the levels of vanilla
flavour compounds where the enzyme could probably catalyze the inter-
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conversions of the flavour precursors resulting in the formation of appropriate
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substrates for the action of the other key enzyme--GLUC. The potential of
A. niger to convert natural precursors of vanilla flavour, i.e., isoeugenol to vanillin
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(Abraham et al. 1988), ferulic acid into vanillic acid (Bonnin et al. 1999) and
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vanillic acid to vanillin (Priefert et al. 2001), is well known. A. niger has also been
described as a ferulic acid-degrading organism (Labuda et al. 1992). Commercial
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exploitation of A. niger for the conversion of ferulic acid to vanillic acid has been
described (Lesage- Meessen et al. 1999). The treatment with the dry cell powder
of A. niger, particularly the acetone-dried powder, significantly enhanced vanillin
on 10th day itself, which was nearly 1.3-fold higher than the blanched and
scarified control, and 2.46-fold higher than the traditional method of blanching
(Sreedhar et al. 2007b). Noteworthy improvements in the other flavour
components were also observed during this period. Lyophilized A. niger powder
was more effective in enhancing vanillic acid and benzoic acid steadily up to 30th
day. A few studies have demonstrated the direct involvement of -GLUC as the
key enzyme for the de-glucosylation of flavour substrates, which is also
controversial due to compartmentalization of the enzyme from the substrate
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Biotechnological Approaches for Curing of Vanilla Beans
(Dignum et al. 2001; Odoux 2006; Odoux et al. 2003; Havken-Frenkel et al.
2004). β-GLUC is also expected to enhance flavour extractability by hydrolysis of
cell wall components (Ruiz-Teran et al. 2001). Nevertheless the present study and
our previous report (Sreedhar et al. 2007b) indicate that the high levels of POD of
the beans and that in the elicitor may also be involved in the quantum turn over of
the flavour molecules, although a deeper study is needed to unequivocally
establish the same. It is interesting to note that there is a decline in the level of
vanillin on 20th and 30th days. These changes may again be attributed to the
catalytic activities of POD followed by -GLUC, where the latter systematically
catalyzes the de-glucosylation whereas the former may be implicated in a cascade
of redox reactions. Plant PODs (POD class III), (both from vanilla beans and red
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beet seedling root-derived) having high redox potential may not only build up but
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also degrade vanilla flavour compounds. Therefore, in a mixture of phenolics, the
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radicals of phenolics that are good substrates for POD can oxidize a poor electron
donor molecule for peroxidase, rapidly. Thus, even the un-preferred substrate is
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indirectly catalyzed by POD, bringing about fluctuations in the flavour profile as
observed in the present study. However, the fungal PODs are known to work the
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other way, where they depolymerize lignin to form vanillin (Hammel et al. 1993;
Kirk and Farrell 1987; Priefert et al. 2001; Ten Have et al. 1998). Thus the highest
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a better sensory profile with high notes of vanilla, sweet, and floral and low
woody, beany and smoky notes which is a characteristic feature of good quality
vanilla (Hariom et al. 2006). Attributes such as fruity, floral, woody and beany
notes did not differ significantly from the others, which might be due to the use of
the higher concentrations of the sample which might have masked the subtle
differences.
4.5 Conclusion
The present study has clearly demonstrated that for developing a process for the
production of vanilla flavour compounds from vanilla beans, one need to carefully
control the process parameters with periodical monitoring for flavour compounds
164
Biotechnological Approaches for Curing of Vanilla Beans
and terminating the reaction as desired because the endogenous enzyme may
divert the flavour molecules towards lignin biogenesis. The pre-treatment methods
developed as a result of the present investigation may find importance for
realizing higher flavour formation in a shorter period since the major quality
parameters and sensorial analysis were found to be comparable and similar with
conventionally processed commercial sample. This study has also shown that the
HPLC profile as well as the sensorial properties of the beans cured just for 10
days with elicitor were characteristically similar to the conventionally cured beans
indicating that the present finding holds great promise for further application in
vanilla bean curing.
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165
Conclusions and Future Prospects
The study on genetic diversity of Indian vanilla using RAPD and ISSR
markers indicated that V. planifolia cultivated in India is likely to share the
same genetic background and therefore, the genetic diversity is extremely
low. This preliminary investigation has determined the absence of genetic
variations in introduced and then commercially cultivated V. planifolia in
India indicating a threat of extinction due to pest and environment
vagaries.
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micropropagated multiple shoots of vanilla developed from axillary bud
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explants established 10 years ago using RAPD and ISSR, it can be
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concluded that the micropropagation system for vanilla can be carried out
for a considerable length of time (for at least 10 years) without any risk of
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genetic instability.
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Vanilla shoot multiplication in semi-solid (SS), complete immersion
system (CIS) and partial immersion system (PIS) were evaluated for
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The present study compares the solid, liquid (referred as CIS) and partial
immersion system (PIS) and substantiates that GrowtekTM bioreactor (as
PIS) appears to be an efficient culture system in all respects. A
combination of equal ratios of red soil: sand: vermicompost was found to
166
Conclusions and Future Prospects
be the best for greenhouse hardening. Field evaluation showed that the
micropropagated plants flowered early with higher bean yield than the
conventionally propagated ones.
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activities of antioxidant enzymes, indicative of shoots’ defensive efforts
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against oxidative stress.
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Thirty one hyperhydricity-associated cDNAs identified by DDRT-PCR
were cloned and sequenced whose electronic homology searches using
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BLASTX analysis resulted in identification of 23 cDNA clones showing
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homology with various stress, apoptosis, DNA repair and carbohydrate
breakdown related proteins to be differentially expressed during HHS.
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which paves the way for a better insight into gene expression during this
common physiological disorder.
shorter period since at this stage the major quality parameters were found
comparable to commercial sample.
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properties. These observations appear useful for developing a rapid
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process for the curing of vanilla beans.
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168
Conclusions and Future Prospects
Future prospects
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be urgently pursued.
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The information on the use of different micropropagation systems for
vanilla and identification of partial immersion system as an easy method
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helps to develop an efficient technology for large scale production of
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micropropagules.
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The study of the use of pre-treatments and the use of food-grade elicitors
in combination with pre-treatments for the accelerated curing of beans
appear useful for developing a rapid process for the curing of vanilla beans
which appears to offer economical advantage.
169
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191
Appendix
Frag.
Acc. No. Sequence (5’-3’)
ID
GACCGACCCAGAGGTAGGGGGCGCCGCCGGGGAGGCGGGAGTGGGTTCCACAT
CGGGAGGAGCAGAAGCCCTCGACAGCGTCGTCGGCGGAGGTGATGATGAGGGC
VP1 GE746280 GATGGCGAAAGGGTTGGTGTTGTTGGAAAGGGCGACGGCGGCGAGGGCGGGAA
GGGCCGAGGAGTGGAGGATGCGGCCGAGGGTGCTGGAAGGGAGGAGGACCTG
AGGGCCGAGGGTGATGGACGGGGGGGATGTGGGTCGGTC
ACAACTGGGGCAATGTGCTGAGAGGTGATGCTTCCCTTGCGAAACTGGCACAGCT
GGAGAGGTTCTCAACCTGCTTGAGAAGAAGAATAGTAGCTCTTGAAGGGACCGTC
GAGTCGCTAAGAGTAGAAGCCGAAGACGACACCTGCTGCTGCATCTGCTACGCCT
GE746281 CCGAGTCCGACACCCAATTCGAGCCTTGTCACCACCAGTCTTGCCTTGGCTGCATTA
VP2
CCAGGCACCTGCTGAATAGTCGAAGATGCTTCTTTTGCAACGCAACTGTTACAGAA
GTGGTTAGGGTTTGCAAAAATTCTAATCAGATTTCTGGAGGATGATTCTCTTGAAC
CCACCTTTTCTGTTTTCTTGAATTGAAAGGTATGCAATATACACACAGGCACCGACC
I
A
R
AGTCAGCCACCAAATCTGTATAACGATGATTCTTATGTCCGAGGTCGTGGACGGG
GCAGAGGAAGAGGAAGAAGTTGGGGTAGAGGCGCATATGGTGGCTACGGCGGA
FT
VP3 GE746282
GGATACGAGCGTTATGGTGGATATAGTGGTTATGGAGGATATTCAAATGATCATG
AAAATGGTGAATGGAATTATAATTGGAATCGAGGCAATGGCCGAGGCAGAGGAA
ATTGGAGTTATCGTGGCTGACT
C
AGTCAGCCACATGGACCAAGGCCACCAAGACAGACTGAGGTCAGGACGGGTGAA
GGAGTGCTAGGATCATCTATCTGGATTTTACAATAAAGAAAAAGAAAGTTACTAAA
@
VP4 GE746283 GATTTTGATTGGTGCAGAAGGGTAAGTGAGCACAGCAGTGTTAGAGAATGACAA
AAAGAAGGGGCTGAGGTGCTAGGGATGAAGTGGGTAGGGGACACTGGAGTGGG
GGGAAACATCCCTCCATCCTTCACTACCCCAGGGCTCTCCTCCAGACCGGGTGCTA
ts
TGGTGGCTGACT
AGTCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTATGGGAGGCAGCAGT
GAGGAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGAA
rin
VP5 GE746284
GAAGGCCCACGGGTTGTGAACTTCTTTTCTCGGAGAAGAAGCAATGACGGTATCT
GAGGAATAAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATG
CAAGCGTTATCCGGAATGATTGGGCGTAAAGCGTCTG
eP
TATGGTGGAGCACGACACTCTGATCAGATCAGAAGATTACCACTTGCCTCAGACG
VP6 GE746285
GAGCGATGATATACCATTGTACACTTGAACTTACCGATGATAAAGCATCATCACTG
GGAAGAGGTCCCGTCTACCCACGACAGGCGGTATTC
VP7 GE746286 TGGTCGGTGCCTGTGTGTATTCTGTTTATCGATCCCCTGCTCTTTGTTCATCGATATC
TATCTTTTTTTCTGTATCGGTCGGCATCTTCTTTCTCAACTCGTCCAGGGCAAGCTA
TAGCGCCATTGCAACGAGGTGTGAGATTAAAAGTTACATCAAAAGGGTGAACAAA
GTTGAAGGTAGAAACTTTGTCATATGATGAGTCATTTTGTAAATAGCATTAGATGG
TACACCCATGTATTGAGATGATCCACTAATGAAACAATATGAAGATAACTGTAACC
VP8 GE746287
TAGTGCAAGGTGACCAAAATTAAAAAATCATTTGATACAATGATAAAGCATATTGT
ATGTTTTGGTGAATGCTTGCTCATAGTCATTTATTTTGAATACTATTGTTTTGATCAT
TATGTATGATTAGACTTTAGGTATCTTAAGATAGTCTAATAGACTTTCTTGCTTTTTC
CATAGCTACAATGGCGCTA
TAGTCAGCCACCTCCTCCAGGTACTACATCATGGAGAGGAAATTTGAGCTCTCCCC
VP9 GE746288 TCTTGCCTTCTCCAAAGGTTTGTAACTTTTCCGTTTTTTTTAATTTTTATGATTATTTT
TTCAGTTTGAACCATTCTTTTTATCAACACTTCTTCTTTCAGCAAAGGCAAAACTAGC
TTTCTTAGAGCTCACCTTGCGATCACATTGGTTCAACTAGTTGGGAGGACTATCATT
192
Appendix
ATATTTGGAAATTTTAGGTTATTAGGCAGGCATTTTGTTGTTTTCTTCAGCCATATC
TGTATTATACACACAGGCACCGACCA
TAGTCAGCCACCTCCTCCAGGTACTACATCATGGAGAGGAAATTTGAGCTCTCCCC
TCTTGCCTTCTCCAAAGGTTTGTAACTTTTCCGTTTTTTTTAATTTTTATGATTATTTT
VP10 GE746289 TTCAGTTTGAACCATTTTTTTTATCAACACTTCTTCTTTCAGCAAAGGCAAAACTAGC
TTTCTTAGAGCTCACCTTGCGATCACATTGGTTCAACTAGTTGGGAGGACTATCATT
ATATTTGGAAATTTTAGGTTATTAGGCAGGCATTTTGTTGTTTTCTTCAGCCATATC
TGTATTATACACACACACACACACACATACACACAGGCACCGACCA
TAGCTTGCCCTGGTACGAGTTTATGATCCTTCCTGTTGGAGCAACCTCGTTTAAGG
AGGCCATGAAGATGGGTGTTGAAGTATATCACAATCTAAAGGGTGTGATTAAGAA
VP11 GE746290 GAAGTATGGTCAGGATGCTACCAATGTTGGAGATGAAGGTGGCTTTGCACCTAAT
ATTCAGGAGAACAAGGAGGGACTAGAATTGCTGAAGATTGCTATTTCTAAGGCTG
GATATACTGGCAAGGTTGTAATTGGAATGGATGTGGCTGACTA
ACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATTAGTCAGCCAC
ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGAGGAATTTTC
VP12 GE746291 CGCAATGGGCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGAAGAAGGCCCAC
GGGTTGTGAACTTCTTTTCTCGGAGAAGAAGCAATGACGGTATCTGAGGAATAAG
I
CATCGGCTAACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATGCAAGCGTTATC
R
CGGAATGATTGGGCGTAAAGCGTCTGTAGGTGGCTGACTAATCACTAGTGAATT
CGCGGCCGCCTGCAGGTC
FT
AACAACTTAACAATAATCAAATCTCACAAATGAAAAATACCAGAATACAAGTCCAA
TACATAACGAACGAAATAAAGATATGAACAATAACACTGTAATTAGAACAAGTAC
VP13 GE746292 CCAAAACAACATATGTTGGAGAGCGAAATAAAGGGAGAGGAGGAATGGGGGGC
C
CGTTGGGTGGTGGCTGACTAATCACTAGTGAATTCGCGGCCGCCTGCAGGTC
ACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATTAGTCAGCCAC
@
AGACAAGACGGCAAAGCAAAGGGTGGACTTGTGGTTTTGGAGAGGTGAGTTGAT
VP14 GE746293 GGAATCTTGTAGATGAGATCAATAGGGAAGCTACATGTGATGATGATGATGATTG
GGAGAACTGAGAAGCCGCATGCAAAATTGGTGGGATTGATGCAAGTATGGCTTGT
ts
GAAGAGAGGTGGCACGGTAAGGAGGGGAGGAGGATATTGAAGGCATGGTGGCT
GACTAATCACTAGTGAATTCGCGGCCGCCTGCAGGTC
rin
TAGTCAGCCACCGCAAACGTGAGTAGTTTGTGTGATTTCATACGAACCGTGTCCTT
GGTGAGTAGCTACGTTCTAACTTTGATCGTTTTGGTCATTCGCTGAATGGCAACGG
VP15 GE746294 CGTGCACTGGATGGTGTGTGCGTGAGTCGGGTTGAGTTCGTTATCGACCATGTGG
CTGACTAA
eP
TTGGACCGGTGCAGCAGAGACGAAGAAAACTCGGGCCAGAGCGATCCCAAACTG
TGGAAGAGCAAGTGCAGGCACTGTTGGAAGCCGAATTTATAAGAGAAGTCAAAT
VP16 GE746295 ACCCACTATGGCTAGCCAACGTCGTTTTGGTGAAAAAATCAAATGGGAAGTGGCG
GATGTGCACCGATTACACAGATCTCAACAAAGCTTGCCCAAAAGATCCATACCCAC
TCCCGAGTATTGACGCCCTGGTAGATGCTTCCTCTGGGTATAAATATCTCTCGTTTA
TGGATGCGTATTCGGGATACAACCAAATCCCATGTACCCACCGGTCCA
TGGTCGGTGCCTGTGTGTAGTATGTACTTGGTTGGTTAAGGGTATAGGGAACTTG
TGGGCGAACGTCAAGAGATGGAGGTGCAGGTTGTTGCAAAGGTAGCTCTTTGTAA
VP17 GE746296 GACTGGTTTTGATCGGGAGTTCTGGGTCGTGAGGTTGAAGAGGTTATTTGGTGAG
GAACCAGCGCTAATTGCTGATTTGGGATTTCGGTGTAATTGCTCTGGGTTTTTGTTT
CAGGAGGTGTGGCTGACTA
GACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATTAGTCAGCCA
CCAATAATAAAAAAGAAAATAGGAAAATAAAAAAAAAAAAAGCTAAACAAATGG
VP18 GE746297
AAAAATTACGTCGCACCGGTAATGCGTTGACTTCAACGGACGCTCAAATGTCCATC
GTCGGCGGTCGTGGCTGACTAATCACTAGTGAATTCGCGGCCGCCTGCAGGTC
193
Appendix
ACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATTAGTCAGCCAC
AGCAGTATCAGCAGCCACAGCAGTATCAACAGTCACAACAGTATCACCAGCAGCC
VP19 GE746298 ACATCGACAGCCACCAAATCTGTATAACGATGGTGTGGCTTTGTTAGCTTGTGTTG
ATTACTAGTGCTGTATAGAATATTGTGGTTATTTTTTCCTTTAGGTTATTGTTTCCCA
CTCATTGGTATTATATATTGGTGTTAAGATTCTTATGTCCGAGGTCGTGGACGGGG
CAGAGGAAGAGGAAGAAGTTGGGGTAGAGGTGCATATGGTGGCTGACTAATCAC
TAGTGAATTCGCGGCCGCCTGCAGGTC
AGCGCCATTGGGTATAGGCATAATGGGTTTTAACAACGTGGCAACAGCTTTAAAA
GTACCGGGTGTTGAACTGGTAGCTGTGGCAGATTTATATACCGGAAGGCTTGAGA
GAGCTAGGGAGTTATATGGTCAGCAGCTCTTCACGACGAAAGACTACGAAGAGAT
TTTAAATAGAAAAGATATTGATGCAGTGATTGTTTCCACTTCAGATAATTGGCACG
ATAGGATTGTAATTGAAGCTTTGAGAAAAGGCAAAGCAGTGTACTGCGAAAAGCC
AATGGTTCATAAAATTGAACAAGGTTTGGATGTCGTTAAAGCGCAAAAAGCAAC
GGCTGGCATACGCAAGTGGGTAGTCAGAGAGTGAGCAGCATTGTATACGCCAAA
VP20 GE746299 GCCAGAGAGCTTTATAAAACAGGAGAGATTGGTCAATTGAATTGTATTGAGGCTT
CCTTTGACCGTCATTCTGCATTGGGTGCCTGGCAGTATACAATGCCAATGGATATTT
CCGAAAAAACAGTGGCCTGGGAAAAATATGTGCGTTCGAAAAAAGATGTTCCCTT
I
CGATCCGAAACAATTTTTCTGGTGGCGGAATTATAAAGAATTCGGAACAGGTGTA
R
GCTGGCGATTTATTTGTTCATCTTTTATCGGGGATACATTTTATTACTGGTTCGCTT
GGTCCTTCTCAGATATTTGCTACAGGTGATATCAGTTATTGGAAAGATGGCCGTAA
FT
CGTGCCTGATGTAATGACCGGTGTCATGCAATATCCCGCTACTAACCAGCAT
AGCGCCATTGCCACAACAGATGCCTGCAGAATCATGACTGAATTAGGGGAACTTC
TGCAGGAAAATAATGCAGAATATGCATATGATGCTGAAATGAATGGAAGCCTGGT
TAATAACCCATTAGCATTTGGCTGTGATGACCCCTCCTTGCAAATTTTTCTTCCAACT
C
CAACCGGATAGCCGAACTGTGCATGCTGGCTTGGGAGAACAATCAAAATTGACTA
GTGTGTTTGAAGACGGTTGGATCTCTCTTGCTCCTGCTGCTGATGGAGTTCAAACT
@
GCTGCAACTCGAGAATGTAGGCCAAATTCAAGAAACCATTTGTTTCTACATGAAAA
VP21 GE746300 CTGGCCAGAGAGCATGGAAAATGATTCTGGTTCTCCCCTCCTGTCTCTGAATAGAC
AACATGAATTGAAAGCAACTTTGAATGGCCAAAAGCCTGATGCATCCTTGCGCCAT
ts
TCCCATCAGCAAAGGTCTATGAGATCCAGATTTATGTTTACACTTGATATAGACTCA
GATAGCCAGTGAATGTAGTATCTGGTGATGAGATTTTGTGGGTCATTTCTATTTCA
rin
CCGTCCTGTTAATAACCTTGGTTACACGAAGAAAAGGCTAAAGAGGAGGTATAGA
AGTATCTAGAAATGAACCTTGTTTGTTCATGTAAATTTTATCTTTCTTTTACCTTTCA
ACAATGGCGCT
eP
AGCGCCATTGCTCAGGGCGAAGCTGGAGGAGTTTTTGGCTGACGATCGTCGTACG
CTCGAGACGCTGGTCATGGCTGTGCGCGATCAGGTGGTTTTGGGGTACGAGGAGT
TTTTCGATGGGTGGGTTGAGCAGCTGGGCGGTGAGAGGAAGGTTAGTCGGAAAG
GCAAGGGCAGAGAGTCGGATGTACTTGGGCCGGAGGTGTTTGGCGATGTCGTTG
GGCGGGTCTTTGAGACTAAGGCGCTTGAGGATGTTGATGATGGAGATAACTCTTG
AGGTTGTGAGGTCCGTGTGGGAGGTTGCAGCGCCTGTGGGCGTGCTTTCTTGGGA
TCTTCAAGAAACCCATGGGAAAATAGCATTTGAACATTTCCTTGGACGGTCCTACC
VP22 GE746301 ATGTGGTCCTTTCCCCCACGTCTGCGTGCTACTCGGCGTGTTTATCGCTGGTTTCTC
TCATGACGTCGAGACGGACGATCTCATACCAAAGAGTTCAAACAGACCGCGTCCA
CAACCCTGCCAGTCCGCGAACAGTTACTAGATGTCTGTGAACACATGTTTTGTTAT
ATGTTCACTTTCAAACAATGACCCACGTTTTCTTGTTTTTCACGTTATTGCCACAATG
GCGCT
194
Appendix
CCAGCATGGGCAACAAGAGTGAAACTCTATCTCAAAAAAAAAAAAAAAAAAAAAG
GAAACTCATTAATGCTGGCTCAGGAGTTCTGACAACATTACCTTTGTTTTTATAGGT
TCTTACAGTAGAGATAAGTTTGCAAAGGCCAGTGTTTGCCTATTTTTCTAATAATTA
ATTCCTTTCCTAATACCAGATGTGATCTCAATCATTCCATAATGCCACACATTTTATT
VP23 GE746302 AGCTTTGCCTCTCATATAGGGATTACTTATTTGTTTTTTAGAAGGGCTGAACGAAAT
AGAAGTTTAATTCCTTCACACGTGGCCTGTAAGAGAGTCACCATTCTTACTGAAGC
TGAGAAGGAAGTTCAAGAATGAGAAGGAAAACTATCTCTGATCTTCTCCAAAGAC
GATAATTTATTTACAGCTGTCTCTTCTCTTGTCTTTACCTTTATTATTAAACACACAC
AGGCACACGCACGCACACAGGCGCACACACACATATACACACAGGCACCGACC
ACGGTTCCACCCTCCTTGGGGATCCTTTCCACCTCCGAGTGCTCGGATGGTAGTGT
AATCTTCCGGTTCGGAGATGCTGCGGAGGAGTCCTGGAAGGATTCGGTTGGAGA
GATGGAACCGACGGGGTCTAGTGAAGATGTTGGGATACTGGAGGAGAATGCAGA
AGATAGCGGACTTGAGACAAGGAATCGAATTCTTGCGTCACTGTCGAGAGTGAAG
CCGGCGTGTGCTCCGATTCTGAGTTTTTTGGTGCTGGAGATGACTTCACAACCGAC
CGTTTTGGTTTTGGTATTGTCACAGAACAAGATTTACGTGATGAAGGAAGCCGGG
I
VP24 GE746303 GTGCGCAGGCTATTGATAAGATGTTGGGCAGCGATGGGATGGAGGATGTCCTTG
R
AAGAGGAATTTTCTGAAACATGGTCAGGAGAGCTGCAATCGGAGGAATCTCACAA
TGAAGATGGTTTGATTGCCGTGGAAGGCCGTGAAACTTCACCATATGGCGGGTAC
FT
AGTAGTTTTGTTTCAACTGAAGTTGGTATGCTGCAGTTGCCCGAGAGTGAGAGCA
AGATTAAGGCAGATTCATTGCAGTCTGATGAGGTTTCATCAAACATTGTAGAAAAT
GATTCCGATGTCGAACAAGATCAAAATGTTGGTTCATCTAACGAGCTTCTTCTTGG
TTCCGAATCTTTATCAAATGCTGAAGCTGAAGCTCTTCAAGTGGAAGACACTCAAA
C
ACCCAGAAGCAT
GCTCGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCATTCTTGCGAACGTACACCCCA
@
GGCGGGATACTTAACGCGTTAGCTACAGCACTGCACGGGTCGATACGCACAACAC
CTAGTATCCATCGTTTACGGCTAGGACTACCGGGGTCTCTAATCCCATTTGCTCCCC
TAGCTTTCGTCTCTCAGTGTCAGTGTCGGCCCAGCAGAGTGCTTTCGCCGTTGGTG
ts
GGAGGACTTGAAAAGCCACCTACAGACGCTTTACGCCCAATCATTCCGGATAACG
CTTGCATCCTCTGTCTTACCGCGGCTG
GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGAGGAATTCTCCGCAATGG
eP
GCGAAAGCCTGACGGAGCAATGCCGCGTGGAGGAAGAAGGCCCACGGGTTGTGA
ACTTCTTTTCTCGGAGAAGAGGCAATGACGGTATCTGAGGAATAAGCATCGGCTA
ACTCTGTGCCAGCAGCCGCGGTAAGACAGAGGATGCAAGCGTTATCCGGAATGAT
TGGGCGTAAAGCGTCTGTAGGTGGCTTTTCAAGTCCTCCGTCAAATCCCAGGGCTC
AACCCTGGACAGGCGGTGGAAACTATCAAGCTGGAGTGCGGTAGGGGCAGAGG
GAATTTCCGGTGGAGCGGTGAAATGCGTAGAGATCGGAAGGAACACCAACGGCG
VP26 GE746305 AAAGCACTCTGCTGGGCCGACACTGACACTGAGAGGCGAAAGCTAGGGGAGCAA
ATGGGATTAGAGACCCCGGTAGTCCTAGCCGTAAACGATGGATACTAGGTGTTGT
GCGTATCGACCCGTGC
CAGCCACCGCGCCGAGGTAGGGGTCGTCCGCCGAAGGCAAAGGTCTCTGGGACA
GAAGGGGCGGGATCTTCGTTAGCCGTTACTAAATTTTGTTCTGAGATGCCCCGAAC
VP27 GE746306 CATGGTGAAGCGGGGACGAGGGCGGCCGCCGAAGGCCAAGATCTCTGGGACAG
AAGGGGCGAGATCTTCCGCCCCCAAGGGTACAGAATCCTCCATTTCTGCCGGAGG
AAAAGATCCCGGTATGTCTCCGAAGAAGAACCAAGATGGGAATGTTGCATCACCA
TCTTCGTTAGCTGGTACTAGTTTTGGTTCTGAGATACCCCAGGCCGGGGTGAAGCG
GGGACGAGGGCGGCCGCCGAAGGTGAAACCCTTTGGGGCGGATGTTGCCGGCAG
195
Appendix
GTGAATTGACTCCAAAGATTTTGTTTTTTTATTGGACTTGTAGTTCTTGGGAAATAT
TTGTCTTGAGTTGGCGATGCGATTTATAAATCTGGGGAACTATAGCTTTGTTGCTG
TTTCAGCTGTGTTTGCTCTTCGTGGTGCATTGTATATGATCTCTACTTGTTGTGGCT
GACT
ACTCTTCCGTTACCGCGCTTAATCACCTCGGGGATTCATAATAATGTTGCAAAAAGT
TTTAATTGCAAACCGTGGTGAAATTGCCCTGCGCATCACCCGAGCTTGCAAAACTT
TAGGAATTAAAACTGTCGGTGTCTATTCAGATGCTGACAAGGACTTGATGCATCTG
CGTTTCTGCGATGAAGCAGTATGTATCGGCCCTGGCGCAAGCAGTGACAGTTACCT
AAATATTCCAGCAATGATCACAGCAGCTGAGATTACTGGTGCGGATGCGATCCAC
VP28 GE746307
CCAGGTTATGGTTTCCTGTCTGAAAATGCTGAATTTGCTGAAATTGTTGAAAGTTCT
GGCTTTATTTTTATTGGCCCACGTCCTGAACACATTCGCCTGATGGGTAAGAAGGT
TTCTGCCATTATTGCCATGAAAAAAGCCGGCGTACCAACAGTACCAGGCTGTGATC
ATGCAGTAACCATCCACAATGCCCTTGCTGAAGCCAAAGAAATCGGTTTCCCGCTG
ATCGTAAAAGCTGCTGCGGGTGGTGGCTGACT
CCAAAAAAGACGTTTCCGGTGCAGAATCACTACGATTAATCGCTTGAATAGAGTTC
GTAAGCGCTTTCGTCTGGATATTGACAACAATGGCGTTTTTTAAACCAGCTGCAGT
AGCCCCCGGTTCTAATAATATATACGAGCATACATTAGCCGATTGCGTTGCTTCATC
I
TACAGATGCCACAATAGGACGGCCATGATACGCTGATACAAAAGCTTTGTCGGTT
R
VP29 GE746308 AACAACTTGGCAATCGCTTGATCTGAAAATGCAGATGCTGGTTGGCGCTCACCGCT
CTGTGAGGAGTAAAACAAGTCCAAACCCGCATTGTAAAGATACACACTGTCGATA
FT
TCTTTCAGTGTCAGAAGATGCTTGTCAATGACTTTGCTTGTTAAGAGAGCCACGCG
CTCATCATTTTTATTCATATTTAAAAATTCAATGATCTGATTGTCGCTATAAAGAGCC
CTCTCTAATTTTTTTGCGGTCTCGTCAAGATAGGTGATACTGTAGTTCGACTGCGCA
ATCAGTTTATCGTTATAGTGGCTGACT
C
AGCCACGCCATATCGTCGAGGGGAGAGGTGCATTCGTAACGTCTCTGTTATGGAT
GATTATGCAGCAATAGGTTCTCCACTTGATCCAGAGGAAGTCTCAGAAGGTGGAA
@
TGTTATCCAGGATGCGCATTGCTCGTTGCGATGACAACCAAAGGCGGCCGAGCTTT
VP30 GE746309
CCAGAGAGTGTTTGTGATGTTGTTTTTGGAGGAAGTGAAATCATGGTCGATGACG
TGAAGTTTACTGTTTCGCACGTAGTGGAGCCAATGGAAAGAGGAATCGCTAGGCT
ts
AAACAAAACGATTCATCAGATCTCAGAAAACATAAAGCAGCATGAAATGCGCAAG
AGAAAAGCCAAGGATGATTCTGATCTAATCCCTTTGCGTACTTGGACAAAAGAGTT
rin
TTCTGAAGCACGCGACCATGTGGCTGACT
TAGTCAGCCACTCGGATGGAACTATTAAGGTTTGGGATATTGGGAAGAGGGGTTG
GCAGTTAGTTCAGGAAGTTCAAGAACATCTGAAGGCTGTTACGGGTCTTTACATTC
eP
196
Appendix
CATAGCTGTTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA
GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACAT
TATTTGCGTTGGCGCTCACTTGCCGCTTTCAGTCGGGGAAACTGTCGTGCCAGCTG
CATTAATAATTCGGCACGCCCGGGAAAAGGGGTTGGTTTTGGGGCTCTTCCTTTCC
CCTCCACAATCCTCCCGGCT
I
R
FT
C
@
ts
rin
eP
197
Publications
Publications
Papers published
I
2: 1007-1013
R
3. Sreedhar RV, Roohie K, Maya P, Venkatachalam L, Narayan MS,
FT
Bhagyalakshmi N (2008) Biotic elicitors enhance flavour compounds
during accelerated curing of vanilla beans. Food Chemistry 112: 461-468
198
Publications
I
3. Sreedhar RV, Venkatachalam L, Kaunain Roohie, Thimmaraju R and
R
Bhagyalakshmi N (2005) Molecular analysis of genetic fidelity in long-term
micropropagated shoots of vanilla (Vanilla planifolia). Poster presented at
FT
National symposium on Plant Biotechnology: New Frontiers, held
between 18-20th November at Central Institute of Medicinal and Aromatic
Plants, Lucknow, India
C
@
ts
rin
eP
199