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Effect of Amino Acids on the Growth and Production of Steroids in Date Palm Using Tissue
Culture Technique
Sherif El-Sharabasy1, Mai Ahmed Farag2, Gehan A.E.El-Emery3, Gehan Safwat2,4 and Ayman Diab1,5
1
2
The Central Laboratory for Date Palm Research and Development
Faculty of Biotechnology, October University for Modern Sciences and Arts (MSA).
3
Institute of Efficient Productivity, Zigzag University, Egypt
4
Horticulture Research Institute, Agriculture Research Centre, Egypt
5
Agricultural Genetic Engineering Research Institute, Egypt
gigi@msa.eun.eg
Abstract: The present investigation studied the effect of amino acids (Glutamine, Spermidine and Asparagine) with
different concentration (50, 250.500 mg/l) used as precursors to produce secondary metabolites (steroids) and
growth development during different stages (callus, embryoids and shooting) of date palm (Malakaby cv.). In
Embryogenic callus stage, callus volume was the highest (4.00) when treated with any of the three amino acids, 50
mg/l of Glutamine or Asparagine showed no effect compared to the control giving the lowest callus volume (3.00).
Total steroids in callus tissues clearly showed that using Glutamine 250 mg/l in medium gave the highest steroid
content 0.662 mg/g and percentage (336% of control), while the lowest (0.111mg/g) was found with Asparagine
(500mg/l) and 56.35% of control. Glutamine at 250 mg/l resulted in the highest weight of embryos (2.100 gm). As
well as, 500 mg/l Spermidine seemed to be the best amino acid used in order to stimulate steroid biosynthesis
resulting in 202.1% of control (0.782 mg/g). In shooting stage, according to the number of shoots, the highest
number of shoots (2.33) was achieved with Glutamine and Spermidine at 500 mg/l. In shoot weight, the highest
weight (7.267 gm) was achieved by using 500mg/l Glutamine , as to steroid biosynthesis in shooting stage, the best
result obtained, were by using Glutamine at 500 mg/l which gave highest steroid biosynthesis (0.534mg/g), 206.0%
of control.
[Sherif El-Sharabasy, Mai Ahmed Farag, Gehan A.E.El-Emery,Gehan Safwat and Ayman DiabEffect of Amino
Acids on the Growth and Production of Steroids in Date Palm Using Tissue Culture Technique. Researcher.
2012;4(1):75-84]. (ISSN: 1553-9865). http://www.sciencepub.net.
Keyword Glutamine, Spermidine, Asparagine
alkaloids and terpenes for pharmaceutical industry
(Bohm, 1980; Staba, 1980; Barz and Eills, 1981;
Deus and Zenk, 1982). El-Sharabasy (2004)
indicated that the precursors have great effect in the
biosynthesis of steroids in date palm callus and
embryogenic callus cells.
The present investigation was planned to study
the effect of some amino acids used as precursors to
produce secondary metabolites (steroids and sterols),
and its effect on plant development during different
stages (callus, embryoids and shooting) of in vitro
date palm cultivation (Phoenix dactylifera L.)
Malakaby cultivar
1. Introduction
Date palm (phoenix dactylifera) is a
monocotyledonous dioecious plant. Dates are
considered the main fruit in Arab nations such as
Saudi Arabia, Iraq, and Egypt. Date palms are not
only used as fruit producing trees, but are also used in
landscape decoration. The major problem in date
palm cultivation is the poor method of vegetative
propagation. (Badawy et al., 2005). During ancient
times, Egyptians recognized the growth of the date
palm (phoenix dactylifera) and was recognized as
being a fertility symbol (Puchoaa and Ramburn,
2004).
Cholesterol, Cholestanol and Coprostanol are
the animal sterols, while, -sitosterol, Compesterol,
Stigmasterol, Ergosterol and Brassicasterol are the
principle plant sterols (Bailey, 1964). Cholesterol,
one believed to be the typical animal sterol has
recently been found to be rather widely distributed
among plants. So far, cholesterol has been identified
in the pollen of many plants including date palm
(Bennett et al., 1966) and oil palm (Slover et al.,
1983). Plant tissue cultures have long been regarded
as a source of commercially important steroids,
2. Materials and Methods
This investigation was carried out in the
laboratory of Plant Cell and Tissue Culture
Department, Biotechnology Division, The Central
Laboratory for Date Palm Research and
Development, 2010.
Plant materials
To test the effect of Glutamine, Spermidine, and
Asparagine as precursors on the growth, development
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and secondary products synthesis (steroids) in
embryogenic callus, somatic embryo and shooting
stages of date palm.
In this experiment, embryogenic callus, somatic
embryo and shooting stages of date palm (Phoniex
dactylefera L.) were treated with 0, 50, 250 and 500
mg/l of Glutamine, Spermidine and Asparagine.
Statistical analysis
The experiments were carried out using
completely randomized blocks design and with three
replicates. The results were analyzed using analysis
of variance and the means compared using L.S.D. at
the 5% level, all obtained data were subjected
analysis of variance completely randomized blocks
according to (Snedecor and Cochtan, 1980).
Tissue culture experiment
MS solid medium and 0.1 NAA were used for
embryogenic callus and somatic embryo, while in
shooting stage 0.05 mg/l BA + 0.1 NAA were used.
Nine groups of jars having 25 ml medium for every
treatment have been set. The pH value was regulated
to 5.7- 5.8 before autoclaving. The cultures were then
incubated at 27 ± 2°C temperature and 16 hours
light/day photoperiod. The following data were
analyzed after one month such as, Measurements on
the callus tissues of embryogenic callus
stage.(Volume of callus, Callus weight (gm),Total
concentration of steroid hormones were calculated
and verified by the spectrophotometer due to the
methods illustrated by Pharco in 1993). ,
Measurements on the embryos tissues of embryoids
stage (Number of embryos,Weight of embryo
(gm),Total concentration of steroid hormones and
Measurements taken on the shooting tissues of
shooting stage (Shoot number, Weight of shoots and
Total steroids)
3. Results
Effect of different amino acids as precursors on
the embryogenic callus volume of embryogenic
callus stage resulting from shoot tip of date palm
(Phoenix dactylifera L.) Malakaby cultivar
Data shown in table 1 and figure 1 demonstrates
the effect of the different precursor concentration on
embryogenic callus value derived from shoot tip of
date palm (Malakaby cultivar). These results
illustrated that the greatest results (4.00) attained with
embryogenic callus grown on medium contained 500
mg/l Glutamine, Spermidine or Asparagine. These
three precursors with concentration 500mg/l showed
the same best effect on callus volume, followed by
medium treatment with 250 mg/l Spermidine (3.67).
Using Glutamine and Asparagine with concentration
250mg/l showed the same effect on callus volume
(3.33).
Alternatively, there was no observed change in
the volume of callus when grown on medium
contained 50 mg/l, which was shown in Asparagine
and Glutamine as they were similar to the control
(3.00), but in Spermidine there was a slight
difference compared to the control (3.33).
Overall there was no examined decrease due to
the addition of precursors with the concentration
applied.
Chemical analysis
Preparation of the unsaponfiable matter (U.S.M)
according to Sharabasy (2001)
Quantitative Determination of Total Steroids in
the unsaponifiable fraction by Spectrophotometer:
The total steroids were determined in the
unsaponifiable fraction by the reaction with Denigee
reagent according to (Pharco 1993).
Table 1: Effect of different amino acids as precursors on the embryogenic callus volume of the embryogenic callus
stage of date palm (Phoenix dactylifera L.) Malakaby cultivar.
Precursors
(A)
Glutamine
Spermidine
Asparagine
Mean
L.S.D at 0.05%
0.0
3.00
3.00
3.00
3.00 B
A =0.7750
(B)
Concentration (mg/l)
50
250
3.00
3.33
3.33
3.67
3.00
3.33
3.11 B
3.44 B
B = 0.8949
Mean
500
4.00
4.00
4.00
4.00 A
AB =1.550
76
3.33 A
3.50 A
3.33 A
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Figure 1: Effect of different amino acids as precursors on the embryogenic callus volume of the embryogenic
callus stage of date palm (Phoenix dactylifera L.) Malakaby cultivar.
the most suitable amino acid to stimulate steroid
biosynthesis 0.662 mg/g and the highest
percentage (336% of control) followed by
Asparagines 250m / l which gave steroid
biosynthesis 0.261 mg/g and the percentage
(132.5% of control) where Spermidine gave the
lowest result in this stage. The data also showed
that using high concentration of different amino
acids specially 500 mg/l had negative effect on
steroid biosynthesis which was 0.159, 0.135, and
0.111 with Glutamine, Spermidine, and
Asparagine respectively.
Effect of different amino acids as precursors on
the production of total
steroids as secondary
metabolite in the embryogenic callus tissues of
embryogenic callus stage of date palm (Phoenix
dactylifera L.) Malakaby cultivar.
Data in Table 2 and Figure 2 shows the
variations in steroid biosynthesis from the
embryogenic callus tissues of date palm,
Malakaby cultivar, under the effect of different
amino
acids
(Glutamine,
Spermidine,
Asparagine) concentration used in this study.
These results clearly showed that using
Glutamine 250 mg/l in MS medium seemed as
Table 2: Effect of different amino acids as precursors on the production of total steroids as secondary
product in the embryogenic callus tissues of embryogenic callus stage of date palm (Phoenix
dactylifera L.) Malakaby cultivar.
Type of precursors
Control
Glutamine
Spermidine
Asparagine
Concentration
(mg/l)
0.0
Total steroids
Mg/g
0.197
% of control
100
50
250
500
50
250
500
50
250
500
0.223
0.662
0.159
0.250
0.161
0.135
0.219
0.261
0.111
113.3
336.0
80.71
127.1
81.85
68.63
111.2
132.5
56.35
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Figure 2: Effect of different amino acids as precursors on the production of total steroids as secondary
product in the embryogenic callus tissues of embryogenic callus stage of date palm (Phoenix
dactylifera L.) Malakaby cultivar.
contrast, the lowest increase in weight of embryos
with concentration 50mg/l was observed in
Glutamine (1.633 gm).
Glutamine added with concentration 250 mg/l to
the medium showed the best increase in weight of
embryos (2.100 gm), and then comes Spermidine
(1.33 gm) and then Asparagine (1.267 gm).
In Glutamine, Spermidine and Asparagine with
concentration 500 mg/l, there were no observed
increase in weight (1.133 gm, 1.067 gm, and 1.30
gm) compared to the other concentration 50 and 250
mg/l, but actually a significant decrease.
In general, the highest weight of embryos
(2.100 gm) in somatic embryo stage was achieved
with Glutamine at 250 mg/l concentration while the
lowest weight of embryos was obtained with
Asparagine (1.067 gm).
Effect of different amino acids as precursors on
the weight of embryos (gm) of somatic embryo
stage of date palm (Phoenix dactylifera L.)
Malakaby cultivar.
Data presented in Table 3 and Figure 3
illustrates the effect of Glutamine, Spermidine and
Asparagine as precursors on embryos weight of date
palm Malakaby cultivar. These results indicated that
there were significant difference between different
precursor’s concentration and their interactions.
Statistical analysis of variance showed that MS
medium supplemented with the low Asparagine
concentration (50 mg/l) and the low Spermidine
concentration (50 mg/l) had a significant stimulating
effect to increase the weight of embryos derived from
shoot tip. In Asparagine compared to the control, the
weight of embryos increased from 0.400gm--1.67gm
and in Spermidine, the weight of embryos compared
to the control increased from 0.400gm--1.733gm. In
Table 3: Effect of different amino acids as precursors on the weight of embryos (gm) of somatic embryo stage of
date palm (Phoenix dactylifera L.) Malakaby cultivar.
(B)
Concentration (mg/l)
Mean
Precursors
(A)
0.0
50
250
500
Glutamine
0.400
1.633
2.100
1.133
1.317 A
Spermidine
0.400
1.733
1.33
1.067
1.333 A
Asparagine
0.400
1.867
1.267
1.300
1.208 A
0.400
C
1.744
A
1.567
AB
1.167
B
Mean
L.S.D at 0.05% A = 0.3971 B = 0.4586
AB =0.7942
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Figure 3: Effect of different amino acids as precursors on the weight of embryos (gm) of somatic embryo
stage of date palm (Phoenix dactylifera L.) Malakaby cultivar.
Glutamine , 128.2% of control (0.496 mg/g) for 250
mg/l Glutamine .
The recorded data indicate that using of 500
mg/l Spermidine seemed to be the best amino acid
used in order to stimulate steroid biosynthesis
resulting in 202.1% of control (0.782 mg/g) where of
control (0.611 mg/g) for 250 mg/l and 83% of control
(0.325 mg/g) for 50 mg/l. Whereas steroid
biosynthesis showed negative correlation responses
when Asparagine concentration reached 500mg/l
which stimulate steroid biosynthesis by about 87.9%
of control.
Effect of different amino acids as precursors on
the production of total steroids as secondary
metabolites in the embryo tissues of somatic
embryo stage of date palm.
Data in Table 4 and Figure 4 records that
steroid biosynthesis responded differently to the
different amino acids (Glutamine, Spermidine and
Asparagine) concentration used in this study.
The obtained results showed that MS medium
supplemented with Glutamine 500 mg/l stimulated
the process of steroid biosynthesis and increased it by
about 132.6 mg/g of control (0.513mg/g) comparing
with 80.62% of control (0.496 mg/g) for 50 mg/l
Table 4: Effect of different amino acids as precursors on the production of total steroids as secondary
products in the embryo tissues of somatic embryo stage of date palm.
Type of
precursors
Control
Glutamine
Spermidine
Asparagine
Concentration
(mg/l)
0.0
50
250
500
50
250
500
50
250
500
Mg/g
0.387
0.312
0.496
0.513
0.325
0.611
0.782
0.270
0.414
0.340
79
Total steroids
% of control
100
80.62
128.2
132.6
83.0
157.9
202.1
69.8
106.0
87.9
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Glutamine
Spermidine
0.782
0.8
Asparagine
0.7
0.6
Total
0.5
steroids
0.4
mg/g
0.3
0.2
0.1
0
0
50
250
500
Concentration mg/L
Figure 4: Effect of different amino acids as precursors on the production of total steroids as secondary
products in the embryo tissues of somatic embryo stage of date palm.
Giving Glutamine the highest value with this
recorded concentration (50 mg/l).
Glutamine added with concentration 250 mg/l to
the medium showed the best increase in shoot
formation (2.33), and then comes Spermidine (1.667)
and then Asparagine (1.373).
In general the highest number of shoots (2.33)
was achieved with Glutamine and Spermidine at
concentration 250 mg/l, while the lowest number of
shoots (1.33) was obtained with Asparagine and
Spermidine at concentration 50 mg/l. On the other
hand, the best concentration achieved with 500 mg/l
was (2.222). Glutamine and Spermidine showed the
same number of shoot formation per embryo with
concentration 500 mg/l, while Asparagine showed the
lowest value (2.00).
Effect of different amino acids as precursors on the
number of shoots during shooting stage of date
palm (Phoenix dactylifera L.) Malakaby cultivar.
Data presented in Table 5 and Figure 5
illustrates the effect of Glutamine, Spermidine and
Asparagine as precursors on number of shoot embryo
of date palm Malakaby cv.From the presentation of
data in Table (5) and Figure (5), it appears that shoot
formation responded differently to the different
precursors used in this study. As in Glutamine with
concentration 50 mg/l added to the medium showed
2.00 shoot/embryo compared to Spermidine and
Asparagine with the same concentration (50 mg/l)
added did not show any increase in shoot/embryo
value (1.333) compared with untreated explants.
Table 5. Effect of different amino acids as precursors on the number of shoots of shooting stage of date palm
(Phoenix dactylifera L.) Malakaby cultivar.
(B)
Precursors
Concentration (mg/l)
Mean
(A)
0.0
50
250
500
Glutamine
1.333
2.00
2.33
2.333
2.00 A
Spermidine
1.333
1.333
1.667
2.333
1.667 A
Asparagine
1.333
1.333
1.373
2.00
1.50 A
1.333 B
1.556AB
1.778 AB
2.222A
Mean
L.S.D at 0.05% A = 0.6821
B = 0.7876
AB =1.364
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Figure 5: Effect of different amino acids as precursors on the number of shoots of shooting stage of date palm
(Phoenix dactylifera L.) Malakaby cultivar.
Spermidine (7.233 gm). On the other hand, the lowest
weight of shoot formed was by explants grown on
medium supplemented with 50 mg/l of Asparagine
(4.133 gm), followed by control (4.00 gm).
Data of table (8) shows that there were a
significant difference between different precursors
and concentration.
Concerning the effect of concentration of each
precursor added under test, it could be mentioned that
increasing precursor level up to 500 mg/l increased
shoot formation and weight.
3.1
Effect of different amino acids as
precursors on shoot fresh weight (gm) of shooting
stage of date palm (Phoenix dactylifera L.)
Malakaby cultivar.
Data presented in Table 6 and Figure 6 clearly
shows the effect of Glutamine, Spermidine and
Asparagine precursors on shoot fresh weight (gm) of
date palm (Malakaby cv.)
The highest weight of shoot was recorded for
MS basal medium supplemented with 500 mg/l of
Glutamine (7.267 gm), followed by 500mg/l of
Table 6: Effect of different amino acids that as precursors on shoot fresh weight (gm) of shooting stage of
date palm (Phoenix dactylifera L.) Malakaby cultivar
Precursors
(B)
Mean
(A)
Concentration (mg/l)
0.0
50
250
500
Glutamine
4.00
6.133
7.00
7.267
6.100 A
Spermidine
4.00
4.167
5.167
7.233
5.142 A
Asparagine
4.00
4.133
4.167
5.967
4.567 A
4.00 B
4.811 AB
5.44 AB
6.822 A
Mean
L.S.D at 0.05% A = 2.079 B = 2.400
AB =4.157
biosynthesis processes in shoot tissues comparing
with control in the most treatments.
Steroid biosynthesis positively correlated with
increasing Glutamine concentration. The values
recorded in this case were 0.157 mg/g (60.85% of
control), 0.423 Mg/g (163.9 of control) and 0.534
Effect of amino acid as precursors on the
production of total steroids as secondary products
in the shoot tissues of shooting stage of date palm.
Data of Table 7 and Figure 7 clearly shows that
the different amino acids stimulate steroids
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Mg/g (206.0% of control) for 50, 250 and 500 mg/l
Glutamine
respectively.
However,
steroid
biosynthesis in the shoot tissues was responding
differently to Spermidine concentration, whereas it
was decreased by increasing Spermidine level from
50mg/l to 500 mg/l which gave 0.393mg/g (152.3%
of control), 0.273 Mg/g (105.8% of control) and
0.260 Mg/g (100.8% of control) respectively.
Increasing Asparagine concentration from 50mg/l to
500 mg/l decreased steroid content in shoot tissues.
In general the best result, were in the usage of
Glutamine at 500 mg/l which gave highest steroid
biosynthesis content (0.534mg/g), 206.0% of control.
The lowest (0.157 mg/g) was obtained with
Glutamine 50 mg/l, 60.85% of control.
Table 7: Effect of amino acid as precursors on the production of total steroids as secondary products in the
shoot tissues of shooting stage of date palm.
Type of precursors
Concentration
Total steroids
(mg/l)
Mg/g
% of control
0.0
0.258
100
Control
50
0.157
60.85
Glutamine
250
0.423
163.0
500
0.534
206.0
50
0.393
152.3
250
0.273
105.8
Spermidine
500
0.260
100.8
50
0.390
151.2
250
0.316
122.5
Asparagine
500
0.285
110.5
Glutamine
Spermidine
Asparagine
0.6
0.534
0.5
0.4
Total
steroids
mg/g
0.3
0.2
0.1
0
0
50
250
500
Concentrations mg/L
Figure 7: Effect of different precursors on the production of total steroids as secondary product in the shoot
tissues of shooting stage of date palm (Phoenix dactylifera L.) Malakaby cultivar.
found to play a vital role on tissue culture techniques
of certain species (Benson, 2000; Liu, 1993).
Amino acids have been applied in medium of
numerous species as alfalfa, maize, sorghum,
pineapple, rice and other monocots as a nitrogen
supply in in vitro cultures to improve somatic
4. Discussion
Amino Acids are the essential components in
the procedure of protein synthesis. A lot of studies
have verified that amino acids can directly or
indirectly control the physiological actions of the
plant. Amino acids added to medium have been
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embryogenesis growth (Skokut et al., 1985; Claparols
et al., 1993; Rao et al., 1995; Hamasaki et al., 2005;
Grewel et al., 2006).
In Embryogenic callus stage there are a lot of
causes that could lead to the increase or decrease in
embryogenic callus volume and wasn’t enclosed only
in the amino acids that was added as precursors to
examine growth rate of callus volume. For instance,
Thomas and Rao (1985) found on oil palm that after
several subcultures on MS medium with sequentially
reduced 2.4-D the callus formed nodular callus
masses which were maintained on a continuous
source for the induction of embryogenesis.
Abou El-Nil (1989) reported various amino
acids that resulted callus growth in plants and
categorized them as follows: first Glutamine, second
Asparagine, third Arginine, fourth Serine, fifth
Glycine and sixth Alanine. Glutamine caused
doubling callus growth compared to control.
El-Sharbasy in 2004 indicated that the highest
value of callus volume in Sewi cultivars was
achieved with callus grown on medium contained
0.01mg/l cholesterol, while lowest value was
observed in case of adding 0.01mg/l of Squaline, and
the highest callus weight was obtained when MS
basal medium supplemented with 1 mg/l Squaline
while the lowest was in 10 mg/l of Pyruvic acid.
Fermandes-Ferreira et al. (1991); FermandesFerreira (1992) found that the sterols Compesterol, βsitosterol, Avenasterol and the Triterpenal -amyrin
are the main constituents of the unsaponifiable
fraction obtained from calli and suspended cells of
Euphorbia characias. Squalene and trace amounts of
hydrocarbons,
namely
nonacosane
and
hentriacontane, were also identified into the
sunsaponifiable fractions obtained from these in vitro
cultures.
El-Sharabasy 2004 indicated that the highest
value of steroids diffused by the callus in the medium
was Pyruvic acid in the concentration 10mg/l and
recorded (609.1%) higher than control. While the
lowest ones were those 0.01mg/l Pyruvic, 10mg/l
Squalene and 0.01 mg/l Cholesterol.
El- Sharbasy 2004 indicated that the highest
embryos weight was recorded with cholesterol
treatment 0.1 mg/l and the lowest was that of
pyruivic acid 10 mg/l.
Amino acids showed effects on the production
of secondary metabolites, for instance, Walton in
1988 made experiments using transformed roots
instead of cell suspensions, it showed that feeding
Putrecine at 1-5 mM caused a 100% increase in the
nicotine content of transformed roots of Nicotiana
rustica compared to controls.
Acknowledgements
We wish to express our sincere gratitude to
Professor. Abd El-Menem El-Bana, the director of
the Egyptian Central Laboratory for Date Palm
Research and Development for giving the chance to
second author for accomplishing her graduation
project. We would sincerely like to thank Dr. Zainab
El- Sayed and Dr. Waleed Ahmed for their kind cooperation toward the completion of this work
Corresponding author
Gehan Safwat2,4
2
Faculty of Biotechnology, October University for
Modern Sciences and Arts (MSA).
3
Institute of Efficient Productivity, Zigzag
University, Egypt
gigi@msa.eun.eg
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