Soybean [Glycine max (L.) Merr.] is an important summer crop in Egypt. The
crop has high seed protein content ranged from 30 to 35% and about 29% seed oil
content. The crop area was about 100,000 feddan in 1991, but it has reduced to be
17,000 feddan only in 2006. The crop usually attacks by several leaf-feeding insects,
amongst, cotton leaf worm (Spodoptera littoralis, Boisd) is considering the main leaf
feeding insect. Cotton leaf worm causes extensive defoliation in plant leaves of
susceptible varieties, which affect photosynthesis processes and hence reduce yield.
The farmers still prefer to grow susceptible varieties such as Clark, Giza 82 and Giza
22. Hence, insect infection still exists in soybean fields and insecticides still widely
used.
Molecular marker generally refer to assays that allow the detection of sequence
differences between two or different individuals. It contain three level isozymes or
other protein based, DNA based and RNA based. Molecular markers are useful tools
for developing detailed linkage maps of species that previously were very poorly
mapped. These maps have obvious utility for the identification of markers linked to
genes of agronomic or insect resistance importance. Beyond this practical utility,
molecular markers and molecular maps can provide detailed information regarding.
The potential benefits of using markers linked to genes of interest in breeding
programmes have been identified for many decades. Random amplified polymorphic
DNA (RAPD) markers were first described in 1990. The analysis for RAPD markers
is quick and simple, although results are sensitive to laboratory conditions. Restriction
Fragment Length Polymorphisms (RFLPs) are markers detected by treating DNA with
restriction enzymes (enzymes that cut DNA at a specific sequence). RFLPs were the
first molecular markers to be widely used. Their use is, however, time-consuming and
expensive and simpler marker systems have subsequently been developed.
2
CHAPTER (I)
FIELD PERFORMANCE AND VARIATIONS OF
SOYBEAN GENOTYPES
INTRODUCTION
Soybean [Glycine max (L.) Merr.] is an important summer crop in Egypt. The
crop has high seed protein content ranged from 30 to 35% and about 29% seed oil
content. The crop area was about 100,000 feddan in 1991, but it has declined sharply
during last decade and reached 50,000 feddan in 1998, then reduced to be 30,000
feddan only in 2004. The main reason behind this reduction is the competition from
other summer crops as corn, rice and cotton. In addition, soybean has higher
production cost and lower net return comparing with corn. Another important reason is
the insect infestation. The crop usually attacks by several leaf-feeding insects,
amongst, cotton leaf worm (Spodoptera littoralis, Boisd) is considering the main leaf
feeding insect. Cotton leaf worm causes extensive defoliation in plant leaves of
susceptible varieties, which affect photosynthesis processes and hence reduce yield.
Management of insect control is important in increasing soybean production. Several
cotton leaf worm resistant soybean varieties have been released at Agricultural
Research Center in Egypt.
3
REVIEW OF LITERATURE
Kulvir et al. (2000) studied the relationships among soybean traits. The results
indicated positive correlation of No. of pods/plant, grains/pod and harvest index with
seed yield. Straw yield was positively correlated to pod bearing, branches/plant and
biological yield. Biological yield was positively correlated to 100-seed weight, pod
bearing branches/ plant but was negatively correlated with harvest index. Plant height,
dry matter and leaf area were positively correlated to biological and straw yields but
negatively correlated to No. of seed yield. Harvest index showed negative correlation
with almost all the characters under study except grains/pod.
4
variance components was 43.4% for leaf area, 63.2% for leaf shape, and 29.1% for
maturity, which were lower than values for days to flowering and flowering period
due to large error variances (sigma superscript 2E) caused by field environmental
factors.
Sudaric et al. (2002) estimated the efficiency and reliability of soybean yield
components as selection criteria for grain yield and to evaluate the agronomic value of
14 domestic cultivars (from maturity groups 0 to II) as potential parents for further
genetic improvement of grain yield. Mean values, coefficient of variation and broad-
sense heritability were calculated for grain yield and the following yield components:
plant height (cm), numbers of fertile nodes, pods and seeds/plant, seed weight/ plant
(g), harvest index (%) and 1000-seed weight (g). Path-coefficient analysis was used to
determine the contribution of the yield components to total grain yield. Seed weight
and number of seeds/plant had the lowest variability, the highest heritability and the
highest positive direct effect on grain yield. Among the tested cultivars, Ika had the
highest mean for both yield components. The obtained results suggested that the
indirect selection for higher soybean grain yield using seed weight and number of
seeds/ plant was more efficient and more reliable than selection using the other yield
components. Among the tested cultivars, Ika appeared to be the most suitable as a
parent in future hybridizations to achieve further genetic advance in soybean grain
yield.
5
maturity and number of grains/pod were also correlated. Days to maturity was
significantly correlated with plant height and days to 50% flowering at the phenotypic
and genotypic levels. The number of days to 50% flowering was positively and
significantly correlated with days to 50% flowering but negatively with number of
pods/plant and 100-grain weight at the genotypic level. The 100-grain weight did not
show any correlation with grain yield. Path coefficient estimates showed that the
number of grains/pod, days to maturity, number of pods/plant and plant height
positively affected grain yield.
Alghamdi (2004) evaluated 5 soybean genotypes (Giza 35, Crawford, Giza 82,
Clark and Giza 111) in six sowing dates (25th of February, March, April, May, June
and July) in Saudi Arabia. The response of seed weight and seed yield varied from
genotype to another across different environmental conditions. Giza 111 and Clark
had high mean performance and phenotypic stability and they could be grown over a
wide range of environments. Giza 111 and Clark had the highest, while Giza 82 and
Giza 35 had the lowest, mean values over all environments and poorly adapted. The
results suggested that to maximize seed yield potential, genotypes which have a
consistently high yield performance across diverse growing environments should be
selected, and more than one year of evaluation.
Mukhekar et al. (2004) path analysis and correlation studies were carried out
using 65 genotypes of soybean. Seed yield was significantly and positively associated
with the number of pods/ plant, days to 50% flowering, mean internodal length, plant
height, days to maturity and the number of branches/ plant. Pod length had significant
and negative correlation with the seed yield. Pods/ plant had the highest direct as well
as final contribution to the seed yield and can be considered as most reliable yield
indicator in soybean.
6
genotypes. Mean values, broad-sense heritability, genetic gain and relative genetic
gain from selection were calculated for grain yield components, grain yield, protein
and oil content in grain. The results of quantitative genetic analysis indicated progress
through breeding in grain quantity and quality traits that will contribute to further
improving and increasing soybean production in the region. Likewise, recent breeding
materials represent good genetic basis for future hybridizations aimed at advancing the
quantity and quality of grain yield in soybean genetically. Grain yield components
were determined as more reliable criteria for selection of superior genotypes than
grain yield/se due to higher heritability (61.87-82.31%) and better progress in
selection (10.63-22.78%). Grain quality traits had medium heritability (60.04-65.89%)
and better progress in selection (6.95-8.94%) compared to grain yield that had less
heritability (29.87%), and the relative genetic gain from selection was 0.43%.
Mello et al. (2004) evaluated the effects of selection for high protein on seed
physiological quality and grain yield of soybean in Brazil. Four populations of BC1F4
and 4 of F4, each from a cross between a commercial variety and a line bearing high
protein seeds were used. The high protein content selection has a tendency to affect
negatively the seed physiological quality. Estimates of correlation coefficients
between protein content and grain yield were mostly negative but varied among
populations. It is possible to obtain lines with high protein content, keeping the grain
yield and the seed physiological quality of their respective recurrent progenitors.
Turkec (2005) estimated the relationships between seed yield and some yield
components as well as the direct and indirect effects of these characters on seed yield
in soybean. Seed yield was significantly and positively correlated with pods/ plant and
with branches/plant. The highest positive correlation was found between seed yield
and pods/plant. Path coefficient analysis indicated that pods/ plant expressed the
highest positive direct effect, followed by 100-seed weight, on seed yield.
Oh et al. (2005) released a new soybean cultivar Bosug in Korea. Bosug has a
semi determinate growth habit, purple flower, grayish brown pubescence, brown
hilum, and rhomboidal leaflet shape. The maturity date of Bosug is 4 days earlier than
the control cultivar, Pungsan. It has a good seed quality and high isoflavone
(3.891micro g/g) and amino acid contents (396 mg/g) for soybean sprout. It has a 100-
seed weight of 8.6 g and exhibits resistance to lodging and soybean mosaic virus. The
average yield of Bosug was 2.62 t/ha the old cultivated.
Liu et al. (2005) investigated the dynamics of dry matter accumulation, Leaf
area index (LAI) and, leaf area duration (LAD) during the reproductive period for the
high-yielding 16 genotypes. Majority of the seed yield and components were
positioned in the middle and upper part of the plant. Both pod number and seed
number were higher in high-yield genotypes in each group. Higher accumulation of
dry matter, higher LAI and LAD during reproductive stages were found to be closely
7
related to high-yield genotypes in each group. No relationship was found between
harvest index (HI) and seed yield.
Khalil (1988) the major constraints faced by Egyptian soybean producers are
germination/emergence problems largely as a result of soil capping, non-adoption of
seed inoculation despite lack of Rhizobium japonicum in Egyptian soils and lack of
resistance to leaf-feeding insects, e.g. cotton leaf worm (Spodoptera littoralis), in early
maturing cv. Changes taking place during seed development and maturation and
factors affecting seed quality such as field environment, seed-borne diseases and
cultural practices are discussed.
Afifi (1990) conducted field studies in Egypt during 1986 and 1987 to
determine the relative distribution of egg-masses and larvae of Spodoptera littoralis in
soybean fields. On average, 95.5% of egg masses were laid on the lower surface of
leaves with 4.5% laid on the upper surface. The larval population consisted of 1st- and
8
2nd-instar larvae (59-62%), 3rd- and 4th-instar larvae (27-28%) and 5th- and 6th-
instar larvae (12-15%).
Awadallah et al. (1990) tested soybean genotypes in the laboratory for their
resistance to Spodoptera littoralis. Plants descended from the hybrid H2 were the most
resistant. The cultivar Celest was resistant and the cultivar Crawford was of
intermediate resistance. Other hybrids were less resistant.
Rizk et al. (1991a) collected eggs of Spodoptera littoralis from the field in
Egypt and reared for 3 generations in the laboratory on soybean, cotton, grapes or
sweet potato. The esterase activity in individuals from each of the colonies was
investigated and compared to that of a laboratory strain. The most specific esterase
activity was recorded in the tissues of larvae reared on soybean and the least in the
laboratory strain, followed by those reared on sweet potato. The larvae reared on sweet
potato were the most susceptible of the non-laboratory strains to deltamethrin,
alphamethrin [alpha-cypermethrin], cypermethrin, flucythrinate and cyhalothri]n.
Rizk et al. (1991b) investigated the effect of food plants on the biology and
susceptibility to insecticides (deltamethrin, cypermethrin, alphamethrin [alpha-
cypermethrin], flucythrinate and cyhalothrin) of Spodoptera littoralis in the
laboratory. Larvae were reared on cotton, soybean, grapes and sweet potato for 3
generations. The different food plants affected the development periods, pupation,
emergence, number of eggs laid and longevity of the insect. Larvae reared on sweet
potato were the most susceptible to the synthetic pyrethroids.
9
compared with F1. Significant difference between the mean weight gain of larvae that
fed on leaves of F1 and F2 was also observed. There was no significant difference
between the mean weight gain of larvae that fed on the leaves of F1 and F3
populations. Chi superscript 2 analysis of F2 and F3 populations gave a good fit to the
1 resistant: 2 segregating: 1 susceptible ratio expected for incomplete dominance at a
single locus. Broad-sense heritability estimates of 46.80 and 58.44% for F2 and F3
populations, respectively, showed that resistance to defoliation in soybean, as
indicated by larval weight gain, is reasonably heritable.
Sun and Gai (1999) studied the resistance of 6 soybean varieties (the resistant
varieties Wujiangqingdou-3, Tongshanbopihuangdoujia and Gantai-2-2, and the
susceptible varieties Wan82-178, Shandongdadou and Morsoy) to Prodenia litura
[Spodoptera litura] under laboratory conditions. There were significant differences
between the levels of leaf consumption, but not of Oviposition antixenosis, of S. litura
on the resistant and susceptible varieties. Following feeding of S. litura larvae on
resistant varieties, larval leaf consumption, and larval and pupal body weight
decreased, while mortality and the duration of development of larvae alone increased.
On the basis of these results, it is concluded that the resistance of soybean to P. litura
is mainly antibiotic.
10
MATRIALS AND METHODS
Fourteen exotic and Egyptian soybean genotypes [Glycine max (L.) Merr.]
selected from the germplasm collection of Soybean Breeding Program at ARC were used
in this study. The genotypes included resistant and susceptible genotypes as indicated in
Table (1). Two experiments were carried out at Giza research station, ARC, at Giza
Governorate in the two successive summer seasons 2003 and 2004. Planting take place
on 25 may in both seasons. In each experiment a randomized complete block design,
with 3 replicates and 7.2m2 plot size (4 ridges, 3 m long and 0.60 m wide, with
33 plants /m2) was used. Fertilizer, irrigation and all agronomic practices were applied as
recommended.
In all experimental plots the following characters were recorded on 10 randomly
selected competitive plants: plant height (cm), number of branches/plant, first pod height
(cm), number of pods/plant, number of seeds/plant and seed yield/plant. The following
characters: days to 50% flowering, days to 90% maturity, maturity period (days to 90%
maturity - days to 50% flowering) and 100-seed weight were recorded on the plot basis.
At harvest, soybean plants in the central 3 m2 in each experimental plot were pulled by
hand (the remaining plot area was discarded to avoid border effect), placed in cotton
sacks, air dried, weighed, then threshed by hand and clean seeds weighed. Seed protein
content was estimated by using the micro Kjhldahl method, where total protein was
determined as total nitrogen in the seed. Percentage of seed protein content was
calculated by multiplying the total sample-nitrogen by 6.25 according to the method of
A.O.A.C., 1990). The total seed-oil (lipids) content was estimated by the method
described in A.O.A.C. (1990), and then the percentage of seed oil content was calculated.
Evaluation of the tested genotypes for their resistance to leaf cotton warm infection
was made in both under field conditions and under laboratory conditions. In the field
experiments under natural insect infection, the evaluation was done using a visual rating
scale as a percent of leaf defoliation levels suggested by Smith and Brim (1979) and
applied in soybean at ARC in Egypt by Habeeb (1988) and Lutfallah et al. (1998). In
this scale, the average of three readings/each plant in every experimental plot (every 7
days) started at two weeks after flowering were recorded. The following levels of leaflet
area consumed by insect were used: (1) 1-10% (resistant); (2) 11-20%; (3) 21-30%
(intermediate); (4) 31-40%; (5) 41-50% (susceptible) and (6) >50% (highly susceptible).
In laboratory evaluation, a survival technique was used (Meisner and Moshe, 1983).
11
In this experiment 10 young larvae (1-8 days old) were placed in a Petry dish and
allowed to feed on fresh soybean leaflets collected from the upper third of a plant taken
randomly from each field experimental plot in each replicate. Other experiment was also
made using similar procedures but with adult larvae (9-17 days old). The first laboratory
experiment called survival 1 and the second laboratory experiment called survival 2. In
both experiments, number of survival and died larvae was counted after 3 days, and
percentage of survival number of larvae was estimated. The large number of survival
indicated high susceptibility to the insect infection.
The analysis of variance of field experiments was made for each season separately,
and then a combined analysis of variance was performed for the two seasons (Gomez
and Gomez, 1984). For laboratory experiments, the analysis of variance was made for
each of experiment (survival 1 and survival 2) separately. Simple correlation
coefficients among all studied characters were performed. The variance components
and coefficients of variation were estimated by the formulae suggested by Burton
(1952). The broad sense heritability and genetic advance were estimated using the
formulae suggested by Allard (1960).
12
RESULTS AND DISCUSSION
The single analysis of variance for each season showed that significant differences
among genotypes were existed for all studied characters. The combined analysis of
variance indicated significant differences among genotypes for all characters, while
the season had significant effects on plant height, no. of pods/plant, no. of seeds/plant,
seed yield/plant and harvest index. The genotype x season interaction effect was
significant in days to 50% flowering, days to 90% maturity, maturity period, plant
height, seed yield/plant and 100-seed weight.
Seasonal effects:
The data showed that number of pods/plant, no. of seeds/plant, seed yield/plant
and harvest index had higher values in the first season than in the second season
(Table 4 and 5). For example, seed yield/plant increased from 67.14 g in the second
season to 71.82 g in the first season. These data reveal that the growth conditions at
Giza were more favorable in the first season than in the second season. The average
maximum and minimum air temperatures in the first season (May-October) were
33.85 and 19.88 oC respectively, while the corresponding temperatures in the second
season were slightly warmer and recorded 33.93 and 20.96oC. Moreover, the soil
temperature during the same period was relatively cool in the first season (24.7 oC)
comparing with the second season (30.6oC). It seems that the temperature in the first
season was more favorable for soybean growth and hence increased the yield and yield
components. The importance of seasonal conditions on various plant characters was
reported previously by Hamdi et al. (1991) in lentil.
Performance of genotypes:
Phenological characters:
The performance of genotypes for phenological characters (days to flowering,
days to maturity and maturity period) and morphological characters (plant height, first
pod height and no. of branches/plant in 2003 and 2004 seasons are presented in Tables
(2 and 3). The average days to 50% flowering for all genotypes were 36.98 and 37.27
days with ranges of 31.49 and 31-48.7, in the first and the second seasons, respectively
(Table 2 and 3). Similar trend was observed for days to maturity and maturity period,
where most genotypes were matured earlier in the first season than in the second
season. The earliest genotypes in flowering, maturity and maturity period were L86K-
73 and Giza 82. These genotypes could be exploited as a source of earliness in
soybean breeding program. Most early-flowered genotypes were also earlier in
maturity and had also short duration in maturity. It seems that there are strong
relations between these three characters as confirmed by the highly significant
correlation between them. The correlation coefficient between time to flowering and
maturity is (r = 0.913**), between flowering and maturity period is (r = 0.782**) and
between days to maturity and maturity period is (r = 0.968**), (Table, 8).
Morphological characters:
Plant height exhibited wide ranges of 61- 89 cm in the first season and 63 –
89.31cm in the second season, with averages of 67.29 and 72.19 cm, in both seasons
respectively (Tables, 2 and 3). The tallest genotypes were Giza 111 (89 cm) in the first
season and Lakota (89.31 cm) and Calland (89.10 cm) in the second season. If not
subjected to lodging, tall plants are preferred, since they have more bud bearing nodes
with the potential for higher seed yield and are also suitable for mechanical harvesting.
The average plant height obtained in the present study is within the range of those
found by Zayed (1998) in Egypt. Number of branches/plant ranged from 1.33 to 4.00
and from 1.70 to 3.50 in the first and the second seasons, respectively. In previous
13
studies, the range of number of branches/plant was 3.71 – 10.25 (Habeeb, 1988). The
genotype Lakota had low first pod height, reflecting more bud bearing nodes than
other genotypes.
14
Table 2: Average of phenological and morphological characters for 14 soybean
genotypes evaluated under field conditions at Giza research station in 2003
season.
Days to Days to Maturity Plant height 1st Pod Number
Genotypes flowering maturity period (cm) height of branches
(day) (cm)
L86K-73 31.00 97.00 66.00 61.00 6.00 1.33
Corsay-79 34.00 114.00 80.00 64.00 5.00 2.00
Giza21 37.00 122.00 85.00 70.00 6.00 3.33
Forrest 48.00 135.00 87.00 66.00 7.00 4.00
Hutcheson 49.00 138.00 89.00 `67.00 7.00 2.70
Calland 39.00 123.00 84.00 69.00 7.00 2.00
Lakota 32.33 98.00 66.00 68.00 5.00 2.70
Giza111 39.00 117.00 79.00 89.00 6.33 2.70
Giza83 33. 00 108. 00 75.00 69.00 6.00 3.00
Clark 38.00 122.00 84.00 66.00 5.33 3.33
Giza22 38.00 111.00 72.00 71.00 6.00 3.00
Giza35 34.00 107.00 73.00 69.00 6.00 3.00
Giza82 31.00 98.00 67.00 67.00 7.00 2.00
Crawford 38.00 120.00 82.00 72.00 5.00 3.33
Average 36.98 113.74 76.71 67.29 5.98 2.69
L.S.D.5% 1.820 2.381 2.309 1.728 1.317 1.191
15
Table 4: yield component characters for 14 soybean genotypes evaluated under
field conditions at Giza research station in 2003 season.
Genotype No. of No. of No. of 100- seed Seed yield/ Biological Harvest
Seeds/ Pods/ Seed/ weight Plant Yield/plant index
Plant Plant Pod (g) (g) (g) (%)
L86K-73 306.00 183.00 1.70 13.13 40.19 103.433 38.91
Corsay-79 325.00 201.00 1.63 14.80 48.10 116.300 41.35
Giza21 440.00 247.00 1.80 15.23 67.01 136.83 48.97
Forrest 612.00 269.00 2.30 14.90 91.16 317.400 28.72
Hutcheson 600.00 264.00 2.30 15.10 90.67 310.400 29.20
Calland 588.00 252.00 1.90 15.70 92.40 300.30 30.77
Lakota 335.00 200.00 2.20 15.20 50.92 120.700 42.19
Giza111 590.00 241.00 2.23 16.40 96.75 253.20 38.12
Giza83 490.00 238.00 1.83 16.30 80.62 222.97 36.16
Clark 480.00 273.00 1.93 14.60 70.10 205.57 34.10
Giza22 430.00 212.00 1.87 16.00 68.79 203.57 34.07
Giza35 420.00 245.00 1.73 16.10 67.62 201.27 33.60
Giza82 325.00 180.00 1.80 15.50 50.40 125.40 40.17
Crawford 560.00 220.00 2.60 16.20 90.73 193.33 46.95
Average 464.38 230.36 1.98 15.37 71.82 200.76 37.38
L.S.D.5% 16.30 3.10 0.313 0.209 2.70 4.95 1.003
16
Table 6: Average of seed oil content, seed protein content, leaf area, number of
hairs, and level of resistance as survival 1 and 2 and field scale characters
for 14 soybean genotypes evaluated under field conditions at Giza research
station in 2004 season.
Genotype Oil Content Protein Leaf area Number Level of resistance
(%) content (cm2) of hairs
(%) Surv1 Surv 2 Field
%
L86K-73 22.35 49.58 18.12 99.67 0.33 2.00 10
Corsay-79 25.83 51.92 26.57 50.00 7.70 7.33 20
Giza21 24.63 37.69 70.24 112.00 1.33 2.67 20
Forrest 23.67 22.70 48.95 103.00 1.00 2.67 30
Hutcheson 26.10 31.24 46.20 58.00 8.33 7.67 30
Calland 23.43 36.40 34.03 41.00 5.00 6.67 30
Lakota 24.30 51.90 53.23 48.00 7.70 9.00 15
Giza111 23.27 38.58 38.10 70.67 1.33 2.33 20
Giza83 24.10 57.93 33.71 173.33 2.33 3.67 10
Clark 22.81 56.61 61.23 41.33 8.00 8.00 30
Giza22 23.45 46.90 54.15 82.00 2.00 2.33 30
Giza35 23.43 55.49 68.32 138.33 0.70 2.33 20
Giza82 24.71 41.21 59.51 99.67 1.00 2.33 15
Crawford 24.27 39.19 63.39 61.33 7.70 9.67 35
Average 24.02 43.62 48.27 84.17 3.88 4.91 22.5
L.S.D.5% 0.088 0.515 19.66 27.69 1.77 1.76 3.68
17
CHAPTER (II)
TISSUE CULTURE ORGANOGENESIS
INTRODUCTION
Soybean (Glycine max (L.) Merr.) is important oil and poultry feed crop in Egypt.
The crop has been mainly grown in the Nile valley and the Delta since 1972. The growing
area has declined from 100,000 feddan in 1991 to less than 38,000 feddan in 2004. The
reduction in the area is due to high production cost and lower net return comparing with
corn, the main competitor summer crop to soybean. The crop production faces several
constraints. Insect infestation during vegetative growth stage by feeding insects such as
cottons leaf worm (Spodopetra littoralis, Boisd.) and green cotton leaf worm (Spodopetra
exigua Hubner) usually causes dramatic yield losses. The seed yield reduction in soybean
due to infection by cotton leaf worm in Egypt ranged from 36.6% to 42.7%.
Therefore, insecticides are extensively used in soybean fields, which rising production
cost, causing accumulation of undesirable residues and increasing environmental pollution.
The use of host-plant resistance is necessarily to solve most of those problems. Despite
several cotton leaves worm-resistant soybean varieties have been released such as Giza 21,
Giza 35, Giza 83 and Giza 111 the farmers still prefer to grow susceptible varieties such as
Clark, Giza 82 and Giza 22. Hence, insect infection still exists in soybean fields and
insecticides still widely used.
The first transgenic plants with resistance to insects contained genes for insecticidal
proteins called 8-endotoxins from the soil microorganisms, Bacillus thuringiensis (Bt). Bt
protected cotton, potato, and corn were introduced to the market place in 1996. They
succeeded to regenerate plants from callus, driven from immature embryos. To obtain callus
organogenesis or somatic embryogenesis is depending on the composition of the medium.
Organogenesis resulted when embryos were plated on Murashige and Skoog, 1962 (MS)
medium, which contained high concentration of micronutrients. This technique has been used
successfully to perform callus and plant regeneration from callus in soybean. Despite the fact
that biotechnology offers good option for genetic enhancement of crop plants, little in vitro
work has been done in soybean in Egypt.
Prior to gene transfer, responding soybean genotypes to organogenesis in tissue
culture technique needed to identify. The aim of this study was to initiate and maintain callus
organogenesis cultures of soybean. The differences among soybean genotypes in callus
formation were also evaluated.
REVIEW OF LITERATURE
Barwale et al. (1986) used the callus derived from immature embryos; regeneration of
viable plants was obtained in soybean (Glycine max (L.) Merr. ). Depending on the
composition of the medium, regeneration occurred via embryogenesis or via organogenesis.
Embryogenesis resulted when embryos were plated on Murashige and Skoog (MS) medium
containing 43uM NAA. In his work with the cultivar Williams 82, the addition of 5.0 uM
thiamine HCl increased embryogenesis from 33% to 58% of embryos plated. Addition of 30
uM Nicotinic acid to the MS medium enhanced embryogenesis Further to
76%.Organogensises was obtained when medium containing 13.3 uM 6-benzylamino purine,
.02 uM NAA and four times the normal concentration of MS minor salts was used histological
studies theses culture confirmed the organogenic and embryogenic nature of the culture, by
demonstrating the formation of shoot buds and somatic embryos respectively. Similar
responses were obtained in all54 genotypes tested in manner. The culture retained the ability
to regenerate complete plants for leas 12 menthes and at 12-15 subcultures. Seed have been
obtained from several regenerated plants and when grown in the field theses produced normal
appear in fertile plants.
Parrott et al. (1989) found that the genotypes had a large effect on the ability of immature
embryo soybean cotyledons undergo auxin stimulated somatic embryogenesis.
Widholm et al. (1990) evaluated nineteen genotypes that had low and high level of Fe
efficiency in the laboratory and 5 field locations. Friable callus was induced from epicotyls
section, weighed and placed on modified MS media, on low NAA (0.02mgNAA and 50 um
Fe EDTA in control media) Callus growth was rated as a lack of growth compared to
respective controls.
Yue et al. (1990) cultured immature embryos 6 to13, 14, to 21, 22 to 28, days after
pollination 13 accessions of 8 Glycine species showed that beast callus and root formation
from members of the subgenus G. Soja which included G. max, G. graceless gave better callus
formation, organogenic and plantlet regeneration than G. max.
Yang et al. (1990) compared various explants for organogenesis and plant regeneration in
soybean (Glycine max Merr.). Young cotyledons produced organic calli, from which a
deventituties buds and shoots were produced by culture in vitro. Flowering and pod
development were observed on regenerated shoots even in vitro, but the recovery of plants
was very inefficient. Histological studies revealed weak connection of the regenerated bud
primodia with differentiated tissues recovery in this culture system. Plant regeneration could
also take place on plumule of young embryo explants. The regeneration process started with
the enlargement of the plumule followed bud the production of the adventitious buds.
Adventitious buds regenerated much more readily from cotyledonary nodes and some from
the plumules in mature embryo explants. An improved culture protocol for efficient plant
regeneration in soybean culturing explants from immature embryos and acclimatizing
regenerated plants at the early stage is proposed.
Zhou et al.(1990) published the ability to induced organogenesis from immature embryos
of soybean Glycine max using medium containing 3 ppm 6-benzylamino purine , 0.05ppm a
naphthalene acetic acid and 3to 4 the concentration of MS minor salts. Both increasing and
decreasing concentration of 6-benzylamino purine reduced the frequency of organogenesis.
20
The best length of immature embryo for 5 to 6 mm. genotypic variation in the frequency of
organogenesis was noted.
Amer (1992) demonstrated the modifications of the culture media were made to stimulate
the growth of shoots formed by organogenic soybean (Glycine max (L.) Merr.) The result
revealed the best alterations for stimulating organogenic shoots were found to be a decrease in
the MS mineral salts content to one half with supplements of 0.203mgL-1 (1uM) indolbutric
acids.
Widholm et al.(1993) Found that deficiencies of B and Zn had a greatest effect on callus
weight, while Mn had only a slight effect. Despite this, significant differences in callus weight
reduction were observed on the three different media. The results indicated that genotypic
variation for response to B, Zn and Mn deficiency is present in soybeans at cellular level.
Settu and Kumari (1998) found the plant regeneration was studied in cotyledon explants
taken from in vitro-grown soybean cv. Co.1 seedlings. Callus induction and proliferation was
best on MS medium containing 2 mg NAA + 0.5 mg BAP [benzyladenine] L-1. Shoot bud
formation was maximum in MS medium with 0.5 mg NAA + 3 mg BAPL-1. Regenerated
shoots developed roots when transferred to MS medium containing 2 mg IBA + 2 mg kinetin.
Kosturkova (2000) this paper reviews the research on the induction of somatic
embryogenesis, organogenesis, and plant regeneration of soybean in tissue cultures. Different
factors affecting the process of morphogenesis, such as genotype, plant growth regulators,
nutrients and light, are discussed.
Roy and Maloo (2001) evaluated a six-parent soybean diallel for four in vitro callus
growth and quality parameters. Significant genotypic variation was observed for fresh and dry
weight of callus colonies and callus protein content. These traits appeared to be predominantly
under the control of additive gene effects as evidenced by the higher magnitude and
21
significance of general combining ability compared to specific combining ability mean
squares. Average midparent heterosis was non-significant for the callus growth traits but
significant and low for callus protein content. Results in general indicated the scope of
selection for superior in vitro callus response in soybean.
Tomlin et al. (2002) assessed seventeen breeding lines of soybean, Glycine max, and cv.
Jack, from relative maturity groups 0.3-7.5 for their ability to undergo somatic embryogenesis.
The goal of this study was to determine which lines had high embryogenic capacity. We also
sought to understand the relationship between relative maturity and embryogenesis. Embryos
from immature cotyledons were initiated on solid MS medium with varying levels of 2, 4-
dichlorophenoxyacetic acid (2,4-D). Qualitative and quantitative measures of initiation,
proliferation, differentiation, and maturation were recorded. The breeding lines differed
significantly with respect to percent induction, number of embryos induced, and quality of
induced embryos. After 1 month of proliferation, two early maturing lines, the control, Jack,
and NK-5, had the best overall performance. High percent response of proliferating embryos
was positively associated with lower maturity groups. Relatively high concentrations of 2,4-D
(compared with that used in proliferating medium, e.g., 226 UM; 50 mg l-1) in the initiating
medium reduced numbers of embryo clusters per cotyledon initiated and percent initiation,
and the concentration of 2,4-D affected the proliferation of somatic embryos in a breeding
line-dependent manner. The breeding lines differed significantly in the time to produce mature
somatic embryos. There was a positive correlation between immature embryo quality and
number of differentiated somatic embryos produced.
Yoshida (2002) reported a new system for simple and efficient shoot regeneration of
soybean (Glycine max cultivars Ohsuzu, Kosuzu, Suzukari, Suzuyutaka, Tachiyutaka and NT-
98-236) is using the hypocotyl of mature seeds. Two transverses of 1-mm sections of the
hypocotyl were cut from mature seeds: sections containing a cotyledonary node and sections 1
to 2 mm from the cotyledonary node. The effects of thidiazuron (TDZ) concentrations, plating
methods, and genotypic differences were examined. Shoots were formed from cotyledonary
node ends in all conditions examined. Meanwhile, shoots were formed from the ends of
hypocotyl sections on B5 media containing TDZ. The optimal TDZ concentration for
organogenesis was 2-10 UM. The efficiency of organogenesis varied with the method of
plating of explants. The ends of the hypocotyl sections in contact with the media only
produced adventitious shoots. Adventitious shoots were formed effectively by placing
explants on the media oriented so that the hypocotyl axes were perpendicular to the surface of
the media. The efficiency of organogenesis differed with the position of the hypocotyl ends. It
was important to use the ends that were 1 mm from the cotyledonary nodes. Genotypic
differences were observed in the organogenesis of the hypocotyl ends.
Manoj and Sharad (2003) obtained somatic embryo induction from hypocotyl explants
of 11 soybean genotypes (Bragg, JS 72-280, JS 72-44, JS 75-46, JS 80-21, JS 90-41, JS 335,
MACS 13, NRC 2, Punjab 1 and PK 472) cultured in Murashige and Skoog's medium
supplemented with 30 mg BAP [benzyladenine] + 0.5 mg NAA L-1 (medium A), 1.0 mg BAP
+ 1.0 mg NAA L-1 (medium B), or 0.5 mg BAP + 0.2 mg IAA L-1 (medium C). Callus
proliferation was initially observed on the second week after inoculation. The formation of
embryoid-like structures was also observed during this period. In a few cases, both
embryogenesis and organogenesis were evident on the same callus. Callus induction,
morphogenic callus formation and shoot induction significantly varied with the medium and
genotype. Medium A was superior in terms of overall callus initiation (87.43%) and shoot
induction (35.31%). Medium A and medium B resulted in the greatest formation of
22
morphogenic calluses. Among the genotypes, JS 80-21, JS 335 and JS 90-41 were the most
responsive to in vitro culture. JS 80-21 (89.73%), MACS 13 (88.86%), JS 90-41 (85.41%) and
JS 75-46 (84.42%) exhibited the greatest callus formation. The greatest proportion of calluses
resulting in morphogenesis was observed in JS 335 (56.86%), JS 90-41 (54.03%), JS 80-21
(49.40%) and MACS 13 (48.79%). The interaction between genotype and culture medium was
also significant. Regeneration was most pronounced in JS 90-41 cultured in medium A, JS 80-
21 and Bragg cultured in medium B, and NRC 2 cultured in medium C.
Smolov and Oleinikova (2003) studied the role of light during exogenous assimilation of
nitrate (the only source of nitrogen) by the callus cells of soybean (Glycine max). The nitrate
consumed and assimilated by the photosynthetic (mixotrophic) and nonphotosynthetic cells
(heterotrophic and chlorophyll-containing cells cultivated in the light in the same medium
complemented with diuron) was quantified. The assimilated nitrate was quantified at the final
stage of the growth cycle as the difference between the amount of nitrogen consumed from the
medium and the amount of endogenous nitrate in the cells. Comparison of the assimilated
nitrate quantities per accumulated dry biomass between the photosynthetic and
nonphotosynthetic cells demonstrated that nearly 30% of nitrate is assimilated with the
involvement of photosynthesis in a mixotrophic culture when nitrate is the only source of
nitrogen.
Sairam et al. (2003) developed an efficient protocol for callus induction and plant
regeneration in three elite soybean cultivars (Williams 82, Loda and Newton). The technique
is most novel in that the shoot buds developed from the nodal callus. Callus induction and
subsequent shoot bud differentiation were achieved from the proximal end of cotyledonary
explants on modified Murashige and Skoog (MS) media containing 2.26 UM 2,4-
dichlorophenoxy-acetic acid (2,4-D) and 8.8 UM benzyladenine (BAP), respectively. Varying
the carbon source optimized the regeneration system further. Among the various carbon
sources tested, sorbitol was found to be the best for callus induction and maltose for plant
regeneration.
Reichert et al. (2003) classified in the US, soybean genotypes into maturity groups (MG;
total of 13) that represent areas of adaptation generally correlated with latitude bands. To
determine if one regeneration procedure could regenerate representatives from diverse areas of
adaptation, 18 soybean genotypes representing nine MG were compared for organogenic
adventitious regeneration and plant formation from hypocotyl explants following the
procedure previously tested on representatives from only three MG. Responding explants
were those capable of producing shoots on the acropetal end of the explant from either the
outer edge plus central region or the central region only. This enabled determination of the
contribution of cotyledonary nodal tissue (outer edge) to shoot regeneration and by
discounting those explants; it also enabled estimates of true adventitious regeneration. All 18
genotypes were capable of producing meristemoids/shoots solely from the central region with
responses ranging from 28.5 to 64.3% after 4 weeks in culture. All genotypes were also
capable of producing elongated shoots that could be successfully rooted. No morphological
differences were noted among regenerants, or between them and seed-initiated plants. All
regenerants produced viable seed which germinated and produced morphologically normal
plants. This study confirmed the genotype- and MG-independent nature of this hypocotyl-
based organogenic regeneration procedure and provided conservative estimates for responses
that were truly/solely adventitious in nature.
23
Kim et al. (2004a) established an efficient acclimation system for regenerated plantlets of
soybean; we used various media with hydroponic nutrient solutions before regenerants were
transplanted into soil. The hydroponic nutrient solution was essential for the survival of the
plantlets. The vermiculite with nutrient solution at pH 5.5 was found to be the best medium
with 97-100% survival rate and better growth of regenerants plantlets. Regenerated grew best
in the following order of solutions: Yoshida solution, modified Yoshida solution, Soy I, Soy
II, and MS medium. However, Soy I solution (EC 2.9 mS/cm), developed by the Honam
Agricultural Research Institute proved to be the most effective for acclimation in terms of the
time required for vigorous growth and economical use of chemicals.
Kim et al. (2004b) studied a successful, efficient system for multiple shoot induction and
plant regeneration of soybean (Glycine max) was established. Four soybean genotypes were
compared for organogenic responses on various media cultured under light conditions. The
adventitious shoots (98%, 2.6 shoots per cotyledon) directly from one-day-old cotyledon after
germination induced by the hormone treatment and its efficiency were higher than any other
conditions. The optimum medium for the induction of multiple shoots from cotyledon in
Pungsannamulkong (shoot formation rate, 98%), Lx 16 (83%) and Ilpumgeomjeongkong
(63%) was the MS medium supplemented with 2 mg BAP [benzyladenine]/l, but for
Alchankong (75%), it was the MS medium supplemented with 1 mg zeatin and 1 mg IAA/l,
3% sucrose and 4% Phytagel. Higher root induction (88%) was observed from the shoots
placed on rooting medium (hormone-free MS basal). Plantlets were transferred onto the same
medium supplemented with 1% activated charcoal for further development. With this
treatment, regenerated plantlets were obtained within 7-8 weeks (shoot induction for 4 weeks,
rooting and shoot elongation for 3-4 weeks).
Manoj and Sharad (2004) cultured leaf discs excised from 7- to 10-day-old seedlings of
soybean cultivars Bragg, JS 72-280, JS 72-44, JS-75-46, JS 80-21, JS-90-41, JS 335, MACS
13, NRC 2, Panjab 1 and PK 472 in Murashige and Skoog's (MS) medium supplemented with
30.0 mg 2,4-D, 10 mg PCPA [p-chlorophenoxyacetic acid] + 0.5 mg BA [benzyladenine]
(MS10PB) or 3.0 mg PCPA + 0.5 mg BA/litre for callus initiation. All callus types were
transferred into an MS medium without growth regulators for the germination of somatic
embryos. The MS medium for plantlet regeneration was modified with 0.4 mg BA and 0.5 mg
NAA, and 20 g sucrose/litre. In the absence of rhizogenesis, the shoots were transferred into
an MS medium containing 1.0 mg IBA and 15.0 g sucrose/litre. Callus initiation, evident on
the second week of incubation, was greatest in JS 90-41 (84.34%), MS10PB medium
(75.56%), and MACS 13 cultured in MS10PB (89.04%). The highest percentage of
morphogenic calluses was recorded for JS 90-41 (41.76%), MACS 13 (39.05%) and JS 75-46
(36.82%); MS10PB medium (36.67%); and MACS 13 cultured in MS10PB (50.67%). Most of
the calluses produced plantlets after prolonged culture in the induction media. The number of
shoot-forming calluses significantly varied with the cultivar and culture medium. The highest
percentages of shoot-forming calluses were registered for JS 90-41 (25.11%) and MACS 13
(22.18%), and in MS10PB medium (19.21%). Plantlet regeneration was greatest in JS 90-41
(18.0%) cultured in MS10PB. This medium was also optimum for plantlet regeneration in
other cultivars.
Manoj and Sharad (2004a) cultured Immature embryos of 11 soybean genotypes were
cultured on MS medium supplemented with different levels of growth hormones, namely 8.0
mg NAA/litre (MS8N), 2.0 mg NAA + 0.2 mg benzyladenine/litre (MS2NB) and 3.0 mg
benzyladenine + 0.5 mg NAA/litre (MS3BN). Highly significant differences were observed in
the response of genotypes, culture media and genotype x medium interactions for callus
24
initiation and plantlet regeneration. The combination of benzyladenine at high rates and NAA
at low rates was the most responsive for soybean tissue culture.
Manoj and Sharad (2004b) found high frequency of morphogenic callus induction was
attained in soybean (G. max) from immature cotyledons. Eleven soybean genotypes (JS 72-
280, JS 72-44, JS 75-46, JS 80-21, JS 90-41, JS 335, MACS 13, NRC 2, Panjab 1 and PK
472) and three media viz., MS30D (MS medium supplemented with 30 mg 2,4-D/litre),
MS3BN (MS medium supplemented with 3 mg benzyladenine/litre + 0.5 mg NAA/litre) and
MSPB (MS medium supplemented with 3 mg PCPA/litre + 2.5 mg benzyladenine/litre) were
tested for in vitro morphogenic efficiency. Morphogenesis was dependent on the genotype and
the explant inoculation medium. Plant regeneration efficiency was highest in genotype JS 90-
41 (53.35%) followed by JS 80-21 (41.0%) when in MS3BN culture medium. Phenotypically
normal plants were regenerated from the immature cotyledons explants.
Park et al. (2004) determined the best explant source, culture media and growth regulators
for the regeneration of multiple shoots from cotyledonary node and hypocotyl explants of
soybean cv. Iksannamulkong. More shoots were regenerated from cotyledonary nodes than
hypocotyl explants. Among the 5 culture media tested, MSB medium (MS with B5 vitamins)
was the most effective for obtaining more regenerated shoots. Addition of BA (2.0 mg/litre),
zeatin riboside (0.05 mg/litre) and thidiazuron (2.0 mg/litre) to the MSB medium were
effective for obtaining enough number of regenerated shoots from cotyledonary nodes. Zeatin
riboside was the most effective cytokinin, producing an average of 15.5 regenerated shoots per
cotyledonary node and giving 75% shoot regeneration. Thus, for efficient shoot regeneration
of soybean in vitro, it is recommended to plate cotyledonary nodes onto MSB medium
supplemented with zeatin riboside at 0.05 mg/litre.
Franklin et al. (2004) regenerated soybean (Glycine max cultivars PNP, Dekalb,
Sandusky, CNRR 279 and CB 277) plantlets were efficiently regenerated from mature and
immature cotyledons of five different cultivars by studying various parameters affecting
regeneration. Green organogenic nodules were induced at the proximal end, which
subsequently differentiated into shoot buds on modified Murashige and Skoog (MS) medium.
The presence of 6-benzyladenine (BAP at 13.3UM) and thidiazuron (TDZ at 0.45-22.71 UM)
in the medium exerted a synergistic effect, in that regeneration efficiency was higher than for
either cytokinin alone. The regenerated shoot buds elongated and rooted on MS medium
containing 0.29 UM gibberellic acid (GA3) and 2.69 UM alpha -naphthalene acetic acid
(NAA), respectively. Rooted plants were established in the greenhouse with 87% success and
produced viable seeds. Preliminary studies with Agrobacterium show great promise for
soybean transformation based on the regeneration protocol reported here.
Sharad et al. (2004) cultured anthers of ten genotypes of G. max on four fortified B5
media supplemented with different levels of growth hormones, i.e. B5 DBIG (2.0 mg 2,4-D
litre-1+0.5 mg IBA l-1+100.0 mg myo-inositol l-1+360.0 mg L-glutamine l-1), B5 DB (2.0
mg 2,4-D l-1+0.5 mg BA l-1), B5DK (2.0 mg 2,4-D l-1+0.5 mg kinetin l-1) and B5BKN (0.5
mg BA l-1+0.5 mg kinetin l-1+1.0 mg NAA l-1). All the media were supplemented with 90.0
g sucrose l-1 and 7.0 g agar l-1. Significant differences in the response of genotypes, culture
medium and genotype x medium interactions were observed for callus initiation, formation of
morphogenic calluses and plantlet regeneration. Genotype JS 90-41 was found superior for in
vitro androgenesis. B5DBIG exhibited higher response for androgenic callus formation and
haploid plant regeneration compared to other media.
25
Patil et al. (2005) determined the morph types of callus in soybean (cv. Bragg) and their
potential ability in plant regeneration. In vitro-germinated seedlings (7-10 days old) from
untreated and treated (20 and 25 kR) seeds were used as sources of explants, i.e. cotyledons
and leaf segments. The explants were cultured on MS basal medium fortified with different
growth hormones such as BAP [benzyladenine], IBA, 2, 4-D and IAA in different
concentrations and combinations. To maintain the culture, subculturing was carried out in the
same media after every 2 weeks and finally subjected to different media to obtain
differentiation. The surface of the callus was noticed to be of 3 types, i.e. smooth, rough and
rough granular. The texture of the callus was either compact or friable. Varied ranges of callus
colour were observed for the explants used. The callus obtained was of embryonic, rhizogenic
and non-embryonic morph types.
Tiwari and Tripathi (2005) five separate experiments were conducted for immature
embryonic axes, immature cotyledon, mature cotyledon, and hypocotyl and leaf disc cultures
with 11 soybean genotypes. For each explant culture, at least 3 different fortifications of basal
MS medium were used. Various cultured explants initiated mainly 4 types of calluses: (a)
undifferentiated callus cultures which were cream in colour and soft friable in texture, (b)
compact, dark green in colour with few or many bead-like structures, (c) compact, dark green
with few or many dark green bead-like structures and sometimes partially covered with a thin
layer of white loose callus, and (d) mixture of creamy white and light green calluses which
were dense and glossy in texture. Culture media played an imperative role in the formation of
embryonic calluses. Hypocotyl, followed by mature cotyledon, proved to be the superior
explants for somatic embryogenesis, and mature cotyledon for plantlet regeneration.
26
MATERIALS AND METHODS
Seven exotic and Egyptian soybean genotypes selected on the basis of their
reaction to cotton leaf worm infection, were obtained from Food Legume Research
Program, Field Crops Research Institute, ARC, Giza, Egypt (Table 9). The genotypes
were grown in the field at Giza research station in 25th May 2003 and 2004. Sowing
was done at a crop density of 33 plants/m2 in 3-meter long ridges, 60cm apart and 4
ridges per plot. Fertilizers at 30 kg P2O5/fed and 15 kg N/feddan were added to the soil
prior to planting. All agronomic practices were applied as recommended. In early pod
initiation stage, 40 young pods were collected from the plants of each genotype and
moved to the laboratory immediately.
Immature seeds were taken out from young pods and then surface sterilized by
immersion in a solution containing 30% commercial bleach Clorox with a drop of
Tween 80 (polyethylene sorbitan monooleate) for 20 min. The immature embryos
(with 0.5-10 mm long) were excised from the seeds by taking the seed coat off and
then cutting next to the hilum. The immature embryos were immersed in an
organogenesis medium (MR), which prepared according to Murshige and Skoog
(1962) and Gamborg et al (1968) and presented in Table (10).
Table 9: Origin and main characteristics of the seven-tested soybean genotypes.
Name Origin Pedigree Characteristic
L86K-73 USA L73-4673 X L73-0132 Maturity group no. I, white flower color.
Corsoy-79 USA Corsoy X Lee 68 Maturity group no. II, white flower color.
Forrest USA Dyer X Bragg Maturity group no. V, white flower color.
Hutcheson USA - Maturity group no. VI, purple flower color.
Lakota, USA Selection Maturity group no. II, purple flower color.
Giza 21 Egypt Crowford X Celect Maturity group no. IV, purple flower color.
Giza 83 Egypt Selection from MBB80-133 Maturity group no. II, purple flower color.
The immature embryos were then incubated under complete darkness at 25oC±2 for 4
weeks. After 4 weeks the percentage of callus induction was calculated as (number of
explants performed calli/total number of used explants) x 100, and callus growth rate
was measured as callus weight (g). To perform shoots, callus were put on MSR
medium (Table 2) at 25oC at day and 18oC at night with 16 h day (light source was
from cool white fluorescent lamps 80 um photons m-2s-1). The calli that did not
perform shoots during 3 weeks were sub-cultured to new MSR medium. This
procedure was repeated every 3 weeks till callus perform shoots. The callus that
performed 1-cm long shoots was transformed to glass tubes containing the hormone-
free MS medium (Murashige and Skoog, 1962) for rooting formation. When
reasonable number of roots is grown, usually after 3-4 weeks, the plantlets were
removed from glass tubes and planted in 10-cm diameter- plastic pots filled with
fumigated soil mixture of peat and sand with a ratio of 3:1. The pots were placed in
green house at Giza research station and were covered with polyethylene bags. To
maintain optimum air humidity surrounds plant. Irrigation was done with Hogland
solution (0.25) (Hogland and Arnon, 1950).
27
of variance was made for each character and the simple correlation among all
characters was calculated (Gomez and Gomez, 1984).
Table 10: Composition of nutrient media for callus initiation (OR) and shooting
formation (MSR) as described by Murashige and Skoog (1962) and
Gamborg et al. (1968).
Components OR (Mg/l) MSR (Mg/l)
Macronutrients
NH4No3 1650.00 1650.00
KNO3 19000.00 19000.00
MgSo4.7H2O 370.00 370.00
KH2Po4 170.00 170.00
CaCl2 .2H2O 440.00 440.00
Micronutrients
KI 4X 0.830 0.830
H3Bo3 4X 6.200 6.200
MnSo4.H2O 4X 22.300 22.300
ZnSo4.7H2O 4X 10.600 10.600
NaMaO4.2H2O 4X 0.250 0.250
CoCl2.6H2O 4X 0.025 0.025
Na EDTA 4X 37.250 37.250
FeSO4.7H2O 4X 27.850 27.850
Vitamins B5
Nicotinic acids 1.00 1.00
Thiamin-HCl 10.00 10.00
Pyridoxine-HCl 1.00 1.00
Myoinsitol 100.00 100.00
Amino acids
Proline 1381.00 1381.00
Hormones
NAA 0.0372 -
BAP 2.996 0.383
IBA - 0.0406
Additions
Thiamin-HCl 1.687 -
Nicotinic acids 3.693 -
Sucrose 30000.00 30000.00
Agar 8000.00 8000.00
pH 5.8 5.8
28
RESULTS AND DISSCUSSION
Seven exotic and Egyptian soybean genotypes were used in this study. Several
types of explants have been used in previously reported plant-regeneration studies in
soybean, but immature embryos have been successfully cultured to produce plants
(Christianson et al., 1983). Thus cultures in this study were initiated from immature
embryos at early developmental poding growth stage with length from 0.5 to 10 mm.
Sterilized immature embryos obtained from each of the tested soybean genotypes were
cultured on OR organogenesis medium. During the first week explants were enlarged,
but no calli have been observed. In the end of the second week callus began to initiate
on OR medium, which consisted of 4 times of micronutrients, Proline (1381 mg/l),
NAA (0.0372 mg/l) and BAP (2.996 mg/l). The calli obtained were vigorously
growing, fragile and had greenish color (Fig.1A). Calli obtained were subculture onto
MSR media, which consisted Proline (1381 mg/l), BAP (0.383 mg/l) and IBA (0.0406
mg/l). Within 4 weeks of culturing, shoots were produced (Fig. 1B). The newly formed
shoots growing on the shooting media were translated to rooting medium when shoot
length reached 3-5 cm. Roots were grown well (Fig. 1C) in MS medium with hormone
free. These results are in accordance with those of Ghanem (1995) on hyoscyamus,
who found that free hormone-MS medium was the best among five rooting media and
gave the best root formation. After 3-4 weeks of root formation, the plantlets were
grown enough to be transferred to pots for adaptation (Fig. 1D).
The performance of callus and plantlet characters for all tested genotypes is
presented in Table (11). Callus induction frequencies among genotypes were different
and ranged from 63% for Corsoy-79 to 79% for L86K-73, but with no significant
differences among genotypes (Table, 11). This result indicating that all tested
genotypes had almost equal responses to callus induction with culture method used in
this study. The callus growth rate ranged widely among genotypes. The genotype
L86K-73 gave the highest growth rate value of 1.18 g followed by Corsoy-79 with
0.93 g (Table, 11). The genotype L86K-73 performed also the highest number of
shoots/callus (16.25), while all other genotypes had markedly lower number of
shoots/callus, which ranged from 3.75 to 9.75. No significant differences observed
among genotypes for percentage of plantlets performed roots and diameter of roots.
The genotype L86K-73 had also the longest root of 14.25 cm and laid among the best
three genotypes performed the highest number of roots. These data indicting that the
genotype L86K-73 is the best in response to tissue culture technique in soybean.
29
Figure 1: Illustration of Callus induction (A), Shooting stage (B),
Rooting stage(C) and Adaptation (D)
Table 11: Mean performances of callus and plantlet characters for seven
tested soybean genotypes.
Genotypes Callus Callus No. of Plantlet No. of Length Diameter
induction growth Shoot/ performed roots of root of root
(%) rate callus roots (%) (cm.) (mm)
L86K-73 79.00 1.180 16.25 20.633 6.00 14.25 2.625
Corsoy-79 63.00 0.930 8.75 26.806 7.00 11.75 1.625
Forrest 66.00 0.135 7.00 22.173 4.50 13.00 2.300
Hutcheson 67.00 0.086 3.75 36.250 3.00 11.25 2.525
Lakota 69.00 0.278 4.50 23.750 4.50 7.75 2.825
Giza21 68.00 0.118 9.75 21.023 7.00 6.75 1.950
Giza83 69.00 0.100 8.75 28.819 4.00 12.50 2.375
L.S.D 5% NS 0.247 2.209 NS 2.254 5.164 N.S.
30
Table 12: Correlation coefficients among all studied characters.
Callus No. of plantlet No. of Root Root
Character growth shoot performed roots length diameter
rate root %
Correlation coefficients among all studied characters were calculated and presented in
Table (12). The Data showed that callus growth rate was positively and significantly
correlated with each of number of shoots/callus and number of roots. Therefore, both
characters are considering important indicator for callus growth rate, and could be used
to predict succeeding of callus growth.
31
CHAPTER (III)
Agrobacterium Establishment Transformation System of
Soybean Using Immature Embryos and Cotyledonary Nodes
INTRODUCTION
Soybean (Glycine max (L.) Merr. 2n=40) is one of the world's leading
protein and oil crops. In Egypt soybean is important oil and feed crop and source
of protein in different forms. Soybean production faces several problems, such
as poor establishment, and damage by insects. Several technologies included
transformation and molecular characterizations are already used extensively for
plant improvement to overcome such problems. The early biotechnology
application in soybean was the transformation of the resistant gene to Round up;
herbicide, which has been widely adopted by farmers over the world.
32
REVIEW OF LITERATURE
Byrne et al. (1987) determinates the response of Glycine max, G. soja and G.
canescens genotypes to inoculation with different Agrobacterium strains were
assessed. Percentage visible tumour formation and tumour size varied widely among
species and genotypes. Susceptible genotypes displayed a heightened response to
nopaline strains of A. tumefaciens, relative to octopine, agropine and A. rhizogenes
strains. A nopaline strain engineered to contain a chimaeric neomycin
phosphotransferase II gene conferred kanamycin resistance on soybean tissue at
kanamycin levels as high as 300 microg /ml.
33
(pTiBo542) and strain LBA4404 carrying an octopine type virulence (vir) region and
a binary vector (pBin6) with a chimaeric gene for kanamycin detoxification gave rise
to tumours, of which 25% were both kanamycin resistant and capable of hormone-
independent growth. Single-strain inoculations with LBA4404 (pBin6) failed to give
rise to kanamycin-resistant callus. Syringaldehyde, a compound which induces vir
genes carried on the Ti plasmid, increased the number of galls incited on excised
cotyledons by the weakly virulent octopine type strain A348 (pTiA6). Similar results
were obtained with whole plants treated with this strain in the presence of the vir-
inducing compound acetosyringone. It is suggested that transformed soybean cells
can be recovered after coinfecting with a supervirulent strain or after adding a
phenolic compound to the inoculum.
Nan et al. (1998) the Bacillus thuringiensis CryIAc gene (encoding a protein
conferring insect resistance) was introduced into the protoplasts of soybean cultivars
Heinong 35, Heinong 37, Hefeng 25 and Hefeng 35 using the PEG method.
Following screening with 30 mg/litre hygromycin and differentiation of selected
resistant calli, three regenerated plants were obtained and transplanted successfully.
PCR analysis of the DNA from the transplanted plants showed a positive reaction.
Southern blot analysis of the PCR-positive plants proved that the CryIAc gene had
integrated into the genome of these plants.
34
Agrobacterium-mediated transformation of this tissue has not been demonstrated.
This papers reports the transformation of embryogenic suspension cultures of soybean
using the 'Sonication-Assisted Agrobacterium-mediated Transformation' (SAAT)
technique. For SAAT of suspension culture tissue, 10-20 embryogenic clumps (2-4
mm in diameter) were inoculated with 1 ml of diluted (OD600nm 0.1-0.5) log phase
Agrobacterium and sonicated for 0-300 s. After 2 days of co-culture in a maintenance
medium containing 100 micro M acetosyringone, the medium was removed and
replaced with fresh maintenance medium containing 400 mg TimentinReg. /litre. Two
weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg
hygromycin and 400 mg TimentinReg. /litre and the medium were replenished every
week thereafter. Transgenic clones were observed and isolated 6-8 weeks following
SAAT. When SAAT was not used, hygromycin-resistant clones were not obtained.
Southern hybridization analyses of transformed embryogenic tissue confirmed T-
DNA integration.
Wang et al. (1999) pBinLK carrying two insecticidal genes, pea lectin (P-
Lec) gene and soybean Kunitz trypsin inhibitor (SKTI) gene, was successfully
transferred into 4 upland cotton (Gossypium hirsutum) cultivars (Xinluzao-1,
Xinluzhong-2, Jihe-321 and Liao-9) via Agrobacterium-mediated transformation.
After co-cultivation of hypocotyl segments with A. tumefaciens, kanamycin-resistant
calli were screened, and somatic embryos and regenerated plants were obtained
through various media. Transgenic cotton plants harbouring two insecticidal genes
were confirmed by NPT-II ELISA, PCR and PCR Southern. The results of bioassay
demonstrated that the transgenic plants showed significant resistance to the larvae of
cotton bollworm (Heliothis armigera [Helicoverpa armigera]).
35
were found to have a higher embryogenic potential. Analysis of Agrobacterium and
immature cotyledon explant interactions involved two Agrobacterium concentrations
for the inoculation phase and three co-culture regimes. No differences in explant
survival or somatic embryogenic potential were observed between the two
Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the
shorter co-culture times resulted in higher explant survival and higher somatic
embryo production on the explants, whereas the co-culture time of 4 days severely
reduced survival of the cotyledon explants and lowered their embryogenic potential.
Analysis of selection regimes revealed that direct placement of cotyledon explants on
medium containing hygromycin 25 mg/litre was detrimental to explant survival,
whereas medium containing 10 mg/litre gave continued growth and subsequent
somatic embryo development and plant regeneration. The overall transformation
frequency in these experiments, from initial explant to whole plant, was 0.03%. Three
fertile soybean plants were obtained during the course of these experiments.
Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of
T-DNA and expression of the GUS-intron gene in the three primary transformants.
Analysis of 48 progeny revealed that three copies of the transgene were inherited as a
single Mendelian locus.
Ronde et al. (2001) used a reproducible gene transfer technique for soybean
would be useful for improving cultivars. Several plant transformation methods are
available, but regeneration from cell culture is required, which is a problem in
soybean, as tissue culture procedures have not yet been efficiently coupled to
transformation for all its varieties. We report here a non-tissue-culture
Agrobacterium-mediated transformation of soybean seed using beta -glucuronidase as
a reporter gene. The method involves subjecting partially germinated seed to vacuum
infiltration in the presence of A. tumefaciens. This method is simple and rapid, and
transformed plants can be obtained directly at high frequency. Transformation was
confirmed using PCR and Southern hybridization analysis.
36
the template amount was produced. Using the resultant linear relationships as
standard curves, we were able to determine the zygosity of both soybeans segregating
for the cry1Ac transgene and that of a T1 peanut segregating for the hph transgene. In
the second assay, a relative determination of copy number (referred to as comparative
Ct) was performed on transgenic soybeans by comparing the amplification efficiency
of the transgene of interest to that of an endogenous gene in a multiplexed PCR
reaction. Both methods proved to be sufficiently sensitive to differentiate between
homozygotes and hemizygotes. These assays have numerous potential applications in
plant genetic engineering and tissue culture, including the hastening of the
identification of transgenic tissue, selecting transformation events with a low number
of transgenes and the monitoring of the transmission of transgenes in subsequent
crosses.
El-Shemy et al. (2002) introduced two plasmid vectors into soybean (Glycine
max (L.) Merr.) and azuki bean (Vigna angularis Willd. Ohwi & Ohashi) using
different transformation systems. Azuki bean epicotyl explants were prepared from
etiolated seedlings and co-cultivated with Agrobacterium tumefaciens for 2 days.
Adventitious shoots were developed from the callus of the explants on a regeneration
medium containing hygromycin, and the shoots were excised and transferred to a
rooting medium containing hygromycin at the same concentration. Rooting shoots
were transferred to soil and grown in a glass-house to produce viable seeds. PCR
analysis confirmed clearly the presence of the hpt gene in most of the azuki beans
regenerated under hygromycin selection. A soybean embryogenic suspension culture
was generated from immature cotyledons, and used for the introduction of plasmids
by particle bombardment. Hygromycin-resistant embryogenic clones were isolated
after 8 weeks of hygromycin selection, and then the green clones were matured on the
differentiation medium. After desiccation, the embryos were germinated on the
rooting medium, and the plants were transferred to soil in a glass-house. More than
50% of the regenerated soybean plants tolerant to hygromycin yielded the hpt
fragment on PCR analysis. The azuki bean transformants were obtained more rapidly
and with higher efficiency than the soybean transformant.
37
was completely inhibited when inoculated immature cotyledons were incubated on a
kanamycin selection medium. These findings clearly demonstrated that the tissue
culture protocols for transformation of soybean should be established under both
Agrobacterium and selection conditions.
38
transgenic T0 soybean plants derived by transformation using strain KYRT1
contained TR from pKYRT1 in addition to the uidA gene from the binary construct.
None of the transgenic tissues or T0 plants contained TL DNA. These results suggest
that some function coded for by TR of pKYRT1 influences somatic embryogenesis in
conjunction with exposure of the plant tissues to 2, 4-D. Since the co-transformation
frequency of the undesirable T-DNA sequences from the vir helper plasmid was
relatively low, the partially disarmed strain KYRT1 will likely be very useful for the
production of normal transgenic plants of diverse soybean cultivars.
Wang et al. (2004) reviewed the current research status on transgenic methods
and receptor systems in soybean. The major obstacles of genetic transformation in
soybean and possible approach for solving the problem are also discussed.
Cotyledonary node via Agrobacterium tumefaciens-mediated and immature cotyledon
via particle bombardment were thought to be the efficient systems of genetic
transformation. Three problems exists in genetic transformation of soybean, i.e.
further improvement of tissue culture techniques, low efficiency and difficulty of
genetic transformation and successful transformation of restricted soybean genotypes
as a receptor. The path of solving these problems needs to set a new and highly
efficient system for tissue culture in soybean. Also the number of target genes to be
transformed should be increased from single to several genes simultaneously.
39
canescens G1171, from hard, green, nodular calluses. Accessions unable to regenerate
buds produced only undifferentiated friable callus. For G1171, medium containing 10
mg benzyladenine and 0.05 mg IBA/litre gave the highest frequency of shoot bud
formation with 30% of tissues responding. When transferred to B50 medium, highly
branched, plagiotrophic root systems developed. Silver nitrate-staining compounds,
which comigrated with agropine, mannopine and mannopinic acid standards, were
present in transformed roots and shoot of G1171, whereas tissues from non-
transformed control plantlets lacked opines.
Delzer et al. (1989) evaluated ten soybean lines adapted to Minnesota for their
ability to regenerate in tissue culture. All the lines produced adventitious shoots and
regenerated plants, with an overall average of 23.2 and 6.2 per cotyledonary node,
respectively, and a shoot: plant conversion frequency of 26.4%. Experimental line
HHP had the highest number of shoots (31.5) and plants (9.1) per cotyledonary node
and Hodgson 78 had the highest conversion frequency (40.8%). Agrobacterium-
mediated transformation of Hodgson 78 and Peking, a maturity group III line, was
unsuccessfully attempted.
Luo et al. (1994) inoculated cotyledons from germinating seeds of cv. Peking
with virulent Agrobacterium tumefaciens strain A281:pZA7, carrying the wild-type Ti
plasmid pTiBo542 and the disarmed Ti plasmid pZA7, containing the GUS (uidA)
and NPT (nptII) genes. Tumours were produced on all inoculated explants and 82%
of these tumour lines were co-transformed by the nptII gene from pZA7 as shown by
polymerase chain reaction analysis (18 of 22 lines tested). Of these 18 lines, 11 were
also resistant to kanamycin. Of 11 lines with GUS activity, 6 were also kanamycin
resistant.
40
the 81 regenerated plants survived, 3 of which developed into plants with 7 pods.
PCR and dot hybridization showed that 7 plants had been transformed. Seeds of
transformed plants were germinated and grew into plants of normal phenotype.
Xing et al. (2000) assembled a binary vector, pPTN133 that harbored two
separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a
GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strains
EHA101. Mature soybean cotyledonary node explants were inoculated and
regenerated on medium amended with glufosinate. Transgenic soybeans were grown
to maturity in the greenhouse. Fifteen primary transformants (TO) representing 10
independent events were characterized. Seven of the 10 independent T0 events co-
expressed GUS. Progeny analysis was conducted by sowing the T1 seeds and
monitoring the expression of the GUS gene after 21 days. Individual T1 plants were
subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a
100 mg l-1 solution of glufosinate and scoring the leaf 5 days post application.
Herbicide-sensitive and GUS-positive individuals were observed in four of the 10
independent events. Southern blot analysis confirmed the absence of the bar gene in
the GUS positive/herbicide-sensitive individuals. These results demonstrate that
41
simultaneous integration of two T-DNAs followed by their independent segregation
in progeny is a viable means to obtain soybeans that lack a selectable marker.
Olhoft et al. (2003) increased the efficiency of soybean [Glycine max (L.)
Merrill] transformation from an average of 0.7% to 16.4% by combining strategies to
enhance Agrobacterium tumefaciens-mediated T-DNA delivery into cotyledonary-
node cells with the development of a rapid, efficient selection protocol based on
hygromycin B. Wounded cotyledonary-node explants were inoculated with A.
tumefaciens carrying either a standard-binary or super-binary plasmid and co-
cultivated in the presence of mixtures of the thiol compounds, L-cysteine,
dithiothreitol, and sodium thiosulfate. Transformed shoots began elongating only 8
weeks after co-cultivation. Southern analysis confirmed integration of the T-DNA
into genomic DNA and revealed no correlation between the complexity of the
integration pattern and thiol treatment applied at co-cultivation. All T0 plants were
fertile and the majority of the lines transmitted the beta -glucuronidase (GUS)
phenotype in 3:1 or 15:1 ratios to their progenies.
Wang et al. (2003) used a soybean transformation procedure involving the use
of Agrobacterium cotyledonary node system and the bar gene as the selectable marker
coupled with glufosinate a selective agent, to study the regeneration of 15 soybean
cultivars and their susceptibility to Agrobacterium tumefaciens EHA 101.
Cotyledonary nodes from 5-6 days germinated soybean seeds were used as explants.
The explants were wounded by slicing 5-6 times, inoculated with A. tumefaciens
EHA 101. Three days after co-cultivation, the explants were washed and placed onto
a shoot initiation medium supplemented with 5 mg glufosinate/litre for selection. The
regeneration rate of the different soybean cultivars was observed after 2 weeks, and
their susceptibility to A. tumefaciens was investigated after 4 weeks by beta -
glucuronidase assay. Heinong 35, Zhongzuo 975, Hefeng 35, Zhongzuo 962 cultivars
had higher regeneration than Thorne, while William 82, PI 361066, Heinong 35 and
Zhongzuo 975 had higher transformation than Thorne. The remaining cultivars had
lower regeneration and transformation rates than the control cultivar.
Patil et al. (2004) studied Explants (cotyledonary node, nodal segment and
shoot tips) were obtained from 7- to 10-day-old in vitro-germinated seedlings of
soybean cv. Bragg, and from plants derived from gamma-irradiated seeds (20 and 25
kR). Multiple shoot induction was done in MS media containing BA [benzyladenine]
at 0.2, 0.4, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 5.0 mg/litre; IBA at 0.05, 0.1 and 0.5
mg/litre; and NAA at 0.1 mg/litre. Rooting was done on half MS media containing
IBA at 0.2, 0.4, 0.6 and 0.8 mg/litre, and NAA at 0.1, 0.2, 0.3, 0.4 and 0.5 mg/litre.
Subculturing was done in the same media every 2 weeks. Culture media containing
42
BA at 5 mg/litre and NAA at 0.1 mg/litre was the most effective in inducing multiple
shoots without the callus phase. The media containing MS + BA at 5 mg/litre + IBA
at 0.1 mg/litre took the least number of days to bud break, and produced the
maximum number of buds for both irradiated samples and the control. Rooting was
obtained from excised shoots regardless of the explant type on 1/2 MS media with
IBA at 0.8 and 1 mg/litre and NAA at 0.5 mg/litre. Irradiated materials took longer
time to initiate bud compared to the control. Increasing irradiation dosage resulted in
the increase in the number of days required for bud initiation. Irradiation effects were
more obvious in the explants from cotyledonary node and nodal segment than those
from shoot tips.
43
enhance Agrobacterium infection of soybean. Nonetheless, an undesirable effect of
using these antioxidants is the compromised recovery of transgenic soybean when
combined with the use of the herbicide glufosinate as a selective agent. Therefore, we
optimized both Agrobacterium infection and glufosinate selection in the presence of
L-cysteine for Williams 82. We have recovered transgenic lines of this genotype with
an enhanced transformation efficiency using this herbicide selection system.
44
MATERIALS AND METHODS
The experiments of the present study were conducted at Field Crops Research
Institute (FCRI), Cell Research Study Department, Agriculture Research Center
(ARC), Giza, Egypt.
Plant material
Fourteen Egyptian and exotic soybean genotypes were used in this study (Table13).
The genotypes were provided by Food Legumes Research Department, FCRI, ARC,
Giza. The genotypes were planted in the field at Giza Agriculture Research Station
in 2005. Every genotype was planted in 10 ridges, 3 m long and 60 cm apart. A
month after flowering, 100 pods/ genotype were collected and used in this study.
45
pBI 121 binary vector contain the NPT-II as a selectable marker and GUS as a
reporter gene was provided by Ain Shams University, Faculty of Agriculture,
Department of Genetics and Cytomolecular Laboratory and used for soybean
transformation.
In vitro immature embryo cotyledonary Organanogenesies of soybean
(control):
Immature embryos were used to produce soybean plantlets according to the
method described by Nasr et al. (2005). The MS medium (Murashige and Skoog,
1962) was used to regenerate plantlets (Table 14). The OR medium was used for
callus induction, while MSR medium was used for production of shoots from callus.
The produced shoots were transformed to glass tubes containing the hormone-free
MS medium for rooting. When a reasonable number of roots is grown (usually after
3-4 weeks) the plantlets were removed from the glass tubes in to 10-cm diameter-
plastic pots filled with fumigated soil mixture of peat and sand with a ratio of 3: 1.
To maintain optimum air humidity surrounding plants, pots were covered with
polyethylene bags and then placed in the green house. Irrigation was applied using
(0.25) Hogland solution (Hogland and Arnon, 1950).
Kanamycin sensitivity:
The immature embryos (0.5-10mm in diameter) and cotyledonary nodes (2-5
days old) of soybean genotypes were tested for the sensitivity to Kanamycin
according to the method described by De-Block (1988), in order to identify the proper
Kanamycin concentration to be used in testing soybean genotypes; kanamycin
concentrations of 0, 25, 50, 75,100 and 125 mg/L were supplemented to the media
and soybean plants were allowed to grow. The proper kanamycin concentration is that
kill all tested explants of soybean genotypes. The data were recorded and statistically
analyzed according to Gomez and Gomez (1984).
Agrobacterium preparation:
Agrobacterium tumefaciens LBA4404 containing the pBI121 was plated on LB media
supplemented with Streptomycin 50 µg mL-1 and Kanamycin 30 µg mL-1 then
incubated for 3 days at 28°C. Colonies were tested for the presence of the pBI121
plasmid using Alkaline Lyses method (Sambrook and Russell, 2001). Positive
colonies were grown on liquid LB supplemented with Streptomycin 50 ugmL-1 and
Kanamycin 30 ugmL-1 for overnight at 28°C with shaking (Ausubel et al. 1994).
Production of transformed plantlet by immature embryos
In vitro immature embryos of soybean (0.5-10 mm in diameter) were excised
from the seeds and collected in sterile Petri dishes under aseptic conditions. The
immature embryos and were incubated with 30 ml of an overnight incubated
Agrobacterium tumefaciens culture for 60, 120,180 and 240 second. After incubation,
the excess of bacteria was blotted on sterile filter paper and the immature embryos
were incubated in an organogenesis medium (OR) which prepared according to
Murashige and Skoog (1962) and Gamborg (1968) for 3 days at 25 ± 1 °C in the
dark. The immature embryos were then rinsed with sterile distilled water and blotted
to dry on sterile filter paper and were planted on callus medium OR (MS salts
supplemented with B5 vitamin, NAA 0.0372 mgL-1, BA 2.996 mgL-1,100 mgL-
1
kanamycin monosulfate and 200 mgmL-1cefotaxime sodium salt). The immature
embryos were then incubated under complete darkness at 2SoC±2 for 4 weeks. Calli
from immature embryos were transferred to MSR media (MS salts supplemented with
B5 containing BA 0.383 mgL-1, IBA 0.0406 mgL-1, 100 mgL-1kanamycin
monosulfate and 200 mgL-1cefotaxime sodium salt) and incubated at 25 ± 2°C,
46
with16 h light (3000 Lux.) per day at for 4 weeks. The small shoots were taken and
placed on rooting medium and incubated till root formation.
Production of transformed plantlet from cotyledonary nodes:
In vitro cotyledonary nodes of soybean (2-5 days old) were collected from
seedling and cut in sterile Petri dishes under aseptic conditions, Then transferred to
Petri plates containing 30 ml of an overnight incubated Agrobacterium tumefaciens
culture, and incubated for 2, 4, 8 and 16 second. After incubation, the excess bacteria
was blotted on sterile filter paper and the cotyledonary nodes were incubated in an
organogenesis medium Cotl [MS salts supplemented with B5 and supplemented with
9.9 mgL-1Benzyl Adenine (BA) and 0.2 Indol butyric acids (IBA)]. Shoots from
cotyledonary nodes were transferred into regeneration medium (1.1 BA and 0.2 mgL-
1
IBA (Cot2) containing 100 mgL-1Kanamycin monosulfate and 200 mgL-1cefotaxime
sodium salt. The cultures were incubated at 25 ± 2°C, 16h lights (3000Lux) per day.
After 20-30 days (subculture/15 days), the small shoots were taken and incubated on
rooting medium (Con) supplemented with 2 mgL-1 IBA till roots production (Table
14).Acclimatization of transgenic soybean genotypes and no transgenic (control)
were carried out as follows. Plantlets (3-7 cm), which obtained from in vitro were
washed under running tap water for 1-2 minutes to remove agar traces, and soaked in
fungicide solution (2 gL-1 of Benlate) for 15-20 min, then plantlets were cultured in
plastic pots(15 cm in diameter containing a mixture of peat moss and sand at 2:1 (VI
V), and watered immediately. To keep constant high humidity (90 %), the pots were
covered with polyethylene bags which were gradually removed within one week. The
plantlets were transferred into plastic pots (23 cm in diameter) after 3 weeks for
adaptation. Irrigation by Hogland solution was applied every 10 days up to maturity
(110-120 days).
Detection of GUS gene:
A-Isolation of DNA:
Samples were collected from one month old leaves of both transgenic and non-
transgenic (control) soybean genotypes and control (non-transgenic leaves) from in
vitro cultures grown on MS medium. Isolation of DNA was performed according to
method described by Dellaporta et al. (1983).
Agarose gel preparation and DNA detection:
DNA was visualized on 1g/100 ml agarose gel on TBE buffer and stained with Ethidium
Bromide (Sambrook and Russell, 2001). DNA bands were visualized using a short UV
light source (254 nm) and photographed by gel documentation system using the
Polaroid instant image film from photo dyne.
B-PCR detection:
PCR reaction was carried out using 200 ng of DNA in the presence of 1 x PCR
buffer, 10 pmol of primer GUS 1 GGTGGGAAAGCCGTT ACAA, 10 pmol of
primer GUS2 GTTTACGCGTTGTTCCGCCA. 1.5mM of MgCI2, 10 mM of dNTPs
and 2 unites of Taq DNA polymerase. Samples were subjected to 35 cycles of PCR
with 1 min of denaturating at 94˚C, 1 min of annealing at 56 ˚C, and 2 min at 72 ˚C.
The series of cycles were preceded by 3 min initial denaturating at 94 ˚C and
followed by 3 min at 72 ˚C.
C-GUS assay:
Soybean explants derived from both transgenic and control genotypes were analyzed
using GUS assay. One ml of GUS buffer containing X-glue was added to the explants
in Petri dishes. Explants emerged in the GUS buffer were incubated at 37°C for 4-24
47
h. on a shaker (Janssen and Gardner. 1989). GUS assay were applied in explants,
shoots and plantlets. Callus, shoots and plantlets of soybean were collected from
transgenic and non transgenic genotypes for the detection of B-glucuroindase gene.
Samples were incubated with X-glue substrate and its buffer at 37°c for 4-24 h.
Table 14: Composition of nutrient media for callus initiation (OR), Shooting
formation (MSR), Cot1, Cot2and Cot3as described by Murashige and Skoog (1962)
and Gamborg et al. (1968).
48
ori V traF
ColE1 ori
kilA
NPT III
tetA
IS1
NPTIII
trfA Rb
tetA
NOSp
Lb
pBI121
14.758 bp
NOSt
pBI121
nptII
Sac I
GUS
CaMV35S
NOSt
Sna BI 4173
Hin dIII
Sph I
Bam HI Pst I
Xba I
Sma I
49
RESLUTS AND DISCUSSION
The immature embryos of the 14 genotypes were tested for their sensitivity to
LBA4404- pBI121. The results of evaluation for transient GUS expression are
presented in Figure (2). The results show that genotypes L86K -73 (no. 1), Giza111
(no.8) and Clark (no.11), had the highest percentage of gene expression of 6, 5 and
4%, respectively, indicating that they are the most responded to the applied technique.
Hence these three genotypes were used for transformation in immature embryos
method. Another three genotypes were chosen randomly to be used in cotyledonary
nodes method.
Agrobacterium strain and helper plasmid are the main factors affecting
transformation efficiency. In this work the vector of pBI121 in the helper strain of
LBA4404 allowed the production of several stable transformed soybean plants. The
binary vector pBI121, which has the GUS and the Kanamycin gene, as a selective
marker, were introduced into soybean genotypes L86K-73, Giza111 and Clark by
immature embryos method, and Giza35 and Giza21 and Calland by cotyledonary
nodes method.
Time of inoculation on Agrobacterium media
Ten plates for each genotype containing 10 ex-plants/plats (explants developed by
immature embryos method) were co cultivated with LBA4404-pBI121 for different
inoculation time as 60, 120, 180 and 240 seconds. The presence of transient GUS
expression was investigated after co-cultivation for 4h (over night) in MS media
supplemented with B5 vitamins and containing 100 mgL-1 kanamycin. The data
indicated that 180 sec. is the best time for incubation. No significant difference was
observed among genotypes (Table 15). Yan et al. (2000) found that the shorter co-
culture times resulted in higher explant survival and higher somatic embryo
production on the explants, whereas the co-culture time of 4 days severely reduced
survival of the cotyledon explants and reduced embryogenic potential.
50
7
usexpressedex-plants
transf ormed ex-plant
4
ercentageof G
1
P
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Trans formed genoty pes
Varieties(V)
Time(T)
Mean
(Second)
L86K-73 Giza111 Clark
51
A similar technique was used with explant developed by cotyledonary nodes. Data in
(Table 16) revealed that four seconds showed the best time for co-cultivation
cotyledonary nodes with Agrobacterium. There was no significant difference among
genotypes regarding the co cultivation time. The highest mean of successful
cotyledonary nodes (27%) was obtained using the genotype Giza35 after 4 seconds as
inculcation time. Harold et al. (1997) used 2 second for inoculation of
Agrobacterium with cotyledonary nodes. The difference in inculcation time between
studies could be attributed to the type of cotyledonary nodes used.
Kanamycin concentration:
52
120
Giza 35 Giza 21 Calland 120 L86K-73 Giza 111 Clark
100
Percentage of successful
100
Percentage of successful
80
80
60
60
40 40
20 20
0
0
0 25 37.5 50 62.5 75 87.5 100 125 0 25 50 75 100 125
Kanamycin Concentration (mg/1l)
Kanamycin Concentration (mg\1L)
Korban (2004).
53
Table 17: Percentage of successful transformed soybean genotypes.
Varieties Successful Successful Callus Successful Successful
Immature % Shoots plantlet
embryos % %
%
54
Table 18: Percentage successful of variance transformed
Organs of three soybean genotypes.
.
Varieties cotyledonary nodes%
(Kanamycin)
Giza35 9.00
Giza21 8.00
Calland 12.00
Mean 9.67
L.S.D.5% 3.693
55
Fig. (6): Agarose gel electrophoresis for plasmid pBI 121.
: M DNA/Ready load TM 1K Plus (marker).
No. 1-6 : pBI 121 plasmid DNA.
56
Plate 1: Illustrated immature embryo showing the results of GUS assay; control
(A), different degree of GUS expression different soybean explants (B, C, D, E, F
and G) and callus in (H) control and (I).
57
Plate 2: Immature embryo organogenesis callus induction (1), shoots in (2) and
plantlet in (3).Cotyledonary nodes (A), shoots in (B) and plantlets in (C).
58
CHAPTER (IV)
Molecular Characterization of Soybean Genotypes
Resistant /Susceptible To Cotton Leaf Worm
INTRODUCTION
59
specific DNA sequences. The analysis for RAPD markers is quick and
simple, although results are sensitive to laboratory conditions.
60
REVIEW OF LITURETIER
A-Diversity of soybean
1-Isozymes
Yoon et al. (2000) studied the variation and geographical distribution of beta -
amylase isoenzymes by isoelectric focusing (IEF) within Korean, Chinese and
Japanese soybean landraces. The amylase of 1152 soybean accessions was separated
into low pI group isoenzymes (Sp1b) and high pI group isoenzymes (Sp1a) in pH 3-10
IEF gel. In pH 4-6.5 gel, isoelectric points were at 5.07, 5.15, 5.25, 5.40 and 5.94, and
h, j and k bands were also found. The distribution of the Sp1a allele (high pI type) was
29.3% in accessions from Korea, 10.1% in those from China, and 6.9% in Japanese
accessions. The frequency of the Sp1a allele was highest in accessions from
Kyungsang province (35%) in Korea, followed by central China (32%) and Honshu
(10%) in Japan.
Tomar and Rajput (2002) tested old and new seeds of three soybean cultivars,
Type-1, Bragg and Clark-63, for viability on the basis of appearance of peroxidase and
esterase isoenzyme markers during seed germination. Enzyme polymorphism was
determined by starch gel electrophoresis. Esterase isoenzymes were observed in old
seeds only for 92 hours of seed germination. Esterase C1 and A1 bands were observed
in germinating new seeds, where as C1 and C2 bands were observed in both old and
new seeds. Activation of esterase in old seeds may be due to degradation of fat, loss of
permeability of membrane, inhibition of respiratory enzyme and non-availability of
cofactors involved in the TCA cycle. The presence of peroxidase isoenzyme was
observed in new seeds only. Peroxidase isoenzyme bands C1, C2 and A4 were
detected at 24 hours after seed soaking, where as bands A3 and A2 were located for
the first time at 48 and 92 hours, respectively. Peroxidase activity in seeds indicated
proper activation of respiratory system leading to the accumulation of toxic
metabolites and photosynthates in the cells, resulting in normal growth. Thus, the
esterase and peroxidase isoenzymes were found to be positively and negatively
correlated, respectively, with the viability of soybean seeds. Thus, these enzymes
could be used as biochemical markers for screening seeds and cultivars for viability.
61
Funatsuki et al. (2003) investigated the isozyme profiles of antioxidant
enzymes in cultivars and lines with different seed productivity in cool climate
conditions as a step towards understanding the physiological and genetical
mechanisms underlying chilling tolerance in soybean. While no difference in
superoxide dismutase, or catalase isozyme profiles was observed among the cultivars
and lines tested, they found polymorphism in the ascorbate peroxidase isozyme
profile; there were two types, with or without a cytosolic isoform (APX1). The
cultivars and lines lacking APX1 proved more tolerant to chilling temperatures, as
evaluated by yielding ability. The genotype-dependent deficiency of APX1 was
consistent in plants and tissues under various oxidative stress conditions including the
exposure to low-temperatures. In addition, the genetic analysis of progeny derived
from crossing between cultivars differing in the isozyme profile indicated that the
APX1 deficiency is controlled by a single recessive gene (apx1), and is inherited
independently of the genes that have previously been identified for their association
with chilling tolerance. Molecular and linkage analyses suggested that the variant gene
of the APX1-absent genotype coding for a cytosolic APX, which contained a single
nucleotide substitution and a single nucleotide deletion in the coding region, is
responsible for the genotype-dependent deficiency of APX1.
Mahmoud et al. (2006) evaluated the extent of the genetic change and its effects
on the seed protein composition of soybean cultivars released during the past 60 years,
representative ancestral cultivars and those derived from selective breeding were
grown in a side-by-side comparison. Total seed protein content, determined by
combustion analysis of nitrogen, revealed a decline in the protein content after decades
of selection and breeding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
comparison of protein profiles of the soybean cultivars indicated that relative
expression of most of the seed storage proteins had not varied substantially from the
ancestral lines to the present commercial cultivars. There was noticeably less â-subunit
of â-conglycinin, a protein devoid of sulfur amino acids, in the modern cultivars
represented by Mustang, Pioneer 93B09, and Asgrow 3602. Comparison of the amino
acid profiles of soybean seed, a benchmark of the protein’s nutritional quality,
revealed that the ancestral progenitor, G. soja, was significantly higher in cysteine,
glutamic acid, histidine, and arginine than either the ancestral or the modern cultivars.
Selective breeding over the past 60 years minimally affected the overall amino acid
composition. The degree of divergence in the DNA sequence of the genes encoding
glycinin and â-conglycinin in the ancestral and modern cultivars was investigated
using Southern hybridization and the polymerase chain reaction. Even though some
restriction fragment polymorphisms could be detected, overall, the banding patterns
were remarkably similar among the ancestral cultivars and those derived from them,
suggesting a high degree of conservation of seed-storage protein genes. The results of
our study suggest that selection and breeding for yield during the past 60 years had no
major influence on the protein composition, ostensibly because of limited genetic
diversity among the parental lines.
Zarkadas et al. (2007a) determined the protein quality of 11 null and 2 tofu
soybean genotypes from their total protein content, their amino acid composition, and
their glycinin and b-conglycinin contents. There were highly significant differences (P
62
< 0.001) in their total storage proteins, and amino acid contents. Total protein among
these genotypes ranged from 33 to 37%, with arginine being the third highest amino
acid (7.4–10.9 g/100 g protein) followed by glutamic and aspartic acids. Methionine
accounted for only 1.6–2.4 g/ 100 g of protein. All genotypes contained a good balance
of essential amino acids (EAA9), ranging from 43.5 to 47.3% of the total protein,
limited only in methionine and possibly threonine and valine. Two dimensional gel
electrophoretic (2-DE) reference maps, using narrow range immobilized pH gradient
(IPG) strips, revealed unique differences in the proteome, and subunit expression of
glycinin and b-conglycinin, among these null genotypes, which can then be correlated
with their protein quality. Out of a total of 111 basic (pH 6–11), and 223 acidic (pH 4–
7) protein spots separated by 2-DE, 41 soybean storage protein spots were excised, and
identified by liquid chromatography on-line with electro spray LCQ DecaXP tandem
quadrupole time-of-flight mass spectrometry (LC/MS/MS). These methods will enable
accurate evaluation of protein quality in soybeans, based on their protein digestibility-
corrected amino acid score, assessment of the genetic variability of soybean genotypes,
and serve as very effective tools for assisting plant breeders in their selection of high
quality soybean varieties.
63
test time but caution was needed during sample preparation and reaction parameters to
make the results reproducible.
Giancola et al. (2002) used the molecular markers such as random amplified
polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and
simple sequence repeats (SSR) as descriptors to characterize and differentiate a set of
100 soybean varieties of commercial use in Argentina was taken as a leading case
study for plant variety protection (PVP) purposes. Sixteen morphological traits were
recorded to compare pedigree relationships among varieties with information derived
from conventional descriptors and molecular markers. Analysis of 109 polymorphic
loci confirmed the rather low genetic variability of commercial soybean germplasm.
Still, genetic fingerprinting of the 100 varieties could be established. Calculated
similarity indexes were dependent on the technique, ranging from 0.262 (SSR), 0.407
(RAPD), 0.400 (AFLP) and 0.574 (morphological traits). Dendrograms generated from
morphological data matrix showed low value correlation with kinship coefficient
matrix (r=0.216). Still, they were suitable to identify and differentiate each of the 100
varieties analyzed which was not possible with RAPD or AFLP markers using
comparable numbers of polymorphic loci. SSR data showed the best fit to pedigree
information (r=0.353), while maintaining an association to morphologically based
separation. Results suggest that the four techniques describe genetic variability in
different and specific ways. A combination of SSR and morphological descriptors
show the best compromise of regarding genetic relationships and the needs of clear
classification for PVP and may help to establish minimum genetic distances for
distinctness within PVP Office definition.
Pham et al. (2003) resolved PCR amplification of total genomic DNA using 20
random primers yielded scorable amplification products. The size of amplification
producted on agarose ranged between 0.3 and 3.0 kb. Seventeen primers detected
polymorphism of all soybean accessions analyzed in the study. The reliability of
RAPD data for the classification of soybean was tested by subjecting the data to
unweighted pair group method analysis of arithmetic means (UPGMA) in order to
explore the possibility of classifying the cultivars using RAPD analysis. The
phenotypic analysis of primers revealed the presence and extent of genetic similarities
among the cultivars. The cluster analysis of RAPD data separated out the cultivars into
five distinct clusters. However RADP data separated out OMDN varieties into three
clusters. Cluster 2 included OMDN43 and OMDN29, cluster 3: OMDN34 and
64
OMDN33, cluster 4: OMDN32, OMDN30,OMDN31. One genotype HL2 was
separated farthest apart in the phenogram. This study confirm a close relationship
between cultivars and evaluated genetic variation in 50 germplasm accessions of
soybean, provides information about amount and distribution of genetic diversity
within and among germplasm.
Kim et al. (2004) used random amplified polymorphic DNA (RAPD) utilizing
1000 operon random primers to identify markers linked to the Ti locus in 94 bulked F2
lines derived from the soybean cross Jinpumkong (with Kunitz trypsin inhibitor (KTI))
x C242 (without KTI). Four RAPD primers (OPAC12, OPAR15, OPO12 and OPC08)
were linked to the Ti locus, with OPO12 linked very closely to the gene for KTI
protein (16 cM).
B-DNA markers and Quantitative Trait Loci (QTL) analysis for soybean
Diers et al. (1992) expanded soybean RFLP map and to identify quantitative
trait loci (QTL) in soybean [Glycine max (L.) Merr.] for seed protein and oil content.
The study population was formed from a cross between a G. max experimental line
(A81-356022) and a G. soja Sieb. and Zucc. Plant introduction (PI 468916). A total of
252 markers was mapped in the population, forming 31 linkage groups.Protein and oil
content were measured on seed harvested from a replicated trial of 60 F2-derived lines
in the F3 generation (F2:3 lines). Each F2:3 line was genotyped with 243 RFLP, five
isozyme, one storage protein, and three morphological markers. Significant (P < 0.01)
associations were found between the segregation of markers and seed protein and oil
content. Segregation of individual markers explained up to 43% of the total variation
65
for specific traits. All G. max alleles at significant loci for oil content were associated
with greater oil content than G. soja alleles. All G. soja alleles at significant loci for
protein content were associated with greater protein content than G. max alleles.
Hnetkovsky et al. (1996) used molecular markers to identify and locate alleles
of chromosomal segments associated with field resistance to SDS in adapted soybean
genotypes. Seventy polymorphic DNA markers were compared with SDS response
among 100 F5-F9 recombinant inbred lines derived from a cross between SDS-resistant
Forrest and SDS-susceptible Essex. SDS disease incidence (DI), disease severity (DS),
and yield were determined in replicated, FSA-infested test sites during 4 yr
encompassing five locations with recombinant inbred lines from the F5-F7 to F5-F11.
Two separate chromosomal segments identified by two RAPD markers, OO05250 and
OC01650, were found to be associated with mean SDS response across five locations
as well as within each of the five locations. These two (QTL) jointly accounted for
34% of total phenotypic variability in mean DI. OC01650 was significantly associated
with mean DS and yield and was putatively assigned to linkage group N. The
beneficial allele was derived from the resistant parent Forrest. OO05250 was not
significantly associated with mean DS or yield and was putatively assigned to linkage
group C. The beneficial allele was derived from the susceptible parent Essex.
Molecular markers can be used to define alleles of chromosomal segments conferring
resistance to SDS in several environments and may allow efficient selection of
resistant genotypes with good yield potential for FSA-infested fields.
66
been mapped to the soybean linkage group D2 and, in conjunction with other QTLs
already identified for SCN resistance, will certainly contribute to understanding of the
genetic basis of resistance of this important disease in soybean.
James et al. (2001) noted that, there has been limited success over the past 30
yr in the development of superior soybean cultivars [Glycine max (L.) Merr] with
insect resistance. Success may be hampered by the quantitative nature of resistance and
by linkage drag from resistant plant introduction (PI) donor parents. Soybean insect
resistance quantitative trait loci (SIR QTLs) have been identified from PI 229358 and
PI 171451 by restriction fragment length polymorphism (RFLP) analysis. The
objective of this study was to tag the SIR QTLs from PI 229358 with simple sequence
repeat (SSR) markers and to determine the extent to which the SIR QTLs have been
introgressed in registered cultivars, germplasm releases, or breeding lines that have
resistance derived from this PI or from PI 171451. Marker analysis defined intervals by
5 centimorgans (cM) or less for a SIR QTL on linkage group D1b (SIR-D1b), and for
SIR-G, SIR-H, and SIR-M. SIR QTLs were tracked through pedigrees by evaluating
the inheritance of PI alleles at marker loci tightly linked to the QTLs during the
phenotypic selection for insect resistance. It was inferred that at least 13 of the 15 SIR
genotypes studied had introgressed SIR-M. PI genome introgression around SIR-M
was measured to assess linkage drag. Some genotypes exhibited a dramatic reduction
in the amount of linked PI genome, which likely occurred in response to phenotypic
selection for agronomic performance as a means of reducing linkage drag. Only a few
genotypes were inferred to possess SIR-G or SIR-H, and no genotypes possessed SIR-
D1b. The results of this study indicate that marker-assisted selection for SIR QTLs is
needed to introgress these loci into elite genetic backgrounds.
Cairo et al. (2002) identified molecular markers linked to the juvenile locus in
soybean. Soybean cultivars carrying the 'long juvenile trait' show a delayed flowering
response under short day conditions. The incorporation of this character into genotypes
of agronomic interest may allow a broader range of sowing dates and latitudes for a
single cultivar adaptation. Experiments were carried out using two pairs of near
isogenic lines (NILs), namely NIP 1 and NIP 2, which differ in the presence of the
long juvenile trait, and RAPD markers. Four hundred primers were first screened to
find polymorphism associated with the trait. Additional differences between NILs
were sought by digesting the genomic DNA with five restriction enzymes.
Polymorphic fragments detected between NILs were tested for linkage to the juvenile
locus in the corresponding F2 segregating populations. Marker bc357-HaeIII was
linked (chi 2L=46.316) to the juvenile locus with an estimated recombination
frequency of 0.13+or-0.03 in one of the genetic backgrounds studied. The fragment
was cloned, sequenced and converted into a sequence characterized amplified region
(SCAR) marker. Moreover, bc357-HaeIII was used as RFLP probe. Both SCAR and
RFLP generated markers linked to the juvenile locus in the two genetic backgrounds
analysed. Results presented in this work can be utilized for both the localization of the
gene associated with the character and for tagging the juvenile trait in soybean
breeding programmes.
Carvalho et al. (2002a) reported that, the soybean stem canker is a serious
soybean (Glycine max) disease caused by the fungi Diaporthe phaseolorum f. sp.
meridionalis/Phomopsis phaseoli f. sp. meridionalis. They have crossed the soybean
67
resistant line UFV 91-61 with the susceptible cultivar Paranaiba, and analyzed the F2
population in order to understand the genetics underlying resistance to this pathogen
(isolate CH8) and to identify molecular markers linked to it. The results indicated that
a single dominant gene controlled resistance to this isolate. RAPD analysis in the F2
population identified two DNA fragments of approximately 1,150 and 1,320 bp of
primer OPAB19 linked in the repulsion and coupling phase at 4.7 cM of the resistance
gene of line UFV 91-61. These markers will be very useful for monitoring the
introgression of this gene into soybean adapted cultivars, and open up the possibility
for a systematic search for markers linked to other resistance genes for stem canker
that could be pyramided into the same genetic background.
Song et al. (2004) developed 391 simple sequence repeat (SSR) markers
designed from genomic DNA libraries, 24 derived from existing GenBank genes or
ESTs, and five derived from bacterial artificial chromosome (BAC) end sequences. In
contrast to SSRs derived from EST sequences, those derived from genomic libraries
were a superior source of polymorphic markers, given that the mean number of tandem
repeats in the former was significantly less than that of the latter (P<0.01). The 420
newly developed SSRs were mapped in one or more of five soybean mapping
populations: 'Minsoy' x 'Noir 1', 'Minsoy' x 'Archer', 'Archer' x 'Noir 1', 'Clark' x
'Harosoy', and A81-356022 x PI468916. The JoinMap software package was used to
combine the five maps into an integrated genetic map spanning 2,523.6 cM of
Kosambi map distance across 20 linkage groups that contained 1,849 markers,
including 1,015 SSRs, 709 RFLPs, 73 RAPDs, 24 classical traits, six AFLPs, ten
isozymes, and 12 others. The number of new SSR markers added to each linkage
group ranged from 12 to 29. In the integrated map, the ratio of SSR marker number to
linkage group map distance did not differ among 18 of the 20 linkage groups;
however, the SSRs were not uniformly spaced over a linkage group, clusters of SSRs
with very limited recombination were frequently present. These clusters of SSRs may
be indicative of gene-rich regions of soybean, as has been suggested by a number of
recent studies, indicating the significant association of genes and SSRs. Development
of SSR markers from map-referenced BAC clones was a very effective means of
targeting markers to marker-scarce positions in the genome.
68
and to identify quantitative trait loci (QTLs) associated with resistance to corn
earworm (Helicoverpa zea Boddie) in a population of 103 F2-derived lines from a
cross of 'Cobb' (susceptible) and PI229358 (resistant). The genetic linkage map
consisted of 128 markers which converged onto 30 linkage groups covering
approximately 1325 cM. There were 11 unlinked markers. The F2-derived lines and
the two parents were grown in the field under a plastic mesh cage. The plants were
artificially infested with corn earworm and evaluated for the amount of defoliation.
Using interval-mapping analysis for linked markers and single-factor analysis of
variance (ANOVA), markers were tested for an association with resistance. One major
and two minor QTLs for resistance were identified in this population. The PI229358
allele contributed insect resistance at all three QTLs. The major QTL is linked to the
RFLP marker A584 on linkage group (LG) 'M' of the USDA/Iowa State University
public soybean genetic map. It accounts for 37% of the total variation for resistance in
this cross. The minor QTLs are linked to the RFLP markers R249 (LG 'H') and
Bng047 (LG 'D1'). These markers explain 16% and 10% of variation, respectively.
The heritability (h2) for resistance was estimated as 64% in this population.
Roger and Walker (2005) reported that, insect resistance in soybean has been
an objective in numerous breeding programs, but efforts to develop high yielding
cultivars with insect resistance have been unsuccessful. Three Japanese plant
introductions, PIs 171451, 227687 and 229358, have been the primary sources of
insect resistance alleles, but a combination of quantitative inheritance of resistance and
poor agronomic performance has hindered progress. Linkage drag caused by co-
introgression of undesirable agronomic trait alleles linked to the resistance quantitative
trait loci (QTLs) is a persistent problem. Molecular marker studies have helped to
elucidate the numbers, effects and interactions of insect resistance QTLs in the
Japanese PIs, and markers are now being used in breeding programs to facilitate
transfer of resistance alleles while minimizing linkage drag. Molecular markers also
make it possible to evaluate QTLs independently and together in different genetic
backgrounds, and in combination with transgenes from Bacillus thuringiensis.
Junyi (2006) suggested the major gene and polygene mixed inheritance model
based on the traditional polygene inheritance model of quantitative traits. The model
was considered as a general one, while the pure major gene and pure polygene
inheritance model was a specific case of the general model. Based on the proposed
theory, the author established the segregation analysis procedure to study the genetic
system of quantitative traits of plants. At present, this procedure can be used to
evaluate the genetic effect of individual major genes (up to two to three major genes),
the collective genetic effect of polygene, and their heritability value. This paper
introduces how to establish the procedure, its main achievements, and its applications.
An example is given to illustrate the steps, methods, and effectiveness of the
procedure.
C-Pest diversity
69
resistant soybean lines from two southern Indiana isolates with different virulence
characteristics and a molecular marker probe was developed for usage with dot blots.
The study demonstrates the feasibility of developing a rapid-based test for use in
diagnosing larger groups of field isolates of nematodes that share common virulence
characteristics toward resistant cultivars.
Silva et al. (2000) found an association with H. glycines eggs and cysts,
although species of Fusarium have been the most prevalent. In many areas of central
Brazil, the sudden death syndrome, caused by F. solani, has become quite frequent and
a serious pathogen of soybean (Glycine spp.). The objective of this work was to
characterize isolates of Fusarium by analysing DNA polymorphisms, using RAPD
70
molecular markers. Eleven isolates of F. solani and F. oxysporum obtained from H.
glycines eggs, five F. solani isolates from soybean plants, and four saprophytic isolates
of F. solani were grown on BDA medium. Following DNA extraction, PCR
amplification was conducted with 17 random primers, for each of the isolates. Cluster
analysis of the genetic distances obtained allowed separation of the isolates into three
groups. Group I represented both the isolates of F. solani which infect nematode eggs
and those which are saprophytic. Group II comprised only the isolates of F. solani that
cause the sudden death syndrome in soybean. Group III comprised isolates of F.
oxysporum. The study also allowed identification of bands which are specific to each
group, enabling the separation of isolates of F. solani with beneficial/neutral or
detrimental traits to soybean.
Fenille et al. (2002) found that Rhizoctonia solani causes pre- and post-
emergence damping-off, root and hypocotyl rot and foliar blight in soybean. Foliar
blight has resulted in yield losses of 31-60% in north and northeast Brazil. The aim of
this study was to characterize isolates of R. solani associated with soybean in Brazil.
Among 73 Rhizoctonia isolates examined, six were binucleate and 67 were
multinucleate. The multinucleate isolates were characterized according to hyphal
anastomosis reaction, mycelial growth rate, thiamine requirement, sclerotia
production, and RAPD molecular markers. Four isolates that caused hypocotyl rot
belonged to AG-4 and using RAPD analysis they grouped together with the HGI
subgroup. Another isolate that caused root and hypocotyl rots was thiamine
auxotrophic, grew at 35 degrees C, and belonged to AG-2-2 IIIB. All 62 isolates that
caused foliar blight belonged to AG-1 IA. RAPD analysis of R. solani AG-1 IA
soybean isolates showed high genetic similarity to a tester strain of AG-1 IA,
confirming their classification. The teleomorph of R. solani, Thanatephorus cucumeris
was produced in vitro by one AG-1 IA isolate from soybean. The AG-4 and AG-2-2
IIIB isolates caused damping-off and root and hypocotyl rots of soybean seedlings cv.
'FT-Cristalina', under greenhouse conditions. The AG-2-2 IIIB isolate caused large
lesions on the cortex tissue, that was distinct from the symptoms caused by AG-4
isolates. The AG-1 IA isolates caused foliar blight in adult soybean plants cv. 'Xingu'
under the greenhouse and also in a detached-leaf assay.
71
compatible with both D. phaseolorum var. meridionalis and D. phaseolorum var.
caulivora isolates; meanwhile, Tracy-M (Rdc1 and Rdc2 genes) was incompatible with
D. phaseolorum var. meridionalis but compatible with D. phaseolorum var. caulivora
isolates. The fact that Rdc1 and Rdc2 together (as in Tracy-M) confer an almost
immune reaction to all assayed isolates of D. phaseolorum var. meridionalis but were
ineffective against the D. phaseolorum var. caulivora isolates evaluated suggests that
the virulence or avirulence genes in D. phaseolorum var. meridionalis and D.
phaseolorum var. caulivora are different. Moreover, physiological races of D.
phaseolorum var. meridionalis were detected by using differential soybean genotypes
carrying distinct single Rdc genes. As far as we know, this is the first report on the
existence of physiological races of D. phaseolorum var. meridionalis in South
America. Selective pressure due to deployment of resistant host cultivars may have
changed the frequency of the virulence or avirulence genes within the population of D.
phaseolorum var. meridionalis. On the whole, our results show that pathogenic
variability of D. phaseolorum in the core soybean-producing area of Argentina is
higher than previously recognized.
Zhu et al. (2006) utilized of native insect resistance genes can be an important
component for managing insects in soybean [Glycine max (L.) Merr.]. A major
quantitative trait locus (QTL-M) for insect resistance from PI 229358, controlling
antibiosis and antixenosis, was previously identified on linkage group (LG) M and was
found to increase the effectiveness of a Bacillus thuringiensis (Bt) transgene in
soybean. The objectives of this study were to fine-map QTL-M using recombinant
substitution lines (RSLs) identified from a ‘Benning’ backcross population, and to
evaluate the main effects and the epistatic interactions between QTL-M and other
resistance QTLs on LGs G and H using near-isogenic lines (NILs) in a Benning
genetic background. The effect of QTL-M was still detectable in the Benning NILs
when they were evaluated for resistance to corn earworm [CEW, Helicoverpa zea
(Boddie)]. The two minor resistance QTLs only provided insect resistance when QTL-
M was also present in the Benning NILs. The QTL-M was fine-mapped to an
approximately 0.52-cM region after the first round of phenotyping the RSLs for
resistance to CEW and soybean looper [SBL, Pseudoplusia includens (Walker)]. These
results should increase the feasibility of cloning QTL-M and help guide the
development of insect resistant soybean cultivars.
72
MATERIALS AND METHODS
1-Stock Solutions:
A-Extraction buffer
50mMTris-HCl buffer, pH7.5 is filtered 0.61g
100%Glycerol 5ml
14mM ß- mercaptoethanol (0.1%V/V) 100ul
H2O(d.w) up to 100ml
B-Acrylamide stock
Acrylamide 29.2g
N,N methylene bis-acrylamide 0.8g
H2O(d.w) up to 100 ml
The stock solution was filtered and kept at 4°C.
2-Gel Buffer:
This buffer was prepared by dilution of one part of electrode
buffer stock (before dilution) with three parts H2O according to Soltis
and Pamela Soltis, 1990. This solution was filtered and kept at 4°C.
3 -Extraction of isozymes:
Freshly tissue of each sample was removed. About 0.5g of each
sample was extracted with 1ml cold extraction buffer (pH 7.5). Each
sample was vortexed for 15 seconds by electric vortex and centrifuged
for 10 min at 10.000 rpm twice at 5°C.The supernatant was transferred
73
to new eppendorf tube and kept at-20°C until use for electrophoretic
analysis.
4-Gel Preparation:
Polyacrylamide standard gel for isozymes at pH 8.7 as follows:
The gel was poured on the plate and 15 well comb was placed immediately
and polymerization was token about 15 min.
5- Application samples:
A volume of 80uL extract of each sample was mixed with 30uL loading
dye(bromophenol blue), then a volume of 100 uL from this mixture
applied to each well.
6-Electrophoretic Conditions:
The gel were completely covered with electrode buffer. The electrodes
were connected to power supply and adjust at 200V for two hours.
7-Isozymes assay:
The gels were stained after electrophoresis according each system and
incubated at 37°C in the dark after adding the appropriate substrate and
staining solution. The staining solutions were listed as follow:
Peroxidase:
0.04M caticol solution 25ml
H2O2 (20volume) 15 ml
Phosphate buffer pH=6(0.1MNaH2PO4 and0.01M Na2HPO4) 50ml
Ingredients were combined and poured over gel. Enzyme activity was
expressed from60-120 second after the substrate was added.
Esterase:
100mMNa-phosphate,pH6(39ml monobasic+61dibasic) 50 ml
ß-Naphthyl acetate(0.05g) dissolve in( 1aceton:1dw) 25mg
α-Naphthyl acetate(0.05g) dissolve in( 1aceton:1dw) 25 mg
Fast blue RR salt 50 mg(1ml)
(0.15g dissolve in 80ml phosphate buffer and filter) then
put the gel then added substrates.
Ingredients were combined and poured over gel, incubated at 37°C in the dark until
brown bands appeared. Rinsed and fixed in 3% acetic acid.
After staining gels were photographed and dendogram were calculated using computer
soft wear statistician.
Malate dehydrogenase (MDH)
It was detected and photographed in situ through the use of specific activity
stains according to Soltis and Pamela Soltis, 1990 as follows:
50 mM Tris-HCl, pH 8.5 50 ml
NAD 10 mg (1ml)
Malic acid 150 mg (1ml)
74
NBT or MTT 10 mg (1ml)
PMS 2 mg (0.4ml)
75
4X Tris HCl/SDS pH6.8 (0.1M) 25.0ml
(4%, W/W) SDS 4.00ml
a- mercaptoethanol 100.00ul
(0.001%W/V) Bromophenol blue 1.00ml
Glycerol 20.0ml
H2O (d.w.) up to 100.00ml
Acrylamide 29.20g
N, N-methelene bis- acrylamide 0.80g
H2O (d.w.) up to 100.00ml
1- Acrylamide and bis- acrylamide were dissolved in 73ml distilled water and shaken
well with magnetic starrier.
2-Then, solution was completed up to 100 ml with distilled water.
3- The stock solution was filtered through filter paper and kept in a dark bottle at 4C
for up to one month.
Staining solution
Coomassie brilliant blue-R-250 staining solution was prepared as follows
Coomassie brilliant blue-R-250 0.50g
Methanol 40.00ml
Acetic Acid Glacial 45.00ml
H2O (d.w.) up to 500.00ml
Gel preparation
1-The gel was papered as shown in Table 19.
76
Table 19: Composition of Resolving gel and stacking gel.
2- Resolving gel was immediately poured into the space between glass plates to height
of1.5 cm bellow bottom of comb (16.50X20 cm).
3-Gels was overlaid with isopropanol and left to polymerize of about 30 min..
4-Stacking gel layer was poured over the resolving gel, and15 min. to polymerize. The
comb was removed and upper buffer tank was filled with running.
Application of samples (Leaf and seed)
1- A volume of 50ul of protein samples were added to the same volume of
SDS sample buffer 2X (pH6.8) in Eppendorf tubes.
2- á- Mercaptoethanol (10ul) was added to each tube and boiled in water
bath for 10 min.
3- A volume of 50ul protein samples was applied to each well by
micropipette
4- Control wells were loaded with standard protein marker.
Electrophoretic conditions
1-lower buffer tank was filled running buffer and attached with upper tank
so that gels were completely covered.
2- The electrodes were connected to a power supply and adjusted at 100V
until the Bromophenol blue reached the bottom of the resolving gel.
77
Tris base 5.40g
Boric acids 2.75g
500 mM EDTA, pH 8.0 0.29g
H2O (d.w.) up to 100.00ml
2-Ethidium Bromide
The stock solution was prepared by dissolving 1g of ethidium bromide in 100 ml
distilled water and mix well with magnetic starrier. Transferred to a dark bottle and
stored at room temperature.
In this study RAPD was used for the identification of markers associated with
the most sensitive and resistance fourteen soybean cultivars for cotton leaf worm
according to Williams et al. (1990)
DNA Isolation
Take leaves from soybean cultivars which were grown in ARC, Giza farm after 2-3
weeks from germination. DNA isolation was done using Dellaporta et al. (1983)
1-Weight 0.5 g leaf tissue and were ground under liquid nitrogen till were as affine
powder.
2- Then the powder was transferred to an appropriately sized tube and the liquid
nitrogen was allowed to evaporate.
RAPD assays:
Four 20 – mer arbitrary primers were used in RAPD analysis. Sequences of all
primers are illustrated in Table (1). For RAPD analysis, PCR amplifications were
carried out in a total volume 25 µl containing 2.5 µl 10 x buffer, 2 µl 25 mM MgCl2, 2
µl 2.5 mM dNTPs, 1 µl 10 pmol primer, 1 µl 50 ng of bacterial genomic DNA and 0.2
µl Taq DNA polymerase (5 units/µl) (Promega, Germany). PCR amplification was
performed in a thermal cycler (Eppendorf, Germany) programmed for one cycle at 95
°C for 5 min. Then 34 cycles were performed as follows: 30 s at 95 °C for
denaturation, 1 min at 45 °C for annealing and 2 min at 72 °C for elongation. Reaction
was then incubated at 72 °C for 10 min for final extension (Istock et al., 2001).
µl of loading dye was added prior to loading of 10 µl per gel pocket. Electrophoresis
was performed at 100 Volt with 0.5 x TBE buffer in 1.5% agarose/0.5 x TBE gels and
then the gel was stained in 0.5 µg/ml (w/v) ethidium bromide solutions and destained
in deionized water (Sambrooke et al., 1989). Finally the gel was visualized and
photographed by using gel documentation system (Alpha ImagerTM 1220, USA).
Presence and absence of RAPD bands produced from the four primers were scored
visually from resulting photographs.
78
Table 20: Sequences of all primers used in RAPD analysis
Primer Nucleotide sequence 5\ to 3\
A1A13 CAGGCCCTTCCAGCACCCAC
A9B7 GGTGACGCAGGGGTAACGCC
A1 CGAGCCCTTCCAGCACCCAC
A7 GAAACGGGTGGTGATCGCAG
Sample preparation
PCR product 15.0 µl
Loading dye 5.0 µl
Gel preparation
Agarose 0.9 g
(1X)TEB pH 8 100.0ml
Ethidium bromide 5.0 µl
Agarose was cooked with (1X) TEB and boiled in water bath. Ethidium bromide was
added to malted gel after temperature become 55˚C. The melted gel were pureed in the
tray of mini gel apparatus and comb was removed when the gel become solid.
DATA Analysis:
The data generated from the detection of polymorphic fragments were analysed.
Specific amplification products were scored as present (1) or absent (0) for each of the
fourteen varieties with four primers. Genetic similarity between all fourteen parents
were estimated by simple matching co-efficient (Sokal and Michener , 1958).
79
(Figure 1). These coding regions are separated from each other by spacers. The larger
and small are separated by the external transcribed spacer (ETS), and the intergenic
spacer (IGS). The 5.8S rDNA is embedded in the internal transcribed (ITS) region.
Different selective forces have led to the evolution of regions with varying
degrees of sequence variation (Olsen and Woese, 1993). Thus, appropriate rDNA
regions can be selected to their best corresponding variations. The large and small
subunits are one of the most highly conserved regions, and therefore, are useful at the
family level and above (Hillis and Dixon, 1991). The smallest rDNA cluster, the 5.8S
rDNA, however, is too short to contain enough phylogenetic information (Hillis and
Dixon, 1991). Due to their high variability, rDNA spacer regions have been used for
identification and determining of phylogenies of closely related species, and
populations (Hillis and Dixon, 1991). Prior to the introduction of PCR technology,
most studies focused on the intergenic spacer (IGS). This spacer consists of
subrepeats, differing in sequence and copy number (Olsen and Woese, 1993). Length
differences in the IGS are detected by Southern blot analysis (Hillis and Dixon, 1991).
Recently, PCR-based analysis of the internal transcribed spacer (ITS) has become the
method of choice to identify and analyze closely related species and populations
(Olsen and Woese, 1993).
Figure 7: Organization of the rDNA and primers proposed in the current study.
NS3 I1
18S 5.8S 25/28S
NS4 I2
Amplification and sequencing both of 16S rRNA and 18S rRNA genes:
Approximately 350 bp from the 16S ribosomal RNA gene and 600 bp from 18S
ribosomal RNA gene were amplified using the universal primers. The detection of a
single amplicon of expected size indicates the specificity of the PCR reaction. I6S r RNA
gene was only detected in samples 1, 2 and 8 respectively. These results suggest that
bacterial association may be only present with these three fungal isolates. Ampilcons with
600 bp from 18S rRNA for the remaining seven isolates were sequenced using 377
automated sequencer and the sequence was analyzed using BLASTn Molecular identities
of these isolates are tabulated (Table 4).
80
investigated on two levels. Firstly, biodegradation assay based on dual serial dilutions of
fungal isolates 1, 2 and 8 respectively with their respective bacterial isolates. Secondly,
molecular search for dyes biodegradation gene(s) in the aforementioned fungal isolates
and their respective bacterial isolates.
81
lab coat, and UV-protective full face shield or glasses when you are exposed to the UV
light of the Transilluminator)
Incubate for at least 1 hour at 37°C. The restriction enzyme can be inactivated by
heating to 65°C for 10 minutes or by adding 1.0 µl 0.5 M EDTA.
82
RESULTIES AND DISCUSSION
There are numerous steps in the development of any novel, desirable plant
germplasm. Plant breeding begins with the analysis and definition of problems and
weaknesses of the current germplasm, the establishment of program goals, and the
definition of specific breeding objectives. The next step is selection of germplasm that
possess the traits to meet the program goals. Here study firstly, report the
characterization of a novel leaf isozymes of soybean and examine its role in defense
responses. Secondly, studied character genotypes at leaf and seed protein profile.
Moreover, extent to DNA level. Thirdly, collected all data to calculate genotypes and
allele frequency to received molecular markers assistant.
Isozymes:
The leaf isozymes polymorphism based on PAGE electrophoreses analysis all
soybean genotypes is presented in (Plate 3, Table 21) three isozymes of soybean
genotypes. The Data in show a qualitative analysis depends on the number of obtained
bands from each tested soybean genotypes. of the different tested genotypes.
83
Table 21: Total number of bands from three isozymes
to soybean genotypes.
Genotypes Isoenzymes
Peroxidase ESTRASE MDH Total
L86k-73 4 2 2 8
Corsay-79 8 1 1 10
Giza 21 6 1 1 8
Forrest 9 0 1 10
Hutcheson 7 0 1 8
Calland 7 1 1 9
Lakota 6 0 1 7
Giza 111 8 0 2 10
Giza 83 6 1 2 9
Clark 7 1 2 10
Giza 22 6 0 2 8
Giza 35 5 1 1 7
Giza 82 7 1 1 9
Crowford 4 1 2 7
84
1 2 3 4 5 6 7 8 9 10 11 12 13 14
1 2 3 4 5 6 7 8 9 10 11 12 13 14
85
Protein PAGE-SDS:
The results of SDS-PAGE for the water soluble proteins leaf in the fourteen
genotypes (Plate 4 and Table 22) revealed a total number of 8 bands with molecular
weights (MW) ranging from about 1263 to144 KDa. Data showed that all bands are
common for the fourteen soybean genotypes (monomorphic), however they differed in
density and intensity. The densitometric analysis (Table 22) of SDS-protein banding
patterns of the leaf of the studied genotypes was found to be not informative in their
work. On the other side, the results of SDS-PAGE for the water soluble proteins in
the seeds of fourteen soybean genotypes (Plate 4 and Table 23) revealed a total
number of 13 bands with molecular weights (MW) ranging from about 220.36 to 9.1
KDa. Data showed that all bands are common for the fourteen genotypes except two
bands 220.36 and 194 KDa. for Calland genotype can be considered as specific bands.
Moreover they differed in density and intensity.
Similarity index among the fourteen soybean genotypes based on protein
analysis, carried out using UPGMA computer program, is shown in Table (24). The
similarity relationships ranged between 1.000 and 0.950. The highest similarity index
(1.000) was recorded between each two of the genotypes; L86K-73,Corsay-79,Giza
21,Forrest,Hutcheson,Lakota,Giza111,Giza 83,Giza 22 ,Giza 35,Giza 82 and
Crowford. However, the lowest similarity index (0.950) was observed between
Calland and each of the thirteen genotypes. The genetic distances relationships among
the fourteen soybean genotypes based on leaves and seeds protein patterns are shown
in (Plate 4). The dendrogram confirmed the close genetic relationship among these
genotypes as appeared in Table (24) for similarity indices.
The overall results of total protein pattern obtained by SDS-PAGE did not show
clear cut in monitoring molecular markers resistance/susceptible to cotton leaf worm
of the 14 soybean genotypes. Similar results were obtained also in soybean by Eman
(2006).
86
SDS-PAGE protein banding patterns for leaves protein (water soluble) of the 14 soybean
genotypes.
SDS-PAGE protein banding patterns for seeds protein (water soluble) of the 14 soybean
genotypes.
Plate4: Protein leaf and seed polymorphism based on SDS-PAGE
electrophoreses analysis the of soybean genotypes.
87
Table 22:SDS-PAGE Leaf protein profile of different genotypes in soybean.
Soybean genotypes
Band M.W
Hutcheson
Corsoy-79
Crawford
L86K-73
Giza111
Calland
Forrest
Giza21
Giza83
Giza22
Giza35
no. (Kda)
Lakota
Giza82
Clark
1 126.3 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2 98.6 1 1 1 1 1 1 1 1 1 1 1 1 1 1
3 73.5 1 1 1 1 1 1 1 1 1 1 1 1 1 1
4 41.6 1 1 1 1 1 1 1 1 1 1 1 1 1 1
5 24.6 1 1 1 1 1 1 1 1 1 1 1 1 1 1
6 17.2 1 1 1 1 1 1 1 1 1 1 1 1 1 1
7 15.2 1 1 1 1 1 1 1 1 1 1 1 1 1 1
8 14.4 1 1 1 1 1 1 1 1 1 1 1 1 1 1
8 8 8 8
Total 8 8 8 8 8 8 8 8 8 8
Soybean genotypes
Band M.W
Hutcheson
Corsoy-79
Crawford
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
no. (Kda)
Clark
1 220.36 1 1 1 1 1 0 1 1 1 1 1 1 1 1
2 194 1 1 1 1 1 0 1 1 1 1 1 1 1 1
3 109.4 1 1 1 1 1 1 1 1 1 1 1 1 1 1
4 64.6 1 1 1 1 1 1 1 1 1 1 1 1 1 1
5 52.14 1 1 1 1 1 1 1 1 1 1 1 1 1 1
6 42.1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
7 33.8 1 1 1 1 1 1 1 1 1 1 1 1 1 1
8 26.7 1 1 1 1 1 1 1 1 1 1 1 1 1 1
9 19.1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
10 15.67 1 1 1 1 1 1 1 1 1 1 1 1 1 1
11 13.4 1 1 1 1 1 1 1 1 1 1 1 1 1 1
12 11.17 1 1 1 1 1 1 1 1 1 1 1 1 1 1
13 9.1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
13 13 13 13
13 11 13 13 13 13 13 13 13 13
Total
88
Figure 8: Dendrogram for the genetic distances relationships among the fourteen
genotypes based on Similarity indices data of Protein SDS-PAGE analysis.
Soybean
Hutcheson
Corsoy-79
genotypes
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
Clark
Corsoy-79 1.000
Giza21 1.000 1.000
Forrest 1.000 1.000 1.000
Hutcheson 1.000 1.000 1.000 1.000
Calland 0.950 0.950 0.950 0.950 0.950
Lakota 1.000 1.000 1.000 1.000 1.000 0.950
Giza111 1.000 1.000 1.000 1.000 1.000 0.950 1.000
Giza83 1.000 1.000 1.000 1.000 1.000 0.950 1.000 1.000
Clark 1.000 1.000 1.000 1.000 1.000 0.950 1.000 1.000 1.000
Giza22 1.000 1.000 1.000 1.000 1.000 0.950 1.000 1.000 1.000 1.000
Giza35 1.000 1.000 1.000 1.000 1.000 0.950 1.000 1.000 1.000 1.000 1.000
Giza82 1.000 1.000 1.000 1.000 1.000 0.950 1.000 1.000 1.000 1.000 1.000 1.000
Crawford 1.000 1.000 1.000 1.000 1.000 0.950 1.000 1.000 1.000 1.000 1.000 1.000 1.000
89
Random Amplified Polymorphic DNA (RAPD):
RAPD-PCR was used to analyze the genetic diversity of the fourteen studied
soybean genotypes, and to assess their genetic relationships using similarity indices
and dendogram tree. Four arbitrary random primers were used to determine RAPD
polymorphism of the fourteen soybean genotypes (Primer OP-A 9 B 7, Primer OP-
A0 7, Primer OP-A0 7, Primer OP-A 1 and Primer OP-A 1 A13). The resulted
amplified fragments are shown in Plate (5) and their densitometric analyses are
illustrated in Tables (25-28). Banding patterns were scored as present (1) or absent
(0). All the 4 primers successfully amplified DNA fragments for all genotypes. A total
number of 62 fragments were visualized across the fourteen investigated genotypes.
The results of RAPD-PCR technique are shown as follows:-
Primer OP-A 9 B 7
The pattern produced by primer OP-A9B7 showed a maximum number of 12
DNA fragments ranging in molecular sizes (MS) between 130 to 1670 bp (Plate 3 and
Table 24). Twelve polymorphic fragments (100%) with numbers from 1 to 12 with
corresponding molecular sizes were observed in (Plate5 and Table 24). Whereas,
fragments were not observed monomorphic. Crowford genotype showed the maximum
number of (7) fragments, while the lowest one (1) appeared in Forrest genotype.
Primer OP-A0 7
Primer OP-A07 showed maximum of five DNA fragments with molecular sizes
ranged from150 to 670 bp (Plate5 and Table 25). Zero polymorphic fragments (0%)
and monomorphic (100%) with molecular sizes were seen in all genotypes.
Primer OP-A 1
Primer OP-A1 exhibited nineteen DNA fragments ranging in molecular sizes
from 50 to 1080 bp (Plate 5 and Table 26). Eleven polymorphic fragments (58 %) with
numbers 1, 2, 3, 4,8,13,14,15,16,17 and 19 with corresponding molecular sizes of
1080,1030,1020,1015, 870, 810, 430,360,320,280,230 and 50 bp were observed, while
the other four fragments were monomorphic.
Primer OP-A 1 A13
Plate (5) and Table (27) showed that primer OP-A 1 A13 gave twenty six
polymorphic fragments with molecular sizes ranging from 270 to 1007 bp. Moreover,
no monomorphic was detected among these genotypes using this primer. Crowford
genotype showed the maximum number of (14) fragments, while the lowest one (5)
appeared in L86K-73 genotype. The genotypes have specific molecular markers in
bands number seven (960bp) for Hetcheson, number fourteen (740bp) for Forrest and
number 24 (360 bp) for Giza, as appositive markers.
90
Plate5: DNA polymorphism based on RAPD-PCR analysis of the soybean
genotypes.
91
The results of the amplified fragments using four 10-mer arbitrary primers for the
fourteen soybean genotypes revealed success in amplifying DNA fragments.
Polymorphism levels differed from one primer to the other, the number of total
amplified fragments (TAF) and polymorphic bands (PB) for each primer, amplified
fragments (AF) and specific markers (SM) for soybean genotypes using the 4 primers
are shown in Table (29).
Primers produced fragments number ranging from four primers. These high
levels of polymorphism served in soybean molecular markers. which can be used to
discriminate each soybean genotypes from the others. There were several specific
fragments either present in only one genotypes and absent in all others(positive
marker) or absent in only one genotype an present in all others(negative marker)
(Table 29). Could be as a follows:-
Primer OP-A9B7 showed specific fragment (520bp) as positive marker for Clark
genotype. Primer OP-A1 showed six specific fragment four of them as negative
markers three for Hutcheson genotype(430, 280 and 50 bp) and one for Lakota
genotype (810 bp) as negative marker, and also two as positive markers (360 and 320
bp) for Hutcheson. Primer OP-A1A13 showed three specific fragments as positive
markers one (960bp) for Hutcheson, one (740bp) for Forrest and one (360bp) for
Giza111. Primer OP-A7 did not showed specific fragment.
Results of cluster analysis (similarity index) based on RAPD-PCR with the four
primers using UPGMA computer analysis is shown in (Fig. 9 and Table 30). The
highest similarity value recorded was 0.885, which was observed between Forrest and
Giza 22 genotypes, while the lowest similarity value recorded was 0.606 between
Hutcheson and Giza35 genotypes. A dendrogram for the genetic relationships among
the fourteen soybean genotypes across the four primers results was carried out and is
shown in Fig. (9). The fourteen soybean genotypes were separated into two clusters;
cluster 1 included two sub cluster, The sub cluster1 were consisted from three groups
.The group 1, group 2 and group 3 were included (Forrest and Giza22);( Calland and
Giza111) and (Giza 35 and L86K-73 )respectively. While, sub cluster 2 comprised
four groups group1 were contained Clark and Giza 82, group 2 have Crowford
genotype only and the genotypes Giza21 and Giza83 were located in group 4. The
second cluster was contained Hutcheson genotype.
The dendogram reflects the performance of genotypes on the basis of their
reaction to cotton leaf worm . For example, the genotypes in sub cluster 1 (Forrest,
Giza 22, Calland, Giza 111 and L86K-73) all are resistance except Calland which is
moderately resistance (See table 28). On the other hand the most susceptible
genotypes Hetcheson is located alone in cluster 2. Therefore, PAPD-PCR technique
was useful as a tool for classification that tested soybean genotypes. PAPD-PCR
technique has been used previously to identify soybean genotypes on the basis of their
performance to insect resistance. For example, Carvalho et al. (2002b) who were
used microsatellites (Satt187 and Satt309) and three RAPD markers (OPAG-05946,
OPF-041038, and OPAQ-011987) to identified molecular markers associated with the
resistance to race 3 of the SCN. Also, James et al. (2001) noted that, there has been
limited success over the past 30 yr in the development of superior soybean genotypes
[Glycine max (L.) Merr] with insect resistance. Success may be hampered by the
quantitative nature of resistance and by linkage drag from resistant plant introduction
(PI) donor parents. Soybean insect resistance quantitative trait loci (SIR QTLs) have
92
been identified from PI 229358 and PI 171451 by restriction fragment length
polymorphism (RFLP) analysis.
Table 25: RAPD profile of different genotypes in soybean using Primer OP-
A9B7
Soybean Varieties
Fragment MS
Hutcheson
Corsoy-79
Crawford
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
No. (bp)
Clark
1 1640 0 1 1 0 0 0 1 0 0 1 0 0 0 1
2 1330 0 0 0 0 0 0 1 0 1 1 0 0 1 1
3 900 0 0 0 0 0 1 0 1 0 1 0 0 1 1
4 810 1 0 0 1 0 1 1 1 0 1 1 1 1 0
5 725 1 0 0 0 0 0 0 0 0 0 0 0 0 1
6 660 0 0 0 0 0 1 0 0 0 1 0 0 1 1
7 600 1 0 0 0 0 1 0 1 0 0 0 1 0 0
8 520 0 0 0 0 0 0 0 0 0 1 0 0 0 0
9 415 0 0 0 0 1 0 1 0 1 1 0 0 0 0
10 375 0 0 0 0 0 1 0 0 0 0 0 0 0 1
11 330 0 1 1 0 1 1 0 1 1 1 0 0 1 1
12 130 0 0 0 0 1 0 1 0 0 0 0 0 0 0
3 2 2 1 3 6 5 4 3 8 1 2 5 7
Total
Table 26: RAPD profile of different genotypes in soybean using Primer OP-
A7
Soybean Varieties
Fragment MS
Hutcheson
Corsoy-79
Crawford
L86K-73
Giza111
Calland
Forrest
Lakota
Giza83
Giza82
Giza21
Giza22
Giza35
No. (bp)
Clark
1 670 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2 550 1 1 1 1 1 1 1 1 1 1 1 1 1 1
3 370 1 1 1 1 1 1 1 1 1 1 1 1 1 1
4 300 1 1 1 1 1 1 1 1 1 1 1 1 1 1
5 150 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Total 5 5 5 5 5 5 5 5 5 5
5 5 5 5
93
Table 27: RAPD profile of different genotypes in soybean using Primer OP-A 1
Soybean Varieties
Fragment MS
Hutcheson
Corsoy-79
Crawford
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
No. (bp)
Clark
1 1080 1 1 1 1 0 1 1 1 1 1 1 1 1 0
2 1030 0 1 0 0 0 0 1 0 0 0 1 0 1 1
3 1020 0 1 1 1 1 1 1 0 1 0 1 0 1 1
4 1015 0 0 0 0 1 0 0 0 0 0 1 0 0 0
5 1000 1 1 1 1 1 1 1 1 1 1 1 1 1 1
6 950 1 1 1 1 1 1 1 1 1 1 1 1 1 1
7 880 1 1 1 1 1 1 1 1 1 1 1 1 1 1
8 810 1 1 1 1 1 1 0 1 1 1 1 1 1 1
9 700 1 1 1 1 1 1 1 1 1 1 1 1 1 1
10 650 1 1 1 1 1 1 1 1 1 1 1 1 1 1
11 580 1 1 1 1 1 1 1 1 1 1 1 1 1 1
12 480 1 1 1 1 1 1 1 1 1 1 1 1 1 1
13 430 1 1 1 1 0 1 1 1 1 1 1 1 1 1
14 360 0 0 0 0 1 0 0 0 0 0 0 0 0 0
15 320 0 0 0 0 1 0 0 0 0 0 0 0 0 0
16 280 1 1 1 1 0 1 1 1 1 1 1 1 1 1
17 230 1 0 1 0 1 1 0 0 0 0 0 0 0 0
18 140 1 1 1 1 1 1 1 1 1 1 1 1 1 1
19 50 1 1 1 1 0 1 1 1 1 1 1 1 1 1
15 15 14 14
14 15 14 13 14 13 16 13 15 14
Total
94
Table 28: RAPD profile of different genotypes in soybean using Primer OP-A 1 A13
Soybean genotypes
Fragment MS
Hutcheson
Corsoy-79
Crawford
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
No. (bp)
Clark
1 1007 1 0 0 0 1 0 1 1 0 1 0 1 0 1
2 1005 1 1 1 0 1 1 1 1 0 1 1 1 1 1
3 1003 0 0 0 1 1 1 1 1 1 1 1 1 1 1
4 1000 1 1 0 1 1 0 0 1 0 1 1 1 0 1
5 990 0 1 1 1 1 1 1 1 1 0 1 1 0 1
6 970 0 0 1 0 1 0 1 0 0 1 1 1 1 1
7 960 0 0 0 0 1 0 0 0 0 0 0 0 0 0
8 950 0 1 0 1 1 1 1 1 0 1 1 0 1 0
9 920 0 0 0 0 1 1 0 0 0 1 0 0 0 0
10 900 0 0 1 0 0 0 0 1 0 1 0 1 0 1
11 870 0 1 0 1 1 1 1 1 1 1 1 0 0 0
12 820 0 1 1 1 1 0 1 0 1 1 0 0 1 1
13 770 0 1 0 0 0 1 0 0 0 1 0 0 0 1
14 740 0 0 0 1 0 0 0 0 0 0 0 0 0 0
15 720 0 0 1 0 0 0 0 0 0 1 0 0 0 1
16 690 0 1 1 0 1 0 1 1 1 0 0 0 0 0
17 650 0 1 0 0 0 0 0 0 0 1 0 0 1 0
18 600 1 1 0 1 1 1 1 1 0 0 1 0 0 0
19 540 1 1 1 0 1 0 1 0 0 1 0 0 1 1
20 490 0 0 0 1 0 0 0 1 0 0 0 1 0 1
21 460 0 0 1 0 1 0 0 0 0 0 0 0 0 0
22 430 0 0 0 1 0 0 0 1 0 0 1 0 0 0
23 385 0 0 0 0 0 0 1 1 0 0 0 0 0 0
24 360 0 0 0 0 0 0 0 1 0 0 0 0 0 0
25 325 0 1 0 0 0 1 0 1 0 0 0 0 1 1
26 290 0 0 0 0 0 0 0 0 0 0 0 1 0 1
5 12 9 10 15 9 12 15 5 14 9 8 8 14
Total
95
Table 29: Number of amplified fragments and specific markers
of the fourteen soybean genotypes based on RAPD-
PCR analysis using 4 primers.
RAPD primers.
OP-A 9 B 7 OP-A 7 OP-A 1 OP-A 1A13 Total
No Genotypes AF SM AF SM AF SM AF SM AF
1 L86K-73 3 - 5 - 14 - 5 - 27
2 Corsay-79 2 - 5 - 15 - 12 - 34
3 Giza21 2 - 5 - 15 - 9 31
4 Forrest 1 - 5 - 14 - 10 (1)+ 30
5 Hutcheson 3 - 5 - 14 (3)-(2)+ 15 (1)+ 27
6 Calland 6 - 5 - 15 - 9 - 35
7 Lakota 5 - 5 - 14 (1)- 12 - 36
8 Giza111 4 - 5 - 13 - 15 (1)+ 37
9 Giza83 3 - 5 - 14 - 5 - 27
10 Clark 8 (1)+ 5 - 13 - 14 - 40
11 Giza22 1 - 5 - 16 - 9 - 31
12 Giza35 2 - 5 - 13 - 8 - 28
13 Giza82 5 - 5 - 15 - 8 - 33
14 Crowford 7 - 5 - 14 - 14 - 40
TAF 12 5 19 26 62
PB 12 0 11 26 59
TSM 1 0 6 3 10
96
Figure 9: Dendrogram for the genetic distances relationships among the fourteen
genotypes based on similarity indices data of RAPD analysis.
97
Table 30: Similarity indexes among the fourteen soybean genotypes
based on RAPD-PCR using 4 primers.
Soybean
varieties
Hutcheson
Corsoy-79
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
Clark
Corsoy-79 0.721
Giza21 0.724 0.800
Forrest 0.737 0.781 0.689
Hutcheson 0.625 0.704 0.706 0.657
Calland 0.742 0.783 0.697 0.769 0.667
Lakota 0.698 0.800 0.746 0.758 0.740 0.704
Giza111 0.750 0.761 0.676 0.806 0.649 0.806 0.740
Giza83 0.667 0.787 0.793 0.807 0.688 0.742 0.794 0.719
Clark 0.687 0.757 0.732 0.686 0.675 0.747 0.763 0.727 0.716
Giza22 0.759 0.800 0.710 0.885 0.706 0.788 0.806 0.794 0.759 0.704
Giza35 0.821 0.667 0.733 0.780 0.606 0.719 0.708 0.818 0.714 0.725 0.800
Giza82 0.700 0.806 0.750 0.730 0.629 0.794 0.783 0.714 0.767 0.822 0.781 0.710
Crawford 0.657 0.757 0.761 0.657 0.623 0.720 0.711 0.701 0.687 0.800 0.676 0.754 0.795
98
Restriction Fragment Length Polymorphism (RFLP) for ITS:
RFLP-PCR was also used to analyze the genetic diversity of the fourteen
studied soybean genotypes, and to assess their genetic relationships using similarity
indices and dendogram tree. Three restrictions enzymes were used to cut ITS PCR
products were used to determine RFLP polymorphism of the fourteen soybean
genotypes (Fig. 7). Restriction site analysis of the internal transcribed spacer (ITS)
region amplified by the polymerase chain reaction (PCR) was used for the specific
identification of fourteen genotypes. PCR amplification using primers based on
nucleotide sequences of soybean ITS regions. Digestion of the PCR products with
endonucleases BamH1,MSPI and Taq1 followed by agarose gel electrophoresis of the
digested products, yielded specific restriction profiles that enabled direct visual
identification of the genotpes analyzed.
Universal primers pair were able to successful amplify the ITS region of all
genotypes tested, providing a single (602pb) PCR product Plate (6). The patterns of the
RFLP analysis were characteristic for each genotype restriction enzyme BamH1
produced for each soybean genotypes was seen identical pattern which showed similar
one band in 285 bp. The enzyme MSPI was also used digestion of PCR products for
each soybean genotypes which give one band (870 bp) for L86K-73 and four bands
(730, 417, 275 and 65 bp) for the rest genotypes. While, Taq1 enzyme give six bands
(580, 430, 90, 70, 50 and 30 bp) in all genotypes except L86K-73 and Corsay-79
which give one band 1400 and 30bp respectively. Data in Fig 10 and table 34 showed
similarity indexes was1.000 in all genotypes except L86k-73 and Corsay give 0.182
and 0.842 respectively. The dendogram that show the genetic distance among 14
soybean genotypes is given in Fig. 10 the similarity indices of tested genotypes based
on PFLP for ITS- PCR using 3 restriction enzymes is presented in( Table 34).The data
indicating that 12 genotypes in group2 are genetically related.
Olsen and Woese, (1993) explained ribosomal DNA (rDNA) sequences have
been aligned and compared in a number of living organisms, and this approach has
provided a wealth of information about phylogenetic relationships. Studies of rDNA
sequences have been used to infer phylogenetic history across a very broad spectrum,
from studies among the basal lineages of life to relationships among closely related
species and populations. The reasons for the systematic versatility of rDNA include the
numerous rates of evolution among different regions of rDNA (both among and within
genes), the presence of many copies of most rDNA sequences per genome, and the
pattern of concerted evolution that occurs among repeated copies. These features
facilitate the analysis of rDNA by direct RNA sequencing, DNA sequencing (either by
cloning or amplification), and restriction enzyme methodologies.
99
Plate.(6) : DNA polymorphism based on PFLP-PCR analysis of the soybean genotypes.
1 -L86K-73 2-Corsay-79 3-Giza21 4-Forrest 5-Hutcheson
6-Calland 7-Lakota 8- Giza111 9-Giza83 10-Clark
11-Giza22 12-Giza35 13-Giza82 14-Crowford.
100
Table 31: RFLP profile of different genotypes in soybean cutting with Bam H1
Soybean Varieties
Fragment MS
Hutcheson
Corsoy-79
Crawford
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
No. (bp)
Clark
1 285 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
Total
Table 32: RFLP profile of different genotypes in soybean cutting with MSPI
Soybean Varieties
Fragment MS
Hutcheson
Corsoy-79
Crawford
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
No. (bp)
Clark
1 870 1 0 0 0 0 0 0 0 0 0 0 0 0 0
2 730 0 1 1 1 1 1 1 1 1 1 1 1 1 1
3 417 0 1 1 1 1 1 1 1 1 1 1 1 1 1
4 275 0 1 1 1 1 1 1 1 1 1 1 1 1 1
5 65 0 1 1 1 1 1 1 1 1 1 1 1 1 1
1 4 4 4 4 4 4 4 4 4 4 4 4 4
Total
Table 33: RFLP profile of different genotypes in soybean cutting with Taq1
Soybean Varieties
Fragment MS
Hutcheson
Corsoy-79
Crawford
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
No. (bp)
Clark
1 1400 1 0 0 0 0 0 0 0 0 0 0 0 0 0
2 580 0 0 1 1 1 1 1 1 1 1 1 1 1 1
3 430 0 0 1 1 1 1 1 1 1 1 1 1 1 1
4 90 0 0 1 1 1 1 1 1 1 1 1 1 1 1
5 70 0 0 1 1 1 1 1 1 1 1 1 1 1 1
6 50 0 0 1 1 1 1 1 1 1 1 1 1 1 1
7 30 0 1 1 1 1 1 1 1 1 1 1 1 1 1
1 6 6 6 6 6 6 6 6 6 6 6 6
1
Total
101
Figure 10: Dendrogram for the genetic distances relationships among the
fourteen genotypes based on similarity indices data of RFLP analysis.
varieties
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
Clark
Corsoy-79 0.182
Giza21 0.143 0.842
Forrest 0.143 0.842 1.000
Hutcheson 0.143 0.842 1.000 1.000
Calland 0.143 0.842 1.000 1.000 1.000
Lakota 0.143 0.842 1.000 1.000 1.000 1.000
Giza111 0.143 0.842 1.000 1.000 1.000 1.000 1.000
Giza83 0.143 0.842 1.000 1.000 1.000 1.000 1.000 1.000
Clark 0.143 0.842 1.000 1.000 1.000 1.000 1.000 1.000 1.000
Giza22 0.143 0.842 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
Giza35 0.143 0.842 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
Giza82 0.143 0.842 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
Crawford 0.143 0.842 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
102
Combined data based on SDS-PAGE protein profile, RAPD and
RFLP:
103
Figure 11: Dendrogram for the genetic distances relationships among the fourteen
genotypes basedon similarity indices data of protein SDS-PAGE, RAPD and RFLP analysis.
Table 35: Similarity indices among the fourteen soybean genotypes based on Protein
SDS-PAGE,RAPD and RFLP-PCR using 3 restriction enzymes.
Soybean
Hutcheson
varieties
Corsoy-79
L86K-73
Giza111
Calland
Forrest
Lakota
Giza21
Giza83
Giza22
Giza35
Giza82
Clark
Corsoy-79 0.772
Giza21 0.754 0.873
Forrest 0.761 0.864 0.848
Hutcheson 0.700 0.818 0.848 0.824
Calland 0.741 0.844 0.828 0.866 0.806
Lakota 0.739 0.870 0.870 0.877 0.861 0.827
Giza111 0.767 0.848 0.833 0.901 0.812 0.881 0.861
Giza83 0.727 0.869 0.902 0.909 0.844 0.855 0.898 0.859
Clark 0.732 0.844 0.859 0.836 0.823 0.847 0.871 0.851 0.855
Giza22 0.772 0.873 0.857 0.944 0.848 0.875 0.901 0.894 0.885 0.844
Giza35 0.804 0.806 0.871 0.894 0.800 0.841 0.853 0.908 0.867 0.857 0.903
Giza82 0.741 0.875 0.875 0.866 0.806 0.877 0.887 0.851 0.887 0.905 0.891 0.857
0.715 0.844 0.874 0.821 0.794 0.832 0.843 0.837 0.840 0.889 0.830 0.872 0.891
Crawford
104
SUMMARY
The experiments of the present study were conducted at Field Crops Research
Institute (FCRI), Cell Research Study Department, Agriculture Research Center
(ARC), Giza, Egypt during the growing seasons 2003, 2004 and 2005.Fourteen
soybean genotypes namely L86K-73, Corsay-79, Giza21, Forrest, Hutcheson, Calland,
Lakota, Giza 111,Giza 83, Clark, Giza 22, Giza 35, Giza 82 and Crowford were used
as plant materials throughout this investigation. The objectives of this study were:
The single analysis of variance for each season showed that significant
differences among genotypes were existed for all studied characters. The combined
analysis of variance indicated significant differences among genotypes for all
characters, while the season had significant effects on plant height, no. of pods/plant,
no. of seeds/plant, seed yield/plant and harvest index. The genotype x season interaction
effect was significant in days to 50% flowering, days to 90% maturity, maturity
period, plant height, seed yield/plant and 100-seed weight.
The results of the evaluation of the genotypes for agronomic characters and their
resistance to leaf cotton warm under field and laboratory conditions, showed narrow
range (22.35 – 26.10%) of seed oil content, while wide range of seed protein (22.70 –
57.93%) was obtained in this study. The average leaf area was ranged from 18.12 cm2
for L86K-73 to 70.24 cm2 for Giza 21, with an average of 48.27 cm2. The number of
leaf-hairs was showed the widest range (41 for Calland to 173.33 hairs for Giza 83)
among these characters. To determine the relations among these characters, correlation
coefficients were made among them. No significant differences have been found
105
among seed oil and protein contents, at one side, and survival (1) and (2) on the other
side. High significant and negative correlation was observed between number of leaf-
hairs and survival (1) and (2). The correlation coefficients among leaf-hairs and
survival (1) and (2) were r = -0.678** and -0.630**, respectively, indicating that dense
leaf-hairs is related with high resistant level. Similar finding was reported by in cotton.
The strong positive correlation between survival 1 and 2 (r = 0.891**) obtained
indicated that one test only, either in young or adult stage of leaf-worm would be
enough for screening.
106
3- Agrobacterium Establishment Transformation System Of
Soybean Using Immature Embryos and Cotyledonary Nodes
107
(Figures 4 and 5). The obtained results are in agreement with those obtained by who
suggested that the Kanamycin concentration, to be used as selectable marker ranged
between 50 and 300 mg/liter in Vitis vinifera. On the other hand, and noted that the
efficient Kanamycin concentration to used as selectable marker is l00 mgL-1, which is
accordance with the results obtained in this study.
As indicated above the transformation was successfully made for (a) and (b)
Agrobacterium mediated immature embryos and cotyledonary node
transformation. The method of cotyledonary node took shorter time than immature
108
embryo method. In addition the somaclonal variation in cotyledonary node is lower
than the other method. Therefore, cotyledonary node is successfully to be better than
immature embryo method. Studied improvements in T-DNA delivery, A. tumefaciens
strain, tissue culture conditions, and selection of transgenic plants have increased the
efficiency of these transformation systems and reported of cotyledonary node method
in approximately.
The second technique was leaf and seed protein analysis by SDS-PAGE
Protein electrophoresis. Bands intensity was much dense in resistance genotypes.
Protein SDS -PAGE didn't show differences between the fourteen genotypes except
the intensity of bands. The results revealed the presence of eight bands of molecular
weight ranging from 1263 to 144 KD. Concerning the differences between genotypes,
it is clear from leaf protein profile that the presence and absence of bands were
assessed with (1) and (0), respectively. The overall results of total protein pattern
obtained by SDS-PAGE were not clear cut in monitoring molecular markers for the
plant insect resistance against the cotton leaf worm spodoptera littorallis (Boisd).
Similar results were obtained also in soybean. This result might be attributed to that
the study of the resistant and susceptible genotypes was done with no cotton leaf worm
infection stress. The results of SDS-PAGE for the water soluble proteins leaf in the
fourteen genotypes (Plate 4 and Table 22) revealed a total number of 8 bands with
molecular weights (MW) ranging from about 1263 to144 KDa. Data showed that all
bands are common for the fourteen soybean genotypes (monomorphic), however they
differed in density and intensity. The densitometric analysis of SDS-protein banding
patterns of the leaf of the studied genotypes was found to be not informative in their
work. On the other side, the results of SDS-PAGE for the water soluble proteins in
109
the seeds of fourteen soybean genotypes (Plate 4 and Table 23) revealed a total
number of 13 bands with molecular weights (MW) ranging from about 220.36 to 9.1
KDa. Data showed that all bands are common for the fourteen genotypes except two
bands 220.36 and 194 KDa. for Calland genotype can be considered as specific bands.
Moreover they differed in density and intensity. Similarity index among the fourteen
soybean genotypes based on protein analysis, carried out using UPGMA computer
program, is shown in Table (24). The similarity relationships ranged between 1.000
and 0.950. The highest similarity index (1.000) was recorded between each two of the
genotypes; L86K-73,Corsay-79,Giza 21,Forrest,Hutcheson,Lakota,Giza111,Giza
83,Giza 22 ,Giza 35,Giza 82 and Crowford. However, the lowest similarity index
(0.950) was observed between Calland and each of the thirteen genotypes. The genetic
distances relationships among the fourteen soybean genotypes based on leaves and
seeds protein patterns. The dendrogram confirmed the close genetic relationship
among these genotypes.
The third technique was Random Amplified Polymorphic DNA (RAPD) which
used four 10-mer arbitrary primers for the fourteen soybean genotypes. The results
showed that polymorphism levels differed from one primer to the other 30-1670 pb. ,
Primers produced high levels of polymorphism fragments number ranging from four
primers.. There were some specific fragments which can be used to discriminate each
soybean genotypes from the other.
These markers as present in following:- Primer OP-A9B7 showed specific
fragment (520bp) as positive marker for Clark genotype. Primer OP-A1 showed six
specific fragment four of them as negative markers three for Hutcheson
genotype(430,280 and 50 bp) and one for Lakota genotype (810 bp) as negative
marker, and also two as positive markers (360 and 320 bp) for Hutcheson. Primer OP-
A1A13 showed three specific fragments as positive markers one (960bp) for
Hutcheson, one (740bp) for Forrest and one (360bp) for Giza111. Primer OP-A7 did
not showed specific fragment.
The results of cluster analysis (similarity index) the highest similarity value recorded was 0.885, which was
observed between Forrest and Giza 22 genotypes, while the lowest similarity value was 0.606 between Hutcheson
and Giza35 genotypes. The dendrogram for the genetic relationships among the fourteen soybean genotypes across
the four primers indicate that the fourteen soybean genotypes were separated into two clusters; cluster 1 included
two sub cluster. The sub cluster1 were conformed from three groups .The group 1, group 2 and group 3 were
included Forrest and Giza22; Calland and Giza111 and Giza 35 and L86K-73 respectively. While, sub cluster 2
comprised four groups group1 were contained Clark and Giza 82, group 2 has Crowford genotype only and the
genotypes Giza21 and Giza83 were located in group 4. The second cluster was contained Hutcheson genotype. The
RAPD was succeeded to distinguish the 14 soybean genotypes on the basis of their resistance cotton leaf worm.
The fourth technique PFLP used The results investigated the PFLP pattern by restriction enzyme BamH1
produced for each soybean genotypes was seen identical pattern which showed similar one band in 285 bp. The
enzyme MSPI was also used digestion of PCR products for each soybean genotypes which give one band (870 bp)
for L86K-73 and four bands (730, 417, 275 and 65 bp) for the rest genotypes. While, Taq1enzymee give six bands
(580, 430, 90, 70, 50 and 30 bp) in all genotypes except L86K-73 and Corsay-79 which give one band 1400 and
30bp respectively. However this technique did not allow differentiating among genotypes on the basis of their
resistance to cotton leaf worm probably due to this narrow genetic distance. The combined dendogram was made
used the results obtained from protein RAPD and RFLP analysis. The dendogram was classified the fourteen
genotypes according to their resistance to cotton leaf worm. The genotypes Forrest, Giza 22, Giza111, Giza 35,
Calland, Clark and Giza 82were the most related and ranked in the dendogram from 1 – 7. Theses genotypes are
resistance/ moderately resistance to cotton leaf worm.
110
REFERENCES
A.O.A.C. (1990). Official Methods of Analysis. Association of Agriculture Chemists,
Al-Janabi, S.M. and R.C. Shoemaker (1992). A rapid method of regeneration and
19: 101-107.
Allard, R.W. (1960). Principal of plant breeding. Wiley and Sons, New York, pp 485.
soybean varieties for the processing and improvement of fermented foods, Part I.
Ausubel,F.M.,Brent,R.,Kingston,R.E.,Moore,D.D.,Seidman,J.G.,Smith,J.A.,Struhl,K.
Awadallah, W.H.; A.F. Lutfallah; Eglal M. Abd Elmonem; M.F. El-Metwally and M.Z.
Hassan (1990). Evaluation of some soybean genotypes for their resistance to the
111
cotton leaf worm Spdopetra littoralis (Boisd.). Agric. Res. Rev., Egypt 68 (1): 121-
126.
Barawale, U.B.; H. R Kerns and J.M. Widholm (1986). Plant regeneration from callus
Planta, 167:473-481.
Bartun, G.W. (1952). Quantitative inheritance in grasses. Proc. VI. Int. Grassland Cong.,
1:222-283.
Byrne,M.C.; R.E. McDonnell; M. S. Wright; M.G. Carnes, (1987). Plant Cell Tissue and
Kiihl (2002). Molecular markers linked to the resistance to race 3 of the soybean
2(1): 77-83.
Carvalho,.G.A.; T. Sediyama; A.L.A. Marin; E.G. Barros and M.A. Moreira (2002).
112
Casas, A.M., E. Igartua, G. Balaguer and M.A. Moreno (1999). Genetic
Chen, L.F.O.; W. C. Yun ; H.Y. Kou and M. H. Chen (1994) Polymorphic distinction of
Chettri, M.; S.Mondal and R. Nath (2003) Studies on correlation and path
Christou, P.; D.E. McCabe and W.F. Swain (1988). Stable transformation of soybean
Colby and Meredith (1990). Kanamycin sensitivity of cultured tissue of Vitis. Plant Cell Rep.,
9: 237-240.
hypocotyl explants. In Vitro Cellular and Develop. Biology Plant, 34(1): 14-21.
tuberosum) using Agrobacterium tumefaciens. Theor. and Appl. Gene., 76: 767-774.
Dellaporta, S. L.; J. Wood and J.P. Hicks (1983). A plant DNA minipreperation.Version III
113
Delzer,B.W.; D.A. Somers and J.H. Orf (1990). Agrobacterium tumefaciens susceptibility
soybean seed protein and oil content. Theor. Appl. Genet. 83: 608 – 612.
19(5): 478-484.
El-Dakroury, F. (1979). Studies on the important cotton insects. Ph. D. Thesis Fac. Agric.
Reproducible transformation in two grain legumes soybean and azuki bean - using
cultivars. M.Sc. Thesis, Fac. Agric. Ain Ahams Univ., Cairo, Egypt.
Fenille, R.C.; N. L. de. Souza and E.E. Kuramae (2002). Characterization of Rhizoctonia
108(8): 783-792.
114
Funatsuki, H.; H. Kurosaki; T. Murakami; S. Matsuba; K. Kawaguchi; S. Yumoto and
Ghanem, S. A. (1995). In vitro propagation of Hyoscyamus. Bull. Fac. Agric. Univ. Cairo
46: 103-112.
Gomez, K.A. and A.A. Gomez (1984). Statistical Procedures of Agricultural research. 2nd
relationships among 10 wild perennial soybean species using specific and arbitrary
Habbeb, B.M. (1988). Genetical studies on soybean (Glycine max (L) Merril). Ph.D. Thesis,
Hamdi, A.; A.M. Khattab and M.K. El-Warki (1991). Genotype x environment interaction
and stability analysis for seed yield of lentil. Fayoum J. Agric. Res. & Dev., 5(2):1-
12.
115
Helms, T.; J. Orf; G. Vallad and P. McClean (1997). Genetic variance, coefficient of
453.
Hoagland, D.R. and D. I. Arnon (1950). The water-culture method for growing plants
Hoekema, A.;P.R. Hirch; P.J.J. Hooykaasand and R.A. Shilperoort (1983). Nature,
(303) 179-180.
Div. Evol.
1, 179–191.
Jagdish S.; R.P. Parmar; and H.S. Yadav (2000). Assessment of genetic variability and
13(1): 227-232.
61-72.
116
Johanson, H.W.;H.F. Robinson and R.E. Comstock (1955). Estimation of genetic and
19.
production, 108-116.
Ko, T.S.; S.M. Lee; S.K. Farrand and S.S. Korban (2004a). A partially disarmed vir
Ko, T .S. and S.S. Korban (2004b). Enhancing the frequency of somatic embryogenesis
soybean [Glycine max (L.) Merrill.]. In Vitro Cellular and Developmental Biology
117
Kosturkova,G.(2000). Development of in vitro regeneration systems of soybean tissue
Li, H.Y.; Y.M. Zhu; Q. Chen; R.L. Conner; X.D. Ding; J. Li and B.B. Zhang (2004).
Production of transgenic soybean plants with two antifungal protein genes via
Li, J. ; J. Faghihi; J.M. Ferris and V. R. Ferris (1996). The use of RAPD amplified DNA
Li-GuiLan; Qiao Ya Ke; Yang Shao Hui; Jin Zhao Xia and Li Ming Gang (2005).
Liu Hai Kun; Yang Chao and Wei Zhi Ming (2004). Efficient Agrobacterium
118
Liu XiaoBing, Wang GuangHua, Jin Jian, Herbert, S. J. and Zhang QiuYing (2005).
Yield components, dry matter, LAI and LAD of soybeans in Northeast China. Field
Luo, G; A. Hepburn and J. Widholm (1994). A simple procedure for the expression of -
119 - genes in transgenic soybean callus tissue. Plant Cell Reports, 13 (11): 632-
636.
Lutfallah, A.F.; Kh. A. Alassily; M.A. El-Nagar; M.S. Ali; M..M. Khewa and E.A.H.
Sherief (2003). Effect of simulated infestation levels caused by leaf feeding larvae on
soybean plant traits and crop yield. Egypt. J. Appl. Sc., 18 (11B): 607-620.
Lutfallah, A.F.; M.Z. Hassan; K. A. Mewafy, Safia T. Abdalla and Kh. A. Alassily (1998).
Studies on soybean genotypes resistant to the cotton leaf worm, Spodoptera littoralis
1860.
Mahmoud, Z.H. (1984). Soybean oil and meal, the focus for production and research in Egypt.
In: Singh S.R., K.O. Rochieand and K.E. Dasniell (eds.). Soybean for tropics. John
soybean (Glycine max L. Merrill) leaf discs influenced by genotypes and plant
119
(Glycine max L. Merr). Plant Cell Biotechnology and Molecular Biology, 5(1/2): 1-
6.
derived cultures of soybean (Glycine max L. Merr). Plant Cell Biotechnology and
Entomologic, 95 (5): 467-470. (C. F. Rev. App. Ent., 71 (11): 7571, 1983).
Mello-Filho, O.L.de; C.S. Sediyama; M.A. Moreira; M.S. Reis; G.A. Massoni; and
N.D. Piovesan (2004). Grain yield and seed quality of soybean selected for high
Mizutani, N.; T. Wada and M. Takahashi (2001). The resistance of the soybean
breeding line Kyukei 279 to the common cutworm, Spodoptera litura, and
Mukhekar, G. D.; N.D. Bangar and D. B. Lad (2004). Character association and path
Murashige, T. and F. Skoog (1962). A revised medium for rapid growth of bioassays with
120
Myong Gi CHUNG; Mi Yoon CHUNG; April D. C. JOHNSON and Reid G. PALMER
Nan Xiang Ri; Liu Wen Ping; Liu Li Yan; Lu Xiao Bo; He Yun Xia and Wei-
protoplasts with Bacillus thuringiensis CryIAc gene. Soybean Science, 17(4): 326-
330.
(9):521 0-5208.
sprout with small seed size, disease resistance and high yield, "Bosug. Korean
Olhoft, P.M.; L.E. Flagel; C.M. Donovan and D.A. Somers (2003). Efficient soybean
Ojo, D.K. and O.J. Ariyo (1999). Inheritance of resistance to the soybean
Jan;7(1):113-23.
121
Owens, L.D. and C. A. Smigocki, (1988). Transformation of soybean cells using mixed
88(3): 570-573.
54-56.
Pham Thi Be Tu, Nguyen Thi Lang and Bui Chi Buu(2003). Soybean
122
Raut,-P-B; N.N.Kolte; T.H. Rathod ;R.S. Shivankar and V.N. Patil (2001). Correlation
and path coefficient analysis of yield and it's component in soybean (Glycine max
Rech, E.L.; T.J. Golds; M.R. Davey and N. Hammatt (1988). Hairy root induction in
canescens. regeneration from soybean Glycine max (L.) Merr. Hypocotyls Plant.
molecular markers linked to quantitative trait loci for soybean resistance to corn
molecular markers linked to quantitative trait loci for soybean resistance to corn
maturity groups. Plant Cell, Tissue and Organ Culture, 75(3): 273-
277.
123
Entomological Society of Egypt, Economic Series,1988-1989 publ
Robertson, J.A.; W.H. Marison and D. Burdick (1973). Chemical evaluation of oil from
Roger Boerma H. and David R. Walker(2005). Discovery and utilization of QTLs for
Ronde, J.A.de; W.A .Cress; Mescht and A van der (2001). Agrobacterium-mediated
transformation of soybean (Glycine max) seed with the beta -glucuronidase marker
Roy,-P-K and S-R- Maloo (2001). Diallel study on some in vitro callus traits of soybean
[Glycine max (L.) Merrill]. Journal of Genetics and Breeding, 55(4): 357-362.
regeneration via organogenesis from soybean cotyledonary nodal callus. Plant Cell
Salama, H.S.; F.N. Zaki; S. Salem and M. Ragaei, (1995). The use of
manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
124
Santarem,E. A. , B. Pelissier and J.J. Finer (1998). Effect of ex-plant orientation, pH,
(Glycine max (L.) Merrill) and peanut (Arachis hypogaea L.) by real-time
Schroeder, H.E.; J.E. Barton; L.M. Tabe; L. Molvig; J.E. Grant; M. Jones and T.J.V.
Higgins (2000). Gene technology for improved weed, insect and disease control
and for seed protein quality. In: Knight R.(ed.). Linking Research and Marketing
Publishers.
a new major QTL associated with resistance to soybean cyst nematode (Heterodera
soybean (Glycine max (L.) Merr). Advances in Plant Sciences. 11(1): 193-197.
Shalini Bajpai; Aparna Sharma and Munishwar Nath Gupta(2005). Removal and
444.
125
Silva, J.F.V.; R.V. Abdelnoor; C.L. Costa and G.S.Baia (2000). RAPD analysis of
Smith, C.M. and C. A. Brim (1979). Resistance to mexican bean beetle and corn earworm in
Soltis, D. E. and Pamela, S. Soltis (1990). Isozymes in plant biology. Dioscorides Press,
PR, - Brazil.
E. Specht and P.B. Cregan (2004). A new integrated genetic linkage map of the
by gel electrophoresis. J. Mol. Biol. 98: 503. supports. Anal. Biochem., 138: 267-
284.
Biol., 79:227-236.
126
Sudaric, A.; M. Vrataric and T.Duvnjak (2002). Quantitative genetic
26(4): 329-332.
Tomar, N.S. and Rajput B.S. (2002). Esterase and peroxidase isozyme markers in seed
Stewart (2002) Screening of soybean, Glycine max (L.) Merrill, lines for somatic
127
Turkec, A. (2005). Correlation and path analysis of yield components in soybean varieties.
(2004). A QTL that enhances and broadens Bt insect resistance in soybean. Theor.
Wang Ping; Wu Ying; Ji Jing; Wang Gang and Yang QingKai (2001). Effect of
23(4): 321-324.
Wang Wei; Zhu Zhen; Gao YueFeng; Shi Chun Lin; Chen Wan Xin; Guo Zhong Chen
Wang, L; T. Clemente; Wang Lian Zheng; S.S. Sun and Huang Qi Man(2003).
Wang, P.; Wang Gang; Ji Jing and Wu Ying(2004). Research progress on system of
Widholm, J. M.; P.A. Stephens and C.D. Nickell (1990). Iron deficiency chlorosis
417-420.
Widholm, J.M.; M.J. Graham; D.L. Meavner and C.D. Nickell (1993). Responses of
soybean genotypes to boron, zinc and manganese deficiency in tissue culture. Plant
128
Williams,J. G. K.;A. R. Kubelik;K. J. Livak;J. A. Rafalski and S. V. Tingey(1990).
J.de. Greef (1985). Dynamics of endogenous IAA and cytokinins during the
growth cycle of soybean crown gall and untransformed callus. Plant and Cell
Xing Ai Qiu; Zhang Zhang Yuan; S. Sato; P. Staswick and T. Clemente (2000). The
use of the two T-DNA binary system to derive marker-free transgenic soybeans.
Xu Xiang Ling; Gao Jing; Liu Wei Hua and Li Ji Lin (1997). Studies on transferring the
B.t. k-delta endotoxin gene into soybeans with Ti-plasmid primaril. Soybean
tendency detected by isoenzyme, RFLP and RAPD markers in wild and cultivated
and Oil from Soybean Northern and Southern Region Uniform Tests. Crop Sci.
42:1504–1515.
Yan, B.; M.S.S. Reddy; G.B. Collins and R. D. Dinkins (2000). Agrobacterium
108.
129
Yencho G.C.; M.B. Cohen and P.F. Byrne (2000). Applications of Tagging and Mapping
Insect Resistance Loci in Plants. Annual Review of Entomology Vol. 45: 393-422.
Yue, S. X.; B. L. Liu; D. Z. Mao;X. B. Li; N. B. Hu; J. H. Fu.; L.C. Li and L.H.
Cober ; Victoria Chang and Stephen Gleddie (2007a). Protein quality and
identification of the storage protein subunits of tofu and null soybean genotypes,
using amino acid analysis, one and two-dimensional gel electrophoresis, and
fourteen soybean [Glycine max (L.) Merr.] cultivars using amino acid analysis and
Zayed, M. E. (1998). Estimation of genetic variance of soybean. M. Sc. Thesis, Fac. Agric.,
130
selection in Agrobacterium-mediated transformation of soybean [Glycine max (1.)
Zhang-Zhan Yuan; Xing Ai Qiu; P. Staswick and T. E. Clemente (1999). The use of
Zhang, Z. Y., Banerjee, A. K., Shou, H. X., Wang, K., Paz, M. M., Guo, Z. B(2004).
transformation using the cotyledonary node explant. Euphytica, Vol. 136 (2) 167-
179.
Zhou, S.; G. Yin; B. Lei; Z. He; C. Lu; K. Zhang and H. Qian (1990). Plant regeneration
Science.9:285-291.
131
ﺍﻟﻤﻠﺨﺺ ﺍﻟﻌﺮﺑﻰ
أ ه ا را ث ا
ا – "! ث درا ا – '&آ $ا #ث
ا $را*
)$ة,/ .ل ,-ث 'ا! 2005 ،2004 ،2003 +و ا 67 '8ه
ا را &> 14آ= ورا7 <' 6-ل ا ;?
وه ، L86k-73 6آر$C ،79-6ة، 21
$Cة$C ،22ة $C ،82ة $C ،83ة $C ، 35ة 7 ،111ر ،آ ،H,آ,ركF ،آ>،E
ه"J8ن ،آ&او7رد.
إ ارا
:
>! أداء و> <?
#ا &8اآ= ا را+ -ل ا ;?
ا
و' وا K8ا
و' وا "
-1
ودة ورق ا O' <Kدرا ا
ت ا را.-
> <?Rودرا ا
Rس وإ
8Hج ا
>
#Tت '< ا ?&Kا &S
#ة <?R8ا
P*Qء ،ودرا -2
ا
7,8/Fت '
< ا &8اآ= ا را+ -ل ا ;?
.
أداء
]Hم ا 8ل ا را 6-وإ
8Hج ا
>
#Tت '< زرا* ا ) <Tا XY
H &Zوا ا .+ -3
ا ^8ا )&8 6_?$اآ= ا را+ -ل ا ;?
ا
و' وا K8ا
و' -4
وا "
ودة ورق ا .<K
• أ K' `7 <8&)> ?&Cث ا )$ة – '&آ $ا #ث ا $را* '،2003 6
2004وآ
ن '
د ا $ا* ه ,R ?
' 25ا < .و>! > ا 6* !' R <?
#8
aة .أ'
)8 #"Tرب ا ا
ودة ورق ا 7 <Kأ&Cى > ا <?
#8
; Survival 1 68+و Survival 2ا * <
' #"Tد ا &
ت ا وا 8ودة
ورق ا -,- <Kأ?
م #> ،<+8' <?&* `7أ '< ا ) اQول 68aا ) ا d
e
) (Survival 1و'< ا ) ا &ا 68a Oا "
دس ) .(Survival 2وإر>
+ع *د ا &
ت
ا ? 6Tأن ا &8آ= ا را6-
132
ERROR: syntaxerror
OFFENDING COMMAND: --nostringval--
STACK:
945
12810
12