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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 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.

Advances in biotechnology can facilitate the development of insect-resistant soybean cultivars by means of gene transformation. Successful use of gene transfer requires that the gene for insect resistant is identified, isolated and then reconstructed for expression in relevant organ of the new host. In addition, gene transfer procedures and appropriate tissue culture methods must be developed for each target species to regenerate fertile, transgenic plants. 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. 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. In the first reports of soybean transformation, two different methods were applied. The first one, was Agrobacterium mediated transformation cotyledonary nodes, while the second, was partial bombardment of shoot meristems. Soybean transformation reports following these initial works have been limited and transformation efficiency for soybean has remained low. Later transformation efficiency has been improved.

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.

The purposes of this study aimed to:

(1)

Describe field performance of 14 soybean genotypes susceptible/ moderate resistance/resistance to cotton leaf worm, and study the related genetic parameters.

genotypes susceptible/ moderate resistance/resistance to cotton leaf worm, and study the related genetic parameters.

(2) Initiate and maintain callus organogenesis cultures of soybean. The differences among soybean genotypes in callus formation were also evaluated. (3) Performed a system for soybean transformation and regeneration using immature embryos and cotyledonary nods. (4) Use of DNA markers in particular to their use in molecular characterization for genetic improvement of cotton leaf worm in soybean.

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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.

The inheritance of resistance to cotton leaf worm (Spodoptera littoralis, Boisd) in soybean, based on the three main criteria; hairiness, leaf area consumed and defoliation, was studied. High to moderate values of broad sense heritability for the three characters, with high expected genetic advance from selection for defoliation were obtained. Study the genetic features of characters related to insect resistance could help for selection soybean varieties resistant to cotton leaf worm. The aim of this study was to describe field performance of 14 soybean genotypes, and study the genetic parameters for their characters. In addition, investigation of interrelationships among all studied characters with resistant-related characters was made to facilitate the indirect selection for resistant varieties.

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characters with resistant-related characters was made to facilitate the indirect selection for resistant varieties. 3

REVIEW OF LITERATURE

Agronomic traits and their Correlations:

Jagdish et al. (2000) evaluated 16 F2s and their 14 parents to assess genetic variability and selection parameters for seed yield in soybean. Significant differences among parents and F2s were recorded for all the studied characters. The estimate of genotypic coefficient of variation and phenotypic coefficient of variation were comparatively high for biological yield, seed yield/plant, pods/plant and plant height. All the characters exhibited high estimates of heritability. Seed yield/plant, biological yield, pods/ plant and plant height showed high heritability with high genetic advance as a percentage of mean. Significant positive correlation and high magnitude of correlated response along with relative selection efficiency for biological yield, pods /plant, seeds/pod and 100-seed weight showed that these are major yield component in early generation of soybean.

Jain and Ramgiry (2000) studied field investigation of 56 soybean genotypes which showed significant variation for all 12 yield components. High phenotypic and genotypic coefficient of variability were recorded for seed yield/plant, followed by plant weight, plant height and moderate coefficient of variability for 100-seed weight, No. of seed/pod, pod bearing nodes and days to flowering. High heritability estimates accompanied by high genetic advance as a percentage of mean were noticed for seed yield, plant height and pods/plant. These traits were found major yield contributing traits in soybean.

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.

Suh-Sugkee et al. (2000) reported soybean (Glycine max) cultivars with smaller leaf area and lanceolate leaf shape have shown better light distribution through their canopy and a higher photosynthetic rate than those with larger leaf area and oval leaf shape. However, very little information has been published about leaf characteristics in relation to yield potential and inheritance, which would assist breeding efforts to develop new cultivars with optimum leaf area and leaf shape. Gene action and heritability for leaf area, leaf shape and other reproductive characteristics were studied in a diallel cross involving nine parents with large, medium and small leaf area. Most progenies from crosses among parents with different leaf areas had larger mean leaf area, longer flowering and later maturity than the parents, suggesting transgressive segregation for these traits. General combining ability (GCA) and specific combining ability (SCA) for leaf area and leaf shape were significant. Ratios of GCA to SCA were 0.96 for leaf shape and 0.89 for leaf area, indicating that GCA effects were more important than SCA effects. Genetic gain for leaf area and shape may be possible via selection. Narrow-sense heritability estimated on the basis of

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Genetic gain for leaf area and shape may be possible via selection. Narrow-sense heritability estimated on

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.

Ayub-Khan et al. (2000) estimated heritability and simple correlation among yield determining components in 86 diverse maturity genotypes of soybean in Pakistan. The heritability value ranged from 29.37% for No. of seeds/pod to 98.98% for maturity. Seed yield were positively and significantly correlated with all characters except pod height. Path coefficient analysis revealed that No. of pods/plant had the direct effect on seed yield followed by 100 seed weight. The No. of Pods/plant affected seed yield negatively via indirect effects of plant height, pod height and seeds/pod.

Raut et al. (2001) evaluated 30 genotypes of soybean, sown in India, for correlation and path coefficient analyses. Data were recorded for 10 characters, i.e. days to 50% flowering, days to maturity, plant height, number of branches/plant, number of clusters/ plant, number of pods/ plant, 100-seed weight, oil content, harvest index and seed yield/ plant. Seed yield showed positive significant association with number of clusters/plant, number of pods/plant, 100-seed weight, oil content and harvest index, both at genotypic and phenotypic levels. Path analysis indicated that 100-seed weight exhibited the maximum positive direct effect on seed yield, followed by number of clusters/plant, days to maturity and number of pods/plant.

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.

Antarlina et al. (2002) investigated the physical, chemical and processing characteristics of 14 Indonesian soybean cultivars. The 100-grain weight of Indonesian cultivars ranged from 6.1 to 15.9 g, with a mean of 10.6+or-2.8 g (mean + or-SD). The 100 grain weights of two soybean genotypes imported from USA were 15.8 and 14.8 g. The Indonesian soybean samples contained 42.0+or-1.4% proteins and 18.6+or- 1.2% lipids (on dry matter bases), while the imported samples contained 36.8 and 36.0% proteins and 21.7 and 21.4% lipids.

Chettri et al. (2003) evaluated 18 elite soybean genotypes for correlation of agronomic traits in India. Results showed that grain yield was significantly correlated with days to maturity and number of grains/pod at the genotypic level. Days to

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grain yield was significantly correlated with days to maturity and number of grains/pod at the genotypic

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.

Bangar et al.(2003) studied the correlation among component characters and yield of 16 soybean genotypes. The values of phenotypic coefficient of variation (PCV) were higher than genotypic coefficient of variation (GCV). The GCV and PCV estimates were highest for branch number/plant and plant height among the characters. The GCV and PCV were of moderate magnitude for the pod number /plant, 100-seed weight (g) and seed yield/ plant (g). Days to 50% flowering and days to maturity had very low GCV and PCV estimates. The differences between GCV and PCV magnitudes were very high for 100-seed weight (12.94) and pod number/plant (10.30). Among the characters, days to maturity (97.80%), branch number/plant (91.39%) and plant height (60.82%) showed the highest magnitude of heritability. Genetic advance was high for branch number/plant, plant height and seed yield. The regression of seed yield on seed weight, plant height and pod number/plant was positive and highly significant. However, the regression coefficients of branch number/plant were negative. Correlation coefficients indicated that the seed was positively and significantly correlated with 100-seed weight and followed by days to maturity, plant height and days to 50% flowering. Days to maturity, plant height, pod number/ plant and 100-seed weight among themselves were positively significant.

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.

Sudaric et al. (2004) conducted to evaluate genetic advance in yield components, grain yield and grain quality of soybean throughout analysis of agronomic values of new domestic elite breeding soybean lines in comparison with commercial cultivars. The reliability of the grain quantity traits as selection criteria in soybean breeding on grain yield was also determined. The investigations were carried out in Osijek, Croatia, from 2001 to 2003 and involved 31 domestic soybean

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determined. The investigations were carried out in Osijek, Croatia, from 2001 to 2003 and involved 31

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.

Jyoti and Tyagi (2005) evaluated 31 soybean genotypes in India. The parameters studied included: days to 50% flowering, plant height, number of primary branches/plant, pod length, pod width, days to maturity, biological yield, 100-seed weight, seed yield/plant and harvest index. Results showed high genetic coefficient of variation and heritability along with high genetic gain for the following traits:

biological yield/plant, 100-seed weight and plant height. These traits also showed significant and positive association with seed yield/plant. Biological yield/plant showed high and direct positive effect 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

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in each group. Higher accumulation of dry matter, higher LAI and LAD during reproductive stages were

related to high-yield genotypes in each group. No relationship was found between harvest index (HI) and seed yield.

Cotton leaf worm (Spodoptera littorallis):

El-Dakroury (1979) carried out laboratory tests on the development of Spodoptera littoralis (Boisd.) on two Egyptian cotton strains, Bahtim-110 (free of gossypol) and Bahtim-101 (hairy) and two wild species, G. klotzschanum and G. thurberi, while, the Egyptian variety Menoufi was used for comparison. He found that the highest larval mortality, the longest larval period, the lowest percentage of pupation and emergence, highest Pupal weight, the lowest number of eggs and hatchability were recorded when the newly hatched larvae of the cotton leaf worm were reared on the leaves of Bahtim-101 and G. klotzschanum. In contrast, the best overall results were obtained when the larvae were reared on Bahtim-101, where most of larvae reached the pupal stage early and gave the heaviest pupae resulting in moths which deposited the highest average number of eggs and the best hatchability. He suggested that this result may be due to Bahtim-101 which is free of gossypol, and its leaves are soft and contain the highest amount of protein. On the other hand, Bahtim- 101 and G. klotzschanum are more hairy especially on the lower surface of the leaf. It was found that significantly negative correlation exists between the larvae which reached the Pupal stage and the average number of hairs/cm2 (-0.97).

Nassib et al. (1985) described the major production constraints. Irrigated soybean in Egypt the main constraints are, nodulation and N fixation, stand establishment, and research/extension work on varietal improvement are discussed. Problems with Spodoptera littoralis, Tetranychus telarius [T. urticae] and Etiella zinckenella are considered.

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.

Croxford et al. (1989) studied leaves of soybean and cotton mechanically damaged with a single hole and offered to larvae of the noctuid Spodoptera littoralis in laboratory bioassays at intervals of between 0 and 7 days after injury. The subsequent within-leaf grazing patterns of damage and undamaged areas were compared using an image-analysing computer, and estimations were made by eye of percentage areas grazed at 3 spatial scales. Reduction in palatability of damaged areas of both plant species was detected at time intervals ranging from 0 to 7 days after injury. This effect was strongest for the longer time intervals and the effect became weaker with increasing distance from the site of damage.

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

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laid on the lower surface of leaves with 4.5% laid on the upper surface. The larval

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.

Salama et al. (1995) in a study carried out in Egypt, an area of 50 feddans [1 feddan = 0.42 ha] cultivated with soybean was treated with Bacillus thuringiensis (B.t.) baits against Agrotis ypsilon [A. ipsilon] and 70 feddans were sprayed with B.t. spray against Spodoptera littoralis. When B.T. baits were used, the percentage mortality of A. ypsilon reached 96.1-96.4 compared with 97.4-98.0 when using Hostathion [triazophos] baits. When B.T. was applied to larvae of S. littoralis, 88.3% mortality was obtained. This increased to 97.3% after a second application. On the other hand, percentage mortality reached 96.8 when Lannate [methomyl] was applied once. The average yield was 1.54 t/feddan when B.T. was applied twice against S. littoralis and 1.42 t/feddan when B.T. was applied once. Areas treated with methomyl gave yields of 1.44 t/feddan, while in the untreated area; the yield was comparatively low at 0.83 t/Fadden.

Ojo and Ariyo (1999) studied Inheritance of resistance to defoliation in soybean (Glycine max) caused by a noctuid moth Spodoptera littoralis in a cross between a susceptible cultivar TGx 526-02D from IITA and a plant introduction PI 171444. Seeds were planted in pots in the screen house and on establishment of insect cultures, fully matured leaves of the parent plants, the F 1 , F 2 and F 3 generations were fed to the third stage larvae without restriction. Larvae mortality was observed daily and weight gains were recorded at the prepupal stage. Pupal weight and length of pupal period were also recorded. Mortality level was high among larvae fed on the leaves of PI 171444. Gain in body weight was significantly smaller in larvae that fed on the leaves of PI 171444 than those on TGx 526-02D. All larvae that fed on the leaves of the susceptible parent survived to their pupal stage and produced normal adult insects. The mean weight gain of larvae that fed on F1 plants was intermediate between those of the parents suggesting an incomplete dominance gene action. F 3 and F 2 populations showed a considerably wider distribution of larval weight gains

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dominance gene action. F 3 and F 2 populations showed a considerably wider distribution of larval

compared with F 1 . Significant difference between the mean weight gain of larvae that fed on leaves of F 1 and F 2 was also observed. There was no significant difference between the mean weight gain of larvae that fed on the leaves of F 1 and F 3 populations. Chi superscript 2 analysis of F 2 and F 3 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 F 2 and F 3 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.

Mizutani et al. (2001) selected Kyukei 279 is a soybean [Glycine max] breeding line that detected through laboratory tests to be resistant to the common cutworm, S. litura. The field densities of S. litura larvae and three major species of stink bugs (Riptortus clavatus, Piezodorus hybneri and Megacopta punctatissimum) attacking soybeans were compared among Kyukei 279 and two other soybean cultivars (Soudendaizu, resistant to S. litura; Fukuyutaka, susceptible) [date and location not given]. Kyukei 279 was intermediately resistant to S. litura in the field, because the density of S. litura larvae on it was apparently lower than that on Fukuyutaka and larger than that on Soudendaizu. The abundance of stink bugs on the three soybean lines depended on the species of bug, and no line was notably freer of bugs. However, percentages of soybean injured mainly by soybean stink bugs were significantly lower in Kyukei 279 and Soudendaizu than in Fukuyutaka. In addition, yields of Kyukei 279 and Soudendaizu were much higher than that of Fukuyutaka, Thus, Kyukei 279 was considered to have tolerance to soybean stink bugs.

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were much higher than that of Fukuyutaka, Thus, Kyukei 279 was considered to have tolerance to

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 7.2m 2 plot size (4 ridges, 3 m long and 0.60 m wide, with

Table 1: Origin, pedigree and main characteristics of 14 tested soybean genotypes.

Name

Origin

Pedigree

Maturity

Resistance to

Flower

groups no.

leaf cotton

color

warm *

Calland

USA

CL 253 (Blackhawk Harsoy x Kent)

IV

MS

purple

Clark

USA

Lincoln (2) x Richland Corsoy x Lee 68

IV

MS

purple

Corsoy-79

USA

II

S

white

Crawford

USA

-

III

S

purple

Forrest

USA

Dyer X Bragg Crawford x Celeste

V

MR

white

Giza 21

Egypt

IV

R

purple

Giza 22

Egypt

Crawford x Forrest Crowford x Celect Crowford x Maplebrasto Crowford x Celect Crowford x Celect

III

R

purple

Giza 35

Egypt

III

R

purple

Giza 82

Egypt

II

R

purple

Giza 83

Egypt

III

R

white

Giza 111

Egypt

IV

R

purple

Hutcheson

USA

-

VI

S

purple

Lakoto

USA USA

Selection from MBB80- 133 L73-4673 X L73-0132

II

S

purple

L86K-73

I

R

white

* R: resistant, S: susceptible, MS: moderately susceptible.

33 plants /m 2 ) 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 m 2 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).

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(6) >50% (highly susceptible). In laboratory evaluation, a survival technique was used (Meisner and Moshe, 1983)

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).

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The broad sense heritability and genetic advance were estimated using the formulae suggested by Allard (1960).

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 o C respectively, while the corresponding temperatures in the second season were slightly warmer and recorded 33.93 and 20.96 o C. Moreover, the soil temperature during the same period was relatively cool in the first season (24.7 o C) comparing with the second season (30.6 o C). 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

ranged from 1.33 to 4.00 and from 1.70 to 3.50 in the first and the second

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.

Seed yield and yield component characters:

The overall means of seed yield/plant were 71.82 and 67.14 g in the first and the second seasons, respectively, with ranges of 40.19 - 96.75 and 26.80 – 94.90 g/plant in corresponding seasons, respectively. The genotype Giza 111 gave significantly higher seed yield than Crawford (Tables 4 and 5). In 2003 and 2004 its seed yield/plant was 96.75 and 94.90 g, respectively, that out yielded the check variety Crawford by 6.6 and 91.3%. This genotype can be exploited in soybean breeding program. Giza 111 also showed the highest no. of seeds/plant in both seasons, and ranked the fourth highest genotype in biological yield/plant and the third highest in harvest index (Tables 4 and 5). The range of numbers of pods and seeds/plant obtained in this study were similar to those reported by Zayed (1998).

Degree of resistant for soybean genotypes and related characters:

The results of the evaluation of the genotypes for their resistance to leaf cotton warm infection under field and laboratory conditions, in addition to seed oil and protein contents, leaf area, and number of leaf-hairs are presented in Table (6). Narrow range (22.35 – 26.10%) of seed oil content was found, while wide range of seed protein (22.70 – 57.93%) was obtained in this study. The average leaf area was ranged from 18.12 cm 2 for L86K-73 to 70.24 cm 2 for Giza 21, with an average of 48.27 cm 2 . The number of leaf-hairs was showed the widest range (41for 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 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 El-Dakroury (1979) 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. Genetic parameters of the studied characters:

Estimates of phenotypic, genotypic variances, heritability and genetic advance for most studied characters are presented in Table (7). The highest magnitude of phenotypic variance was observed for biological yield/plant, number of leaf-hairs and leaf area, indicating the possibility for selection for these traits. High heritability estimates were found also in these characters in addition to seed yield/plant, which ranged from 97.99 - 99.80%. The expected genetic advance was high for leaf area (61.88%), seeds/plant (56.94%) and harvest index (30.93%). Johanson et al. (1955) stated that heritability estimates together with genetic advance are more important than heritability alone to predict the resulting effect of selecting the best individuals. Therefore, pronounced progress should be expected from selection between genotypes for seeds/plant, harvest index and leaf area. However, since number of hairs had high estimates of heritability and P.C.V value, selection for this character would be useful as indirect selection for insect resistant.

14

heritability and P.C.V value, selection for this character would be useful as indirect selection for insect

Table 2: Average of phenological and morphological characters for 14 soybean

genotypes evaluated under field conditions at Giza research station in season.

2003

 

Days to

Days to

Maturity

Plant height

1 st 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

Table 3: Average of phenological and morphological characters for 14 soybean genotypes evaluated under field conditions at Giza research station in 2004 season.

 

Days to

Days to

Maturity

Plant

1 st Pod height

Number

Genotypes

flowering

maturity

period

height

of branches

(day)

(cm)

(cm)

L86K-73

31.00

90.00

59.00

63.41

4.50

2.19

Corsay-79

35.00

116.00

81.00

66.25

5.10

1.70

Giza21

36.70

122.00

85.00

71.81

5.50

2.61

Forrest

46.00

133.00

87.00

63.97

6.05

3.03

Hutcheson

48.70

137.00

88.00

63.00

5.20

3.50

Calland

39.00

122.00

83.00

89.10

5.64

2.54

Lakota

33.70

98.00

64.33

89.31

4.70

2.64

Giza111

38.33

116.00

77.70

86.50

6.13

2.90

Giza83

33. 00

107. 00

74.00

68.25

4.50

2.80

Clark

38.70

121.00

82.33

68.83

4.19

2.90

Giza22

37.00

110.00

72.00

71.13

6.10

3.22

Giza35

34.00

109.00

75.00

69.98

5.54

2.90

Giza82

33.70

98.00

64.33

70.03

6.74

3.10

Crawford

37.00

119.00

82.00

68.66

4.28

3.40

Average

37.27

114.14

76.81

72.19

5.29

2.81

L.S.D.5%

1.395

1.662

1.852

4.84

0.898

0.794

15

37.27 114.14 76.81 72.19 5.29 2.81 L.S.D.5% 1.395 1.662 1.852 4.84 0.898 0.794 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

Table 5: yield component characters for 14 soybean genotypes evaluated under field conditions at Giza research station in 2004 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

201.40

131.70

1.50

13.00

26.80

104.00

25.54

Corsay-79

305.70

192.33

1.60

15.50

47.10

117.33

40.29

Giza21

373.90

227.33

1.63

18.80

61.65

137.70

44.81

Forrest

545.05

253.33

2.13

15.83

85.90

318.70

27.02

Hutcheson

536.27

245.70

2.20

15.77

84.38

310.70

27.16

Calland

502.90

232.00

2.13

17.06

85.20

303.33

28.18

Lakota

297.20

186.00

1.60

16.00

47.40

124.33

38.22

Giza111

545.20

231.33

2.40

17.50

94.90

252.00

37.70

Giza83

426.40

221.33

1.93

17.70

75.00

211.70

35.45

Clark

409.70

251.33

1.63

15.90

64.52

205.33

31.56

Giza22

377.80

203.33

1.93

1750

65.98

204.70

32.25

Giza35

378.00

231.70

1.60

17.01

64.10

205.33

31.190

Giza82

291.430

170.00

1.70

16.40

47.60

127.63

37.270

Crawford

548.00

217.33

2.50

16.40

49.60

195.00

45.99

Average

409.96

213.93

1.89

16.47

67.14

201.26

34.48

L.S.D.5%

73.74

26.10

0.174

2.02

7.40

9.90

6.017

16

1.89 16.47 67.14 201.26 34.48 L.S.D.5% 73.74 26.10 0.174 2.02 7.40 9.90 6.017 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

(cm

2 )

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

Table 7: Genetic and phenotypic variances, broad sense heritability, Genetic (GCV) and phenotypic (PCV) Coefficients variation and genetic advance for all studied characters.

Character

Genetic

Phenotypic

Heritability

G.C.V

P.C.V

Genetic

variance

variance

(%)

advances

Broad Sense

(%)

Flowering

24.50

26.41

92.8

13.3

13.8

26.50

Maturity

165.60

193.10

85.76

11.29

12.20

21.56

Maturity period

66.81

85.58

78.10

10.65

12.05

19.39

No. of branches

0.265

0.444

59.70

18.72

25.28

29.80

Plant height

8.603

63.58

13.53

0.042

11.43

3.19

First pod height

0.421

0.730

57.64

11.51

15.16

0.21

Pods/plant

1005.31

1104.33

91.03

14.27

14.96

28.05

Seeds/plant

12409.15

12896.24

96.22

25.48

25.98

5.15

Seeds/pod

0.074

0.1053

70.28

14.04

16.75

24.29

Seed yield/plant

375.11

382.81

97.99

27.88

28.16

56.94

100-seeds weight

0.899

1.419

63.35

0.060

0.075

9.76

Biologic. Yield/pl.

5472.82

5483.30

99.8

36.8

36.8

0.76

Harvest index

35.51

43.32

81.97

16.59

18.32

30.93

No. of hairs

1452.41

1458.90

99.55

30.29

30.50

7.87

Leaf area

213.53

216.80

98.49

30.29

30.50

61.88

17

1458.90 99.55 30.29 30.50 7.87 Leaf area 213.53 216.80 98.49 30.29 30.50 61.88 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.

Advances in biotechnology can facilitate the development of insect-resistant soybean cultivars by means of gene transformation. Successful use of gene transfer requires that the gene for insect resistant is identified, isolated and then reconstructed for expression in relevant organ of the new host. In addition, gene transfer procedures and appropriate tissue culture methods must be developed for each target species to regenerate fertile, transgenic plants.

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.

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, .0 2 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

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.

Dan-YingHui and Reichert (1998) compared of hypocotyl sections of 13 soybean genotypes representing maturity groups IV-VI for organogenic responses on media supplemented with 5.0 or 10.0µM benzyladenine (BA) or 2.5 UM BA + 1.0 UM NAA, and under continuous darkness or a 16-h photoperiod. All genotypes responded by producing adventitious shoots on the acropetal end of the hypocotyl explants, confirming genotype- independence of shoot initiation. Media containing 5.0 or 10.0 UM BA induced the greatest numbers of shoots. Light conditions had no effect over the first 4 weeks. Histological studies confirmed the adventitious nature of the shoots by indicative formation of meristematic zones and shoot primordia from parenchymatous tissues of the central pith and plumular trace regions of the hypocotyl. Incompletely excised cotyledonary buds also contributed to shoot initiation. Cv. Centennial, Epps and Lyon gave the greatest individual responses. Among cultivars across all treatments, the regeneration potential (percentage of explants producing meristem-like structures or shoot primordia) 4 weeks after initiation ranged from 47 to 75%. Four weeks later, regenerative ability (number of shoots produced per responding explant) and regeneration efficiency (number of shoots produced per explant plated) were 1.4-7.1 and 1.0- 5.0, respectively. The optimized protocol included initiation on a medium containing 5.0 UM BA for 4 weeks, then transfer onto a shoot elongation medium (0.36 UM BA) for 4 weeks. For 11 genotypes, 66-100% of excised shoots produced roots after 4 weeks on media containing 12.5-29.2 uM IBA. Of 109 plantlets transplanted to soil, 94% survived and no sterility was observed on those mature enough to flower.

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

traits appeared to be predominantly under the control of additive gene effects as evidenced by the

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

callus initiation (87.43%) and shoot induction (35.31%). Medium A and medium B resulted in the greatest

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

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

differences were observed in the response of genotypes, culture media and genotype x medium interactions for

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

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

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 25 th May 2003 and 2004. Sowing was done at a crop density of 33 plants/m 2 in 3-meter long ridges, 60cm apart and 4 ridges per plot. Fertilizers at 30 kg P 2 O 5 /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 Corsoy X Lee 68 Dyer X Bragg

Maturity group no. I, white flower color. Maturity group no. II, white flower color. Maturity group no. V, white flower color.

Corsoy-79

USA

Forrest

USA

Hutcheson

USA

-

Maturity group no. VI, purple flower color. Maturity group no. II, purple flower color. Maturity group no. IV, purple flower color. Maturity group no. II, purple flower color.

Lakota,

USA

Selection Crowford X Celect Selection from MBB80-133

Giza 21

Egypt

Giza 83

Egypt

The immature embryos were then incubated under complete darkness at 25 o C±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 25 o C at day and 18 o C at night with 16 h day (light source was from cool white fluorescent lamps 80 um photons m -2 s -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).

To measure the response of soybean genotypes to callus induction the following characters were measured and calculated in 4 replicates: Number of shoots/callus, Percentage of plantlets performed roots [(number of plantlets/total number of shoots) x 100], number of root/plantlet, length of root (cm) and diameter of root (mm). Analysis

27

number of shoots) x 100], number of root/plantlet, length of root (cm) and diameter of root

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

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

roots. These data indicting that the genotype L86K-73 is the best in response to tissue culture
Figure 1: Illustration of Callus induction (A), Shooting stage (B), Rooting stage(C) and Adaptation (D)

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

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 %

Callus indication %

0.158

0.157

0.088

0.033

-0.112

-0.050

Callus growth rate

 

0.605

**

-0.083

0.467

**

0.320

-0.220

No. of shoots

   

-0.381

0.253

0.228

-0.026

Plantlet performed root

     

0.335

0.191

-0.259

No. of roots

       

-0.093

-0.226

Root length

         

0.157

** Significant at 0.1 level of probability.

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.

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are considering important indicator for callus growth rate, and could be used to predict succeeding of

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.

In the first reports of soybean transformation, two different methods were applied. Used Agrobacterium mediated transformation cotyledonary nodes, while used partial bombardment of shoot meristems. Soybean transformation reports following these initial works have been limited and transformation efficiency for soybean has remained low. Later transformation efficiency has been improved. Additionally, several methods have been used to genetically transform plants, such as electroporation, Silicon carbide fibers, liposome mediated transformation and vacuum infiltration of whole plants using Agrobacterium. However, these methods have not been optimized for soybean, and they are therefore less efficient and not widely used. In soybean, tissue culture induced indirect organogenesis from immature embryos was successfully used to viable soybean plantlet. The cotyledonary nodes have the ability to produce direct organogenesis shoots in soybean. However, the production of genetically transformed soybean plants needs broad studies. In this investigation, a system for soybean transformation and regeneration using immature embryos and cotyledonary nods was performed.

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a system for soybean transformation and regeneration using immature embryos and cotyledonary nods was performed. 32

REVIEW OF LITERATURE

Transformation by Agrobacterium (LBA4404) in immature Embryos:

Facciotti et al. (1985) successfully transferred soybean cultivar Forrest with A. tumefaciens containing a bacterial kanamycin resistance gene linked to the 5' portion of a soybean small subunit ribulose 1, 5-diphosphate carboxylase gene. Comparison of light-grown versus dark-grown transformed callus demonstrated light induction of the chimaeric gene as measured by increased levels of kanamycin resistance. This was not unexpected as the small subunit genes are under light regulation.

Wyndaele et al. (1985) induced 2 soybean crown gall lines by a nop+ Agrobacterium tumefaciens C58 str., max. Cytokinin (essentially glucosyl-trans- zeatin) levels were attained in the beginning of the exponential growth phase, followed by a drastic decrease just before the stationary phase was reached. Quantitatively the green tumour line showed a 2-3 times higher cytokinin content compared with the pale line. In untransformed callus very little cytokinin was detected. Analysis of endogenous IAA levels showed no difference between the 2 lines and the untransformed callus tissue, all having a low and constant level throughout the entire growth cycle. The relevance of the endogenous accumulation of phytohormones in relation to the hormone autotrophic growth of transformed soybean tissue is discussed.

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.

Christou et al. (1988) accelerated immature soybean embryos from commercially important cultivars the targets of rapidly, DNA-coated, gold particles (fired from an arc discharge gun powered by a 25 kV 2 micro F capacitor). Protoplasts were prepared from these tissues and propagated in culture under selection conditions for the introduced neomycin phosphotransferase II gene (fused to a cauliflower mosaic virus promoter). Kanamycin-resistant calluses were obtained at a rate of approximately 10-5. Enzyme assays and Southern blot hybridization confirmed the expression of the foreign gene and its stable integration into the soybean genome. The results show that particle acceleration can be used for the introduction of foreign DNA into the soybean genome and are taken to indicate that the technique may be useful in the recovery of engineered plants by transformation of regenerable tissues.

Owens and Smigocki (1988) inoculated cotyledon explants from germinated 1-day-old soybean seedlings with single or mixed strains of A. tumefaciens. Mixed- strain infections with the supervirulent L,L-succinamopine type strain A281

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mixed strains of A. tumefaciens. Mixed- strain infections with the supervirulent L,L-succinamopine type strain A281 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.

Zhou and Atherly (1989) introduced binary vectors into soybean by Agrobacterium tumefaciens-mediated transformation. After 10 days growth on B5BA medium containing 250 ml kanamycin/litre, actively-growing callus tissue was removed and stained with x-glcU. Calluses transformed with an intact GUS gene showed even staining of the tissues, those transformed with pZAc1 (a vector containing GUS under the control of the CaMV 35S promoter and including a 5.1 kb fragment of the Ac transposable element (from maize) in the untranslated leader region) showed staining only rarely, while those transformed with a vector containing the Ds maize element in the untranslated leader region of GUS showed no staining. Different-sized fragments to those of the original insertion event were obtained when DNA from the calluses was digested with AcoRI and PstI and hybridized with radioactive Ac DNA, providing additional proof that Ac is active in soybean calluses.

Delzer et al. (1990) excised cotyledons from 1-day-old axenic seedlings of 10 soybean genotypes and Peking, a highly susceptible Maturity Group IV cultivar wounded and inoculated with A. tumefaciens strains C58, A208 or A281. The relative frequency of tumor formation, an indicator of susceptibility, was greatest for strains C58 and A208 on all genotypes tested. Peking and PI180529 were the most susceptible. The same genotypes were evaluated for shoot organogenic tissue culture initiation from the cotyledonary node and plant regeneration via adventitious shoot formation. The genotypes producing the highest frequency of mature plants 30 weeks after culture initiation were Experimental Line HHP, Evans, PI445799, Hodgson 78, Corsoy 79 and PI180529. These may be useful genotypes for whole plant transformation studies.

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.

Trick and Finer (1998) successfully transferred of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue. Although soybean embryogenic suspension cultures have been transformed via particle bombardment,

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of the target tissue. Although soybean embryogenic suspension cultures have been transformed via particle bombardment, 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]).

Zhang et al. (1999) described a soybean transformation procedure using the Agrobacterium-cotyledonary node transformation system and the bar gene as the selectable marker coupled with glufosinate as a selective agent. Cotyledonary explants were derived from 5 day old seedlings and co-cultivated with Agrobacterium tumefaciens for 3 days. Explants were cultured on Gamborg's B5 medium supplemented with 1.67 mg BAP [benzyladenine] per litre and glufosinate at 3.3 mg or 5.0 mg/litre for 4 weeks. After 4 weeks explants were subcultured to medium containing MS major and minor salts and B5 vitamins (MS/B5) supplemented with 1.0 mg zeatin-riboside, 0.5 mg GA3 (gibberellins) and 0.1 mg IAA amended with 1.7 mg or 2.0 mg glufosinate per litre. Elongated shoots were rooted on a MS/B5 rooting medium supplemented with 0.5 mg NAA/litre without further glufosinate selection. Plantlets were transplanted to soil and grown to maturity and set seed in the greenhouse. Primary transformants and their progeny were characterized by Southern

blot analysis and a leaf paint assay.

Yan et al. (2000) investigated Agrobacterium tumefaciens-mediated transformation of soybean (Glycine max cv. Jack) using immature zygotic cotyledons

to identify important factors that affected transformation efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium concentration during inoculation

and co-culture and the selection regime. These results showed that 8 to 10 mm

zygotic cotyledons exhibited a higher transformation rate, as indicated by transient

GUS gene expression, whereas the smaller zygotic cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants

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cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants

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.

Wang et al. (2001) investigated the effects of erythromycin base, cefazolin sodium, cefradine and 2 kinds of cefotaximes on callus induction and growth of transformed soybeans using Agrobacterium LBA4404. The ideal concentration of cefotaximes was recorded at 300 mg/litre using hypocotyl explants, and 500 mg/litre using cotyledon explants. No significant differences were observed in the response of soybean varieties in terms of rate of callus induction, although differences were observed in terms of rate of brown callus formation. Differences in the response of different soybean explants to kanamycin were observed; the young leaves were sensitive while the hypocotyl explants were not. The ideal selection pressure of kanamycin, used as a selection marker, was 50-100 mg/litre in young leaves and cotyledons, and 100 mg/litre in hypocotyl explants.

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.

Schmidt and Parrott (2001) determined quantitative real-time polymerase chain reaction (PCR) assays that enabled the zygosity of transgenes in soybean (G. max) and groundnut (A. hypogaea), were designed. The two zygosity assays, based on TaqMan technology that uses a fluorogenic probe which hybridizes to a PCR target sequence flanked by primers, were both accurate and reproducible in the determination of the number of transgenes present in a cell line. In the first assay, in which TaqMan assays were performed on increasing amounts of a plasmid containing the transgene of interest, a linear relationship between the level of fluorescence and

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amounts of a plasmid containing the transgene of interest, a linear relationship between the level of

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.

KO and Korban (2004) identified a characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation- competent cells/tissues on immature cotyledons of soybean (Glycine max) under hygromycin selection. This highly embryogenic immature cotyledon was characterized with emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5-8 mm in length, a 1:1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-week cocultivation period significantly increased the frequency of transgenic somatic embryos. On the other hand, increasing the infection period of explants or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An optimum selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mg hygromycin l-1 for a 2-week period, and then maintained on selection media containing 25 mg hygromycin l-1 in subsequent selection periods. However, somatic embryogenesis

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on selection media containing 25 mg hygromycin l-1 in subsequent selection periods. However, somatic embryogenesis 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.

Wang et al. (2004) induced Somatic embryogenesis and the regenerated plants were obtained by higher concentrations of auxins with immature cotyledon of 55 genotypes in soybean. Bivalent insect resistant genes were transformed into immature cotyledon of soybean which have high frequency of somatic embryogenesis via Agrobacterium-mediated. The results showed that 14 genotypes possessed high frequency of somatic embryogenesis (more than 40%) among soybean genotypes from Northeast area. A total of 2147 immature cotyledons of 5 different soybean genotypes cultured in Northeast area was inoculated with LBA4404 (including pGBI121S4ABC plasmid). A total of 17 plantlets were obtained under kanamycin selection. A total of 12 plantlets showed positive reaction in PCR and PCR-Southern detection. These analyses confirmed the presence of introduced BT gene in soybean.

Liu et al. (2004) reported the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans [Glycine max (L.) Merr.]. Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on MSB5 medium supplemented with 3.5 mg l-1 N6-benzylaminopurine (BAP) for 24 h, and explants were transferred to MSB5 medium supplemented with 0.2 mg l-1 BAP and 0.2 mg l-1 indolebutyric acid. Use of embryonic tips yielded a higher regeneration frequency (87.7%) than regeneration systems using cotyledonary nodes (40.3%) and hypocotyl segments (56.4%) as starting materials. Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains the binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 78.2% and the transformation efficiency reached up to 15.8%. These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium- mediated transformation.

Ko et al. (2004) studied Agrobacterium tumefaciens strain KYRT1 harboring the virulence helper plasmid pKYRT1 induces transgenic somatic embryos (SEs) at high frequency from infected immature soybean cotyledons. KYRT1 is derived from the highly oncogenic strain Chry5. However, pKYRT1 is not completely disarmed and still contains an entire T-right (TR) and a portion of T-left (TL). In this report, binary strains, each carrying fully disarmed vir helper plasmids including pKPSF2, which is a fully disarmed version of pKYRT1, were compared to strain KYRT1 for their ability to induce transgenic SEs on immature cotyledons of soybean. Six weeks following cocultivation, histochemical GUS assays of cultured explants indicated that all fully disarmed vir helper plasmids transferred their binary T-DNA, containing a GUS-intron gene, into soybean tissues. However, none of these transformed tissues developed SEs on medium with or without 2, 4-dichlorophenoxyactic acid (2, 4-D). On the other hand, immature cotyledons cocultivated with strain KYRT1 exhibited high induction of transgenic SEs, but only on medium supplemented with 2,4-D. Derivatives of strain Chry5 harboring other vir helper plasmids did not induce transgenic SEs under any conditions tested, thus suggesting that the chromosomal background of KYRT1 alone was not sufficient to promote somatic embryogenesis. PCR analysis indicated that 55% of transgenic embryogenic cultures and 29% of

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to promote somatic embryogenesis. PCR analysis indicated that 55% of transgenic embryogenic cultures and 29% of

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.

Li et al. (2004) utilized either Agrobacterium-mediated transformation or particle bombardment; they obtained transgenic soybean (Glycine max) plants expressing the chitinase gene (chi) and the barley ribosome-inactivating protein gene (rip). Six regenerated plants were grown to maturity and set seed. The identification of transgenic soybean plants that co-integrated the 2 antifungal protein genes was determined by polymerase chain reaction (PCR) and Southern blot analysis. Protein detection from the soybean leaves demonstrated the expression of the chitinase (CHI) and the ribosome-inactivating protein (RIP) in the 6 R0 transformants. Soybean cotyledonary nodes were transformed using the bivalent plant expression vector pBRC containing chi and rip both driven by the CaMV 35S double promoter. Following vacuum (0.06 MPa) infiltration treatment of the tissue for 5 minutes, Agrobacterium was co-cultivated with the cotyledonary nodes for 3 days on MSB medium (MS salts and B5 vitamins; pH 5.2), and the transformation frequency reached a maximum of 1.33%. The chi and rip genes were present in all the transgenic plants. Co-bombardment of immature cotyledons with plasmids pBchE (encoding chi) and pARIP (encoding rip) resulted in the greatest transformation frequency of 0.52% with a 50% co-integration rate. Our results demonstrate the efficient co-transformation of multiple genes in soybean.

Transformation by (LBA4404) in cotyledonary node:

Rech et al (1988) infected seedlings of G. canescens and G. clandestina with A. rhizogenes. All accessions responded to infection, the frequency ranging from 10% (G. clandestina G1001) to 70% (G. canescens G1340 and G1240). Limited callus proliferation occurred at the infection site 7-16 days after inoculation, with root emergence 2-3 days later. Cotyledon infection was less efficient in both species, with bases (proximal regions) being more responsive than tips. Growth regulators inhibited root growth and promoted callus formation. Shoots were produced only by G.

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than tips. Growth regulators inhibited root growth and promoted callus formation. Shoots were produced only by

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.

Al-Janabi and Shoemaker (1992) cultured cotyledons of Glycine max cv. Peking in vitro, wounded and inoculated with A. tumefaciens strain A281 containing the binary vectors PZAC1, PZAC1/R or PZA3. They were then transferred successively to shoot induction medium, kanamycin selection medium and rooting medium, producing mature transformed plants in 12-14 weeks. All the regenerated plants were fertile.

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.

Lee and Komatsuda (1994) excised embryos of soybean genotypes Peking 501, American Jellow, Kou 502 (Masshokutou) and Bominori from immature seeds and cultured in vitro. Explants undergoing embryogenesis or organogenesis were cocultivated for 1 day with either EHA101/PSAOR1221 or LBA4404/PTRA415 vectors. PSAOR1221 is a binary Ti plasmid containing the beta -glucuronidase (GUS) gene driven by the CaMV 35S promoter. PTRA415 harbours a tobacco PR1a protein gene which is induced by stress or chemicals. Following selection on kanamycin- containing medium and GUS assays of regenerants, transformants were only identified from the EHA101/PSAOR1221 treatment (0-5.4% transformants via embryogenesis and 4-12.2% via organogenesis).

Xu et al. (1997) transferred the pKT54B7C5 plasmid containing the B.t.k. [Bacillus thuringiensis kurtosis] delta -endotoxin gene into soybean cultivars Heinong 37 and Heinong 39 by Agrobacterium tumefaciens. Adventitious buds and regenerated plants were obtained from hypocotyls and cotyledon nodes. Successfully transferred plasmids were detected by kanamycin and alkaloid selection. Only 30 of

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and cotyledon nodes. Successfully transferred plasmids were detected by kanamycin and alkaloid selection. Only 30 of

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.

Zhang et al. (1999) described a soybean transformation procedure using the Agrobacterium-cotyledonary node transformation system and the bar gene as the selectable marker coupled with glufosinate as a selective agent. Cotyledonary explants were derived from 5 day old seedlings and co-cultivated with Agrobacterium tumefaciens for 3 days. Explants were cultured on Gamborg's B5 medium supplemented with 1.67 mg BAP [benzyladenine] per litre and glufosinate at 3.3 mg or 5.0 mg/litre for 4 weeks. After 4 weeks explants were subcultured to medium containing MS major and minor salts and B5 vitamins (MS/B5) supplemented with 1.0 mg zeatin-riboside, 0.5 mg GA3 (gibberellins) and 0.1 mg IAA amended with 1.7 mg or 2.0 mg glufosinate per litre. Elongated shoots were rooted on a MS/B5 rooting medium supplemented with 0.5 mg NAA/litre without further glufosinate selection. Plantlets were transplanted to soil and grown to maturity and set seed in the greenhouse. Primary transformants and their progeny were characterized by Southern blot analysis and a leaf paint assay.

Donaldson and Simmonds (2000) studied response a short-season adapted soybean (Glycine max) genotypes (maturity groups 0 and 00) were susceptible to Agrobacterium tumefaciens in tumour-formation assays with A. tumefaciens strains A281, C58 and ACH5. The response was bacterial-strain and plant-cultivar dependent. In vitro Agrobacterium-mediated transformation of cotyledonary node explants of these genotypes with A. tumefaciens EHA105/pBI121 was inefficient, but resulted in a transgenic AC Colibri plant carrying a linked insertion of the neomycin phosphotransferase and beta -glucuronidase (GUS) transgenes. The transgenes were transmitted to the progeny and stable GUS expression was detected in the T7 generation. The low rate of recovery of transgenic plants from the co-cultured cotyledonary explants was attributed to inefficient transformation of regenerable cells, and/or poor selection or survival of such cells and not to poor susceptibility to Agrobacterium, since, depending on the cultivar, explants were transformed at a rate of 27-92%, but transformation events were usually restricted to non-regenerable callus.

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

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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.

Kumari et al. (2004) standardized an efficient and reproducible protocol for Agrobacterium-mediated transformation in soybean (G. max) using the binary vector pBI 121. Putative transgenic shoots were selected on kanamycin medium. GUS assay was performed to confirm the presence of GUS gene from putatively transgenic shoots. Transgenic plants were multiplied followed by successful rooting in the media containing 1.5 mg IBA/litre. Plants with well-developed roots were successfully hardened in plastic cups containing sand, soil and sawdust (1:1:1). Stable integration of the transgene (uid A and npt II) was confirmed by PCR analysis. Southern blot hybridization confirmed the presence of npt II gene in putative transformed plants.

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

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at 0.1, 0.2, 0.3, 0.4 and 0.5 mg/litre. Subculturing was done in the same media every

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.

Zhang et al. (2004) investigated conditions affecting Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merr.], including seed vigour of explant source, selection system, and cocultivation conditions. A negative correlation between seed sterilization duration and seed vigour, and a positive correlation between seed vigour and regenerability of explants were observed in the study, suggesting that use of high vigour seed and minimum seed sterilization duration can further improve transformation efficiency. Selection schemes using glufosinate or bialaphos as selective agents in vitro were assessed. Glufosinate selection enhanced soybean transformation as compared to bialaphos. The use of 6 mg L -1 glufosinate during shoot induction and shoot elongation stages yielded higher final transformation efficiency ranging from 2.0% to 6.3% while bialaphos at 4 to 8 mg L-1 gave 0% to 2.1% efficiency. Including cysteine and DTT during cocultivation increased the transformation efficiency from 0.2-0.9% to 0.6-2.9%. This treatment also improved T-DNA transfer as indicated by enhanced transient GUS expression. Shoot regeneration and Agrobacterium infection were attained in twelve soybean cultivars belonging to maturity groups’ I-VI. These cultivars may be amenable to genetic transformation and may provide a valuable tool in soybean improvement programs.

Somers et al. (2004) studied soybean transformation methods based on DNA delivery via A. tumefaciens use proliferating apical meristems, cotyledons of immature embryos and seedling cotyledonary nodes as sources of regenerating cells for the production transgenic plants. Improvements in T-DNA delivery, A. tumefaciens strain, tissue culture conditions, and selection of transgenic plants have increased the efficiency of these transformation systems. Further improvements are required to expand the range of genotypes that can be transformed using these procedures and to shorten the time to produce transgenic plants. Characterization of T-DNA loci produced using the cotyledonary-node method indicate that on average of approximately one simple locus is produced per transgenic plant indicating that A. tumefaciens-based soybean transformation systems produce plants with a high proportion of simple transgene loci.

Zeng et al.(2004) started that modern genetic analysis and manipulation of soybean (Glycine max) depend heavily on an efficient and dependable transformation process, especially in public genotypes from which expressed sequence tag (EST), bacterial artificial chromosome and micro array data have been derived. Williams 82 is the subject of EST and functional genomics analyses. However, it has not previously been transformed successfully using either somatic embryogenesis-based or cotyledonary-node transformation methods, the two predominant soybean transformation systems. An advance has recently been made in using antioxidants to

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methods, the two predominant soybean transformation systems. An advance has recently been made in using antioxidants

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.

Li et al. (2005) studied the factors influencing Agrobacterium tumefaciens- mediated soybean cotyledonary node transformation to improve the transformation frequency of soybean. The transformation frequency was different among soybean cultivars. Jilin 35 was the highest in transformation rate, Zhonghuang 28 was the second followed by Nannong and Tiefeng 29. There was no transformed plant in Tiefeng 30 and Kaiyu 12. The average and the highest transformation rate were 2.16 and 6.12% for Jilin 35, and 1.9 and 3.33% for Zhonghuang 28, respectively. The suitable seedling age was ~4 days old and the appropriate durations of infection and co-culture with A. tumefaciens were ~30 minutes and 3 days, respectively. There was an interaction between germination medium and shoot regeneration medium for transformation frequency. The best combination patterns of germination medium and shoot regeneration medium were the G2+Y1 (with transformation rate of 6.12%) and G1+Y2 (with transformation rate of 5.26%). The screening method had a significant effect on increasing the transformation frequency of the A. tumefaciens-mediated transformation system. Kanamycin was used as the screening agent. A higher transformation rate was obtained by using the screening model S1 in which the kanamycin concentration increased gradually from 60 to 100 mg/l when subculture in the shoot regeneration stage. It was suggested that the lower concentration of the screening agent should be used in the earlier screening stage; the higher concentrations increased gradually in the later stages.

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agent should be used in the earlier screening stage; the higher concentrations increased gradually in the

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.

Table13: Origin and main characteristics of the fourteen-tested soybean genotypes used in this study.

NO.

 

Genotypes

Maturi

Pedigree

Origin

Flower

ES

ty

Color

group

1

 

L86K-73

I

L73-4673 X L73-0132

USA