InTechOpen uses cookies to offer you the best online experience. By continuing to use our site, you agree to our Privacy Policy.

Agricultural and Biological Sciences » "A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen Relationships", book edited by James E. Board, ISBN 978-953-51-0876-4, Published: January 2, 2013 under CC BY 3.0 license. © The Author(s).

Chapter 20

In vitro Regeneration and Genetic Transformation of Soybean: Current Status and Future Prospects

By Thankaraj Salammal Mariashibu, Vasudevan Ramesh Anbazhagan, Shu-Ye Jiang, Andy Ganapathi and Srinivasan Ramachandran
DOI: 10.5772/54268

Article top

In vitro Regeneration and Genetic Transformation of Soybean: Current Status and Future Prospects

Thankaraj Salammal Mariashibu1, Vasudevan Ramesh Anbazhagan1, Shu-Ye Jiang1, Andy Ganapathi2 and Srinivasan Ramachandran1

1. Introduction

Soybean [Glycine max (L.) Merrill], grown for its edible seed protein and oil, is often called the miracle crop because of its many uses. It belongs to the genus Glycine under the family Leguminosae, and is widely cultivated in the tropics, subtropics and temperate zones of the world [1].

Soybean is now an essential and dominant source of protein and oil with numerous uses in feed, food and industrial applications. It is the world’s primary source of vegetable oil and protein feed supplement for livestock. The global production of soybeans is 250-260 million tons per year. The US is the largest producer with 90.6 million metric tons. Other major countries such as Brazil, Argentina, China and India contributing 70, 49.5, 15.2 and 9.6 million metric tons, respectively [2]. The US, Brazil and Argentina are the major exporters of beans; while China and Europe are the major importers. The annual world market value is around 2 billion US dollars, which stands second in world food production.

Recent nutritional studies claim that consumption of soybean reduces cancer, blood serum cholesterol, osteoporosis and heart diseases [3]. This has sparked increased demand for the many edible soybean products. The priority for more meat in diets among the world’s population has also increased the demand for soybean protein for livestock and poultry feed.

Soybean seeds are comprised of 40% protein, mostly consisting of the globulins β-conglycinin (7S globulin) and glycinin (11S globulin). The oil portion of the seed is composed primarily of five fatty acids. Palmitic and stearic acids are saturated fatty acids and comprise 15% of the oil. Soybean is rich in the unsaturated fatty acids like oleic, linoleic and linolenic, which make up 85% of the oil. Soybeans are a good source of minerals, B vitamins, folic acid and isoflavones, which are credited with slowing cancer development, heart diseases and osteoporosis [4].

The productivity of soybean has been limited due to their susceptibility to pathogens and pests, sensitivity to environmental stresses, poor pollination and low harvest index. Among the abiotic stresses, drought is considered the most devastating, commonly reducing soybean yield by approximately 40% and affecting all stages of plant growth and development; from germination to flowering, and seed filling and development as well as seed quality [5]. It suffers from many kinds of fungal diseases, such as frogeye leaf spot and brown spot [6]. As demand increases for soybean oil and protein, the improvement of soybean quality and production through genetic transformation and functional genomics becomes an important issue throughout the world [7].

The main objectives of soybean improvement include increase in yield, development of resistance to various insects, diseases and nutritional quality. Commercial breeding is still very important for the genetic improvement of the crop. However, breeding is difficult due to the fact that the soybean is a self pollinating crop, and the genetic base of modern soybean cultivars is quite narrow [8]. Most of the current soybean genotypes have been derived from common ancestors; therefore, conventional breeding strategies are limited in capability to expand the soybean genetic base. Recent advances in in vitro culture and gene technologies have provided unique opportunities for the improvement of plants, which are otherwise difficult through conventional breeding. The technology of plant transformation is only moderately or marginally successful in many important cultivars of crops, which can be a major limiting factor for the biotechnological exploitation of economically important plant species and the wider application of genomics.

Although numerous methods have been developed for introducing genes into plant genomes, the transformation efficiency for soybean still remains low [9]. Since the first successful transformation of soybean was reported [10], two major methods have been used in soybean transformation: one is particle bombardment of embryogenic tissue and another is Agrobacterium tumefaciens-mediated transformation of the cotyledonary node. Both methods have limitations: the former is highly genotype-dependent, requires a prolonged tissue culture period and tends to produce multiple insertion events, while the latter is labour intensive and requires specially trained personnel to undertake the work [9]

For soybean in vitro regeneration, two principal methods have been identified: somatic embryogenesis and shoot morphogenesis. Each of these systems presents both advantages and disadvantages for production of transformed plants, and each can be used with both of the predominant transformation systems [11]. A better understanding of physiology and molecular biology of in vitro morphogenesis needs focal attention to reveal their recalcitrant nature.

The present review gives an overview on the problems associated with low transformation efficiency, and the research conducted to improve tissue culture and transformation efficiency of soybean during the past (Table 1&2) and also discuss the future prospects, demands of these technologies and upcoming new technologies in soybean improvement.

Year Explant tissue Major contribution Reference
1973HypocotylAdventitious bud development Kimball and Bingham, [13]
1980Cotyledonary nodeShoot morphogenesisCheng et al. [14]
1986Immature embryoPlant regeneration from callusBarwale et al. [18]
1986Cotyledonary nodeMultiple shoot formationBarwale et al. [19]
1986Cotyledonary nodeMultiple shoot formationWright et al. [20]
1987EpicotylCallus induction and shoot regenerationWright et al. [29]
1988Cotyledonary nodeTransfered npt II and gus gene by Agrobacterium mediated transformation Hinchee et al. [10]
1988Immature seedsDeveloped transgenic soybean by Particle bombardmentMcCabe et al. [25]
1989Germinating seedsTransfered npt II gene by Agrobacterium mediated transformationChee et al. [45]
1989Immature seedParticle bombardment of meristemsChristou et al. [62]
1990Immature cotyledonPlant regeneration from protoplast Luo et al. [127]
1990Cotyledon, cotyledonary nodeEvaluated Agrobacterium sensitivity and adventitious shoot formation Delzer et al. [44]
1990Immature cotyledon, plumule, cotyledonary nodeAnalysed plant regeneration efficiency of various explants Yang et al. [32]
1990Immature embryoOrganogenesis and plant regenerationYeh,[128]
1990Primary leaf nodeAdventitious shoot formationKim et al. [27]
1991Immature cotyledonPlant regeneration from protoplastDhir et al. [129]
1992Epicotyl and hypocotylInvestigated the stimulative effect of allantoin and amides on shoot regeneration Shetty, et al. [21]
1993Shoot tipTransfered gus gene via particle bombardmentSato et al. [130]
1994Primary leaf nodeInvestigated the synergistic effect of proline and micronutrients on shoot regenerationKim et al. [40]
1996Cotyledonary nodeDeveloped transgenic soybean resistance to bean pod mottle virus (BPMV)Di et al. [131]
1997Cotyledonary node and hypocotylMultiple shoot induction by TDZKaneda et al. [22]
1998Cotyledonary nodeEvaluation of sonication assisted Agrobacterium mediated
transformation (SAAT) for cotyledonary node
Meurer et al. [50]
1998HypocotylAdventitious shoot regeneration Dan and Reichert, [33]
1999Cotyledonary nodeAssessed the use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybeanZhang et al. [61]
2000Cotyledonary node Agrobacterium two T-DNA binary system as a strategy to derive marker free transgenic soybeanXing et al. [132]
2000Cotyledonary nodeEvaluated the effect of glyphosate as a selective agent for Agrobacterium mediated cotyledonary node transformation systemClemente et al. [60]
2000Embryonic axesUsed of Imazapyr as selection agent for selection of meristematic soybean cells Aragao et al. [47]
2001Cotyledonary nodeInvestigated the use of thiol compound to increase transformation frequencyOlhoft et al. [56]
2001Cotyledonary nodeIncreased Agrobacterium infection using L-cystine Olhoft and Somers, [16]
2001Cotyledonary nodeDeveloped transgenic soybean plants resistant to soybean mosaic virus (SMV)Wang et al. [133]
2001Cotyledonary nodeExpressed oxalate oxidase gene for resistant to sclerotinia stem rot caused by Sclerotinia sclerotiorum Donaldson et al. [65]
2003HypocotylScreened soybean genotype for adventitious organogenic regeneration Reichert et al. [41]
2003Cotyledonary node Assessed the effect of genotype, plant growth regulators and sugars on regeneration from calli Sairam et al. [1]
2003Cotyledonary node Used mixture of thiol compounds and hygromycin based selection for increased transformation efficiencyOlhoft et al. [57]
2004Cotyledonary nodeAssessed glufosinate selection for increased transformation efficiency Zeng et al. [134]
2004Cotyledonary nodeInvestigated the effect of seed vigor of explant source, selection agent and antioxidant on Agrobacterium mediated transformation efficiency Paz et al. [15]
2004Cotyledonary nodeTransferred chitinase gene and the barley ribosome-inactivating protein gene to enhance fungal resistanceLi et al. [6]
2004Mature and immature cotyledonShoot regeneration Franklin et al. [31]
2004Embryonic tipEstablished regeneration and Agrobacterium mediated transformation system Liu et al. [35]
2004Cotyledonary nodeEstablished liquid medium based system for selection transformed plantsYun, [58]
2005Cotyledonary nodeDeveloped repetitive organogenesis system Shan et al. [23]
2005Cotyledonary nodeExpressed Escherichia coli K99 fimbriae
subunit antigen in soybean to use as edible vaccine
Piller et al. [66]
2006Cotyledonary node Agrobacterium mediated transformation efficiency was improved by using half seed explant from mature seedPaz et al. [24]
2007Cotyledonary node Investigated Agrobacterium rhizogen to transform soybean cotyledonary node cells.Olhoft et al. [59]
2007Cotyledonary nodeExpressed synthetic Bacillus thuringiensis cry1A gene that confers a high degree of resistance to Lepidopteran Pests Miklos et al. [135]
2007Cotyledonary node and leaf nodeEstablished organogenic callus induction and Agrobacterium mediated transformation Hong et al ., [43]
2007Half seed Expressed jasmonic acid carboxyl methyltransferase in soybean to produce methyl jasmonate, which resulted in tolerant to water stressXue et al. [67]
2008HypocotylUsed silver nitrate to enhance adventitious shoot regeneration after Agrobacterium transformation and developed transgenic soybean producing high oleic acid content by silencing endogenous GmFAD2-1 gene by RNAiWang and Xu, [7]
2008Cotyledonary nodeImproved transformation efficiency using surfactant Silwet L-77 during Agrobacterium infection and L-cysteine during co-cultivation Liu et al. [136]
2008Cotyledonary nodeDeveloped rapid regeneration system using whole cotyledonary nodeMa and Wu, [2008]
2010Cotyledonary nodeProduction of isoflavone in callus cell lines by expression of isoflavone synthase gene. Jiang et al. [69]
2010Cotyledon and embryoDeveloped shoot regeneration from calli of soybean cv.Pyramid Joyner et al. [39]
2011HypocotylTransgenic soybean with low phytate contentYang et al. [70]
2011CotyledonDeveloped transgenic soybean with increased Vitamin E content by transferring γ-tocopherol methyltransferase (γ-TMT) gene in to seedling cotyledonLee et al. [137]

Table 1.

Major landmarks in soybean organogenesis and transformation

Explant Tissue Year Major Contribution Reference
Embryonic axes1983Embryoids development and plant regeneration via suspension culture Christianson et al.[77]
Immature cotyledon1984Somatic embryo Induction Lippmann & Lippmann, [84]
Immature cotyledon1985Plant regeneration via somatic embryogenesisLazzeri et al. [138]
Immature embryo1985Somatic embryogenesis and assessment of genotypic variationRanch et al. [139]
Immature embryo, cotyledon and, hypocotyl from germinating seedling1986Somatic embryogenesis from callusGhazi et al. [140]
Hypocotyl and cotyledon1986Embryoids development in suspension cultureKerns et al. [141]
Immature embryo and cotyledon1987Investigated the effect of nutritional, physical, and chemical factors on somatic embryogenesisLazzeri et al. [85]
Immature cotyledon1988Investigated the effect of auxin and orientation of explant on somatic embryogenesisHartweck et al. [142]
Immature cotyledon1988Analysed genotype dependency and High concentration of auxin on somatic embryo inductionKomatsuda and Ohyama, [143]
Immature cotyledon1988Investigated the interaction between auxin and sucrose during somatic embryogenesisLazzeri et al. [86]
Immature cotyledon1988Germination frequency of somatic embryo has been improved by reducing the exposure to auxinParrott et al. [87]
Immature cotyledon1988Developed rapid growing maintainable embryogenic suspension cultureFiner and Nagasawa, [82]
Immature cotyledon1988Histological analysis to investigate secondary somatic embryo formation. Finer, [79]
Immature cotyledon1989Demonstrated the effect of genotype on embryogenesisParrott et al. [144]
Immature cotyledon1989Developed primary transformants expressing zein gene by agrobacterium mediated transformation Parrott et al. [105]
Immature cotyledon1989Assayed somatic embryo maturation for conversion into plantletsBuchheim et al. [94]
Immature cotyledon1989Investigated the developmental aspects of somatic embryogenesis Christou and Yang, [145]
Immature cotyledon1990Screened soybean genotypes for somatic embryo productionKomatsuda et al. [146]
Immature cotyledon1991Transformed embryogenic cultures with gus and hpt gene via particle bombardmentFiner and McMullen., [64]
Immature cotyledon1991Analysed the interaction between genotype and sucrose concentration on somatic embryogenesisKomatsuda et al. [147]
Immature cotyledon1991Demonstrated adventitious shoot formation from cotyledonary and torpedo stage embryo Wright et al. [148]
Immature cotyledon1992Somatic embryo proliferation by somatic embryo cycling.Liu et al. [83]
Immature cotyledon1993Improved germination efficiency of somatic embryos of cultivar H7190 by desiccation Bailey et al. [101]
Immature cotyledon1993Demonstrated genotypic effect on induction, proliferation, maturation and germination of somatic embryoBailey et al. [96]
Immature cotyledon1993Investigated the factors affecting somatic embryogenesisLippmann & Lippmann, [149]
Immature cotyledon1993Soybean transformation by particle bombardment of embryogenic culturesSato et al. [130]
Immature cotyledon1994Developed transgenic soybean resistance to insect.Parrott et al. [150]
Immature embryos1995Investigated the effect of glutamine and sucrose on dry matter accumulation and composition of somatic embryo.Saravitz and Raper, [151]
Immature cotyledon1996Demonstrated the significance of embryo cycling for transformationLiu et al.[152]
Immature cotyledon1996Transformed embryogenic cultures with 12 different plansmid via particle bombardmentHadi et al. [115]
Immature cotyledon1996Developed transgenic soybean expressing a synthetic Bacillus thuringiensis insecticidal crystal protein gene (BtcrylAc) which is resistance to insectsStewart et al. [46]
Immature cotyledon1997Investigated the effect of ethylene inhibitors on embryo histodifferentiation and maturation Santos et al. [92]
Epicotyls and primary leaves1997Somatic embryogenesis and plant regeneration from cotyledon, epicotyls and primary leavesRajasekaran and Pello, [153]
Immature cotyledon1997Studied the effect of explant orientiation, pH, solidifying agent and wounding on induction of soybean from immature cotyledonsSantarém et al. [81]
Immature cotyledon1998Studied growth characteristics of embryogenic cultures for transformabilityHazal et al. [113]
Immature cotyledon1998Established sonication-assisted Agrobacterium mediated transformation of soybean immature cotyledonSantarem et al.[48]
Immature cotyledon1998Established sonication-assisted Agrobacterium mediated transformation of embryogenic suspension culture tissueTrick and Finer, [108]
Immature cotyledon1998Improved proliferation efficiency of embryogenic cultures by modifying sucrose and nitrogen content in mediumSamoylov et al. [89]
Immature cotyledon1998Developed liquid medium based system for histodifferentiation of embryogenic culturesSamoylov et al. [154]
Immature cotyledon1998Studied soluble carbohydrate content in soybean somatic and zygotic embryo during development.Chanprame et al. [155]
Immature cotyledon1999Studied the factors influencing transformation of prolific embryogenic cultures using bombardmentSantarem and Finer, [116]
Immature cotyledons1999Developed transgenic plants with bovine milk protein, β-caseinMaughan et al. [114]
Immature cotyledons1999Transformed GFP into embryogenic suspension culture with the aim to improve transformation and regeneration strategyPonappa et al. [156]
Immature cotyledons2000Improved somatic embryo development and maturation by application of ABATian and Brown, [157]
Immature cotyledon2000Screened genotypes for proliferative embryogenesisSimmonds and Donaldson, [97]
Immature cotyledons2000Studied physical factors influencing somatic embryo development from immature cotyledons.Bonacin et al. [99]
Immature cotyledon2000Investigated the factors affecting Agrobacterium mediated transformation soybeanYan et al. [109]
Immature cotyledon1989Investigated maturation of somatic embryo for efficient conversion into plantletsBuchheim et al. [94]
Immature cotyledon2000Developed and evaluated transgenic soybean expressing a synthetic cry1Ac gene from Bacillus thuringiensis for resistance to variety of insectsWalker et al. [158]
Immature cotyledon2001Effect of polyethylene glycol and sugar alcohols on soybean somatic embryo germination and conversionWalker and Parrott, [90]
Immature cotyledon2000Developed integrated bombardment and Agrobacterium transformation methodDroste et al.[159]
Immature cotyledon2001Screened soybean from different location in the US for uniform embryogenic responseMeurer et al. [103]
Immature cotyledon2001Studied the effect of osmotica for their influence on embryo maturation and germinationWalker & Parrott, [90]
Immature cotyledon2001Developed transgenic plant expressing 15-kD zein protein under β-phaseolin seed specific promoterDinkins et al. [125]
Immature cotyledon2001Somatic embryogenesis in Brazilian soybean cultivarsDroste et al. [160]
Immature cotyledon2002Somatic embryogenesis and particle bombardment for south Brazil cultivarsDroste et al. [100]
Immature cotyledon2002Histological analysis of developmental stages of somatic embryogenesisFernando et al. [161]
Immature cotyledon2002Screened soybean genotypes for somatic embryo induction and maturation capabilityTomlin, [162]
Immature cotyledon2003Investigated the effect of proliferation, maturation and desiccation on somatic embryo conversionMoon and Hildebrand, [88]
Immature cotyledon2004Improved transformation efficiently using Agrobacterium strain KYRT1 carrying pKYRTIKo et al. [111]
Immature cotyledon2004Developed transgenic plant containing phytase gene that store (produces) more phosphrous in seed. Chiera et al. [163]
Immature cotyledon2004Developed fertile transplastomic soybeanDufourmantel et al.[117]
Immature cotyledon2004Transferred chi and rip gene to enhance fungal resistanceLi et al. [6]
Immature cotyledon2004Improved transformation efficiency using Agrobacterium strain KYRT1Ko and Korban, [80]
Immature cotyledon2004Analysed media components and pH on somatic embryo inductionHoffmann et al. [80]
Immature cotyledon2005Developed transgenic soybean expressing maize γ-zein proteinLi et al. [124]
Immature cotyledon2005Modified soybean histodifferentiation and msaturation medium with the aim to improve the protein and lipid composition of somatic embryo Schmidt et al. [164]
Immature cotyledon2005Analysed the effect of carbon source and polyethylene glycol on embryo conversionKorbes et al. [91]
Immature cotyledon2006Improved fatty acid contentChen et al. [119]
Immature cotyledon2006Investigated the ontogeny of somatic embryogenesisSantos et al. [165]
Somatic embryo2006Developed transgenic soybean resistance to dwarf virusTougou et al. [120]
Immature cotyledon2006Investigated the influence of antibiotics on embryogenic cultures and Agrobacterium tumefaciens suppression in soybean transformationWiebke et al. [166]
Immature cotyledon2006Developed transgenic soybean for increased production of ononitol and pinitolChiera et al. [167]
Immature cotyledon2007Developed transgenic soybean resistant to dwarf virus
Tougou et al. [168]
Immature cotyledon2007Improved somatic embryogenesis in recalcitrant cultivars by back cross with a highly regenerable cultivar JackKita et al. [104]
Immature cotyledon2007Evaluated Japanese soybean genotypes for somatic embryogenesisHiraga et al. [102]
Immature cotyledon2007Soybean seed over expressing the Perilla frutescens γ -tocopherol methyltransferase gene
Tavva et al. [123]
Immature cotyledon2007Improved protein quality in transgenic soybean transformed with modified Gy1 proglycinin gene with a synthetic DNA encoding four continuous methionines.EI-Shemy et al. [169]
Immature cotyledon2007Analysed the effect of Abscisic acid on somatic embryo maturation and conversion.Weber et al. [170]
Immature cotyledon2007Developed transgenic soybean resistance to soybean mosaic virusFurutani et al. [121]
Immature cotyledon2008Used a new Selectable Marker Gene Conferring resistance to DinitroanilinesYemets et al. [171]
Immature cotyledon2008Developed strategy for transfer of multiple genes via micro projectile-mediated bombardmentSchmidt et al. [172]
Immature cotyledon2009Assessed the effect mannitol, abscisic acid and explant age on somatic embryogenesis in Chinese soybean cultivars Yang et al. [98]
Somatic embryo2009Developed transgenic soybean with increased oil contentRao and Hildebrand, [118]
Embryonic tip2010somatic embryogenesis and plant regeneration from the immature embryonic shoot tipLoganathan et al. [173]
Immature cotyledon2010Developed transgenic soybean with more tryptophan content in seedIshimoto et al. [122]
Immature cotyledon2010Screening of Brazilian soybean genotypes for embryogenesisDroste et al. [174]
Immature cotyledon2011Demonstrated Metabolic engineering of soybean seed coat for the production of novel biochemicalsSchnell et al. [126]
Immature cotyledon2011Investigated developmental profile of storage reserve accumulation in soybean somatic embryosHe et al. [175]
Immature cotyledon2011Improved transformation efficiency by Micro wounding with DNA free particle bombardment followed by Agrobacterium mediated transformation. Wiebke et al. [112]
Immature cotyledon2012Developed vacuum infiltration assisted Agrobacterium mediated transformation for Indian soybean cultivars.Mariashibu et al. [176]

Table 2.

Major landmarks in soybean somatic embryogenesis and transformation

2. Organogenesis and transformation

Organogenesis is characterized by the production of a unipolar bud primordium with subsequent development of the primordium into a leafy vegetative shoot. A successful plant regeneration protocol requires appropriate choice of explant, definite media formulations, specific growth regulators, genotype, source of carbohydrate, gelling agent, other physical factors including light regime, temperature, humidity and other factors [12]. Plant regeneration by organogenesis in soybean was first reported by Kimball and Bingham, [13] from hypocotyl sections followed by Cheng et al.[14] by culturing seedling cotyledonary node segments. Transfer of T-DNA into cotyledonary node cells by Agrobacterium mediated transformation was first reported by Hinchee et al. [10]. Advancement in soybean transformation appears to be slow compared to some of the recent improvement in cereal transformation (Paz et al. 2004). Olhoft et al. [16] stated that the efficiency of soybean transformation has to be improved 5-10 times before one person can produce 300 transgenic lines per year. Soybean transformation efficiency has been improved by optimizing the selection system, enhancing explant-pathogen interaction and improving culture conditions to promote regeneration and recovery of transformed plants.

2.1. Organogenesis

The successful application of biotechnology in crop improvement is based on efficient plant regeneration protocol. Soybean has been considered as recalcitrant to regenerate in vitro. Tissue culture responses are greatly influenced by three main factors viz. whole plant physiology of donor, in vitro manipulation, and in vitro stress physiology [17]. After the first report of adventitious bud regeneration from hypocotyl sections by Kimball and Bingham, [13] researchers have used different parts of the soybean plant as explants for successful shoot morphogenesis in soybean. These include cotyledonary node [10,14,18-24], shoot meristems [25], stem-node [26,27] epicotyls [28], primary leaf [29], cotyledons [30,31], plumules (32), hypocotyls [22,33,34], and embryo axes [25,35]. Plant regeneration via organogenesis from cotyledonary node was found to be the most convenient and faster approach in soybean. However, much improvement is needed for the cotyledonary node regeneration system. This limitation is mainly due to low frequency of shoot regeneration, long regeneration period and explant growth difficulties, which prevent the plant from being regeneration-competent[36].

The nutritional requirement for optimal shoot bud induction from different explants has been reported to vary with mode of regeneration. Media compositions have a key role in shoot morphogenesis, the basal medium MS [37] is most commonly used for soybean organogenesis and the medium B5 [38] are useful in some approaches. Benzylaminopurine (BA) has been the most commonly used plant growth regulator either alone or in combination with a low concentration of cytokinins, kinetin or thidiazuron (TDZ) [22, 39]. TDZ was reported to induce multiple bud tissue (MBT) from cotyledonary node axillary meristem which then gives shoots in the presence of BA [23]. The efficiency of shoot bud formation were enhanced by supplementing media with proline, increased level of MS micro nutrients [40], and ureide in the form of allantoin and amides [21].

Adventitious shoot regeneration from cotyledonary node or leaf node is based on proliferation of meristems. Use of pre-existing shoot meristems in transformation procedures can increase the chance of chimerism, so identifying tissues that can produce shoots in the absence of such pre-formed organs would be important [41]. Adventitious soybean shoots have been induced from hypocotyls [13]; cotyledons [18, 20], primary leaves [29] and epicotyls [28]. Hypocotyls of seedlings have been used as explants for adventitious shoot regeneration by Kaneda et al. [22]. Explants cultured on media supplemented with TDZ induced adventitious shoots more efficiently than BA. Histological analysis of adventitious shoot regeneration from the hypocotyl shows shoot primordias, formed from parenchymatous tissues of central pith and plumular trace regions [33]. Hypocotyls of seedlings have seldom been used as explants, even though the shoot regeneration frequency from hypocotyl segments was found to be higher than from cotyledons [22]. Franklin et al. [31] investigated the factors affecting adventitious shoot regeneration from the proximal end of mature and immature cotyledons. The presence of BAP and TDZ in the medium exerted a synergistic effect, in that regeneration efficiency was higher than for either cytokinin alone.

Indirect organogenesis is important as an alternative source of genetic variation in order to recover somaclones with interesting agronomic traits. Callus regeneration is advantageous over direct regeneration for transformation since effective selection of transgenic cells can be achieved [1]. However, the efforts made to regenerate plants from callus have yielded poor results since plants could not be regenerated from any type of soybean callus [42]. Yang et al. [32] compared different explants excised from immature and germinated seeds for callus mediated organogenic regeneration, although induction of organogenic callus was easily achieved by culture of immature cotyledons, development of adventitious buds from these calluses and the subsequent growth of these buds to shoots were inefficient, suggesting that only part of the callus was competent for regeneration. Sairam et al. [1] developed a rapid and efficient protocol for regeneration of genotype-independent cotyledonary nodal callus for cultivars Williams 82, Loda and Newton through manipulation of plant growth regulators and carbohydrates in the medium. Hong et al. [43] reported organogenic callus induction from cotyledonary node and leaf node explants in media supplemented with TDZ and BA, the system has been successfully utilized for Agrobacterium-mediated transformation

2.2. Genotype

Among the different factors affecting soybean regeneration, the genotypic dependence is ranked quite high. Since there is strong genotype specificity for regeneration of different soybean genotypes, a major limiting factor, it is pivotal to formulate genotype specific regeneration protocols. Genotype specificity for regeneration in soybean is well documented, although organogenesis is less genotype dependent and has become routine in several laboratories [18,20,28,29&33]. Reichert et al. [41] tested organogenic adventitious regeneration from hypocotyl explants excised from 18 genotypes. Plant formation from hypocotyl explants showed that all genotypes were capable of producing elongated shoots that could be successfully rooted. This study confirmed the genotype independent nature of this organogenic regeneration from the hypocotyl explant. Sairam et al. [1] developed an efficient genotype independent cotyledonary nodal callus mediated regeneration protocol for soybean cultivars Williams 82, Loda and Newton developed through manipulation of plant growth regulators and carbon source. Callus induction and subsequent shoot bud differentiation were achieved from the proximal end of cotyledonary explants on modified MS [37] media containing 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA), respectively. Sorbitol was found to be the best for callus induction and maltose for plant regeneration. The genotypic dependence of regeneration from cotyledon explants could be reduced by the use of combinations of cytokinins (Franklin et al. [31]). Though there was no significant difference in shoot bud formation among different genotypes, but there was significant difference in conversion of the number of regenerated plants in each cultivar (Delzer et al. [44]).

2.3. Agrobacterium mediated transformation

Agrobacterium-mediated transformation of soybean was first demonstrated by Hinchee et al. [10] through delivering, T-DNA into cells in the axillary meristems of the cotyledonary-node. After that scientists have attempted to introduce a lot of genes using Agrobacterium [25, 45-47]. The cotyledonary-node method is a frequently used soybean transformation system based on Agrobacterium-mediated T-DNA delivery into regenerable cells in the axillary meristems of the cotyledonary-node [16]. The efficiency of this transformation system remains low, apparently because of infrequent T-DNA delivery to cells in the cotyledonary-node axillary meristem, inefficient selection of transgenic cells that give rise to shoot meristems, and low rates of transgenic shoot regeneration and plant establishment. The development of an effective Agrobacterium transformation method for soybean depends on several factors including plant genotype, explant vigor, Agrobacterium strain, vector, selection system, and culture conditions [48, 49]. Increased soybean transformation efficiency, may be achieved by further optimizing the selection system, enhancing explant-pathogen interaction and improving culture conditions to promote regeneration and recovery of transformed plants. It has been reported that soybean genotype contributed to variation in susceptibility to Agrobacterium and regenerability in tissue culture [50, 51]. In addition, surface sterilization of plant tissue material for in vitro tissue culture and transformation is one of the critical steps in carrying out transformation experiments. While a short time of sterilization cannot completely decontaminate explants, prolonged sterilization may cause damage to explants and consequently affect their regenerability [52]. Antioxidant reagents such as cysteine, dithiothreitol, ascorbic acid and polyvinyl pyrrolidone have been used in plant transformation optimization to enhance either tissue culture response or transformation efficiency [53-55]. Recently, high transformation efficiency has also been reported in soybean by adding cysteine and thiol compounds to the cocultivation media [16, 56,57]. Liu et al. [35] established Agrobacterium mediated transformation using shoot tip explants of Chinese soybean cultivars. It had the advantage over the cotyledonary node by having no necrosis after infection, and showed more transient gus expression as embryonic tips are more sensitive to Agrobacterium because they contain promeristems and procambium. Yun, [58] established liquid medium to select transformed plants from the cotyledonary node. Liquid selection has proven to be more efficient than solid selection due to the direct contact of the explants with the medium and the selection agent in the medium. Olhoft et al. [59] transformed soybean cotyledonary nodes using Agrobacterium rhizogens strain SHA17 for the first time. The transformation efficiency was as high as 3.5 fold when compared with Agrobacterium tumefaciens strain AGL1. Clemente et al. [60] successfully used and evaluated the effect of glyphosate as a selective agent within the Agrobacterium mediated cotyledonary transformation system. Imazapyr is a herbicidal molecule that inhibits the enzymatic activity of acetohydroxyacid synthase, which catalyses the initial step in the biosynthesis of isoleucine, leucine and valine. Aragao et al. [47] used Imazapyr as a selection agent for selection of meristematic soybean cells transformed with the ahas gene from Arabidopsis. The bar gene encodes for phosphinothricin acetyltransferase (PAT) which detoxifies glufosinate, the active ingredient in the herbicide. Zhang et al. [61] successfully used glyphosate to select transformed cells after Agrobacterium transformation of cotyledonary node cells.

2.4. Particle bombardment

Even though particle bombardment is a widely used technique for transforming soybean embryogenic cultures, it was rarely explored for shoot morphogenesis. McCabe et al. [25] was the first to report particle bombardment mediated transformation in soybean. Transforming meristems of soybean bu DNA coated gold particles followed by shoot regeneration in the presence of cytokinin, resulting in the development of chimeras. In subsequent studies, non-chimeric plants were obtained through the use of screening methods for the selection of plants that contained transgenic germ-line cells [32,62&63]. Shoot apex transformation is labour intensive because the meristematic tissue is diffcult to target and, without selection, a large number of plants must be regenerated and analysed [64].

2.5. Genes for trait improvement

Soybean has been improved by Agrobacterium mediated transformation followed by shoot regeneration. Wheat germin gene (gf-2.8) encoding an oligomeric protein and oxalate oxidase (oxo) genes were introduced into soybean to improve resistance to the oxalate-secreting pathogen Sclerotina sclerotiorum [65]. Li et al.[6] successfully utilized Agrobacterium-mediated transformation to transfer chitinase gene (chi) and the barley ribosome-inactivating protein gene (rip) into soybean cotyledonary node cells. Piller et al. [66] investigated the feasibility of expressing the major Enterotoxigenic Escherichia coli K99 fimbrial subunit, FanC, in soybean for use as an edible subunit vaccine. Xue et al. [67] successfully expressed jasmonic acid carboxyl methyltransferase (NTR1) gene from Brassica campestris into soybean cv.Jungery that produces methyl jasmonate and showed tolerance to water stress. Soybean oil contains very low level of α-tocopherol which is the most active form of tocopherol. The tocopherols present in the seed are converted into α- and β-tocopherols by overexpressing γ-tocopherol methyltransferase from Brassica napus (BnTMT) [68]. Jiang et al. [69] transferred isoflavone synthase (IFS) gene into soybean callus using Agrobacterium-mediated transformation and the transgenic plants produced increased levels of the secondary metabolite, isoflavone. Transgenic soybean plant containing PhyA gene of Aspergillus ficuum exhibited a lower amount of phytate in different soybean tissues including the leaf, stem and root. This indicated that engineering crop plants with a higher expression level of heterologous phytase could improve the degradation of phytate and potentially in turn mobilize more inorganic phosphate from phytate and thus reduce phosphate load on agricultural ecosystems [70].

3. Somatic embryogenesis and transformation

Somatic embryogenesis is a process by which a plant somatic cell develops into a whole plant without gametic fusion but undergoes developmental changes as that of zygotic embryogenesis [71, 72]. The first demonstration of in vitro somatic embryogenesis was reported in Daucus carota by Reinert [73]. The concept of embryogenesis has drawn a lot of attention because of its significance in theory and practice. Primarily, somatic embryos can be produced easily and quickly, so that it provides an economical and easy way to study plant development. Secondly, synthetic seeds developed from somatic embryos open the possibility of developing high quality seeds and may allow us to produce seeds from those plants that require a long period for seed production. Somatic embryogenesis is also useful in plant genetic engineering since regeneration via somatic embryogenesis is frequently single of cell origin, resulting in a low response of chimeras and high a number of true transgenic regenerants [74, 75].

3.1. Somatic embryogenesis

The first record of soybean somatic embryogenesis was reported by Beversdorf & Bingham [76], followed by Christianson et al. [77] who regenerated plants through the method. The immature cotyledon is the preferred explant for soybean somatic embryogenesis as it has pre-determined embryogenic cells. Somatic embryogenesis is a multi-step regeneration process starting with the formation of proembryogenic cell mass, followed by somatic embryo induction, their maturation, desiccation and finally plant regeneration [78].

Soybean somatic embryos were induced from immature cotyledon explants cultured on medium containing high levels of 2,4-D [79]. Even though NAA induced somatic embryogenesis from immature cotyledons, the mean number of embryos produced on 2,4-D was significantly higher [80]. Explant orientation, pH, solidifying agent, and 2,4-D concentration have a synergic effect on somatic embryo induction [81]. The early-staged somatic embryos can be maintained and proliferated by subculturing the tissue on either semi-solid medium [79] or liquid suspension culture medium [82]. Somatic embryos incubated in a medium containing NAA do not proliferate so well as those produced on a medium containing 2,4-D [83]. Somatic embryos initiated on NAA are more advanced in embryo morphology than those induced on 2,4-D and the efficiency of somatic embryo induction was highest with a medium containing 2-3% sucrose. Cultures initiated on lower sucrose concentrations tended to produce a higher amount of friable embryos, while increased concentrations of this sugar impaired embryo induction [80,84-86]. Histodifferentiation and maturation of somatic embryos doesn’t need exogenous auxin or cytokinins [87]. Indeed, poorly developed meristem or swollen hypocotyls may be an undesired outcome of the application of exogenous auxins and cytokinins, respectively. Moon and Hildebrand, [88] investigated the effects of proliferation, maturation, and desiccation methods on conversion of soybean somatic embryos to plants. Somatic embryos proliferated on solid medium showed a higher regeneration rate when compared with the embryos proliferated in liquid medium. The growth period of somatic embryo development can be reduced one month by culturing in a medium devoid of 2,4-D and B5 vitamins. Carbon source is critical for embryo nutritional health and improves somatic embryo maturation. The effects of carbohydrates on embryo histodifferentiation and maturation on liquid medium were analyzed by Samoylov et al. [89]. FNL medium supplemented with 3% sucrose (FNL0S3) or 3% maltose (FNL0M3) were compared. Data indicated that sucrose promotes embryo growth and significantly increases the number of cotyledon-stage embryos recovered during histodifferentiation and maturation. However, the percentages of plants recovered from embryos differentiated and matured in FNL0S3 was lower than those grown in FNL0M3 (Samoylov et al. 1998b). The quality of somatic embryos can be positively influenced by a low osmotic potential in maturation medium [90, 91]. Carbohydrates can act as an osmotic agent. Polyethylene glycol 4000, mannitol and sorbitol were tested as supplements to a liquid Finer and Nagasawa medium-based histodifferentiation/maturation medium FNL0S3, for soybean (Glycine max L. Merrill) somatic embryos of ‘Jack’ and F138 or ‘Fayette’[90]. Overall, 3% sorbitol was found to be the best of the osmotic supplements tested. The ability of histodifferentiation and conversion of somatic embryo have been improved by the use of ethylene inhibitor aminoethoxyvinylglycine [92]. The effects of ethylene on embryo histodifferentiation and conversion were genotype-specific. The germination frequency of soybean embryos is very low [93], and therefore, partial desiccation of somatic embryos was emphasised with a view to improving the germination frequency in soybean [87,94&95]. Desiccation induced a physiological state there by increase the germination ability of somatic embryos [87].

3.2. Genotype

Soybean somatic embryogenesis is highly genotypic when compared to organogenesis. The existence of strong genotype specificity in the regeneration capacity of the different cultivars represents a major limiting factor for the advancement of soybean biotechnology. The embryogenic efficiency of soybean was shown to be different among cultivars at each stage (induction, proliferation, maturation, germination) of somatic embryogenesis [92,96] and it is very challenging to identify genotypes highly responsive to all stages. Simmonds and Donaldson, [97] screened 18 short season soybean genotypes for proliferative embryogenesis. Five genotypes produced embryogenic cultures which were proliferative for at least 6 months. Yang et al. [98] screened 98 Chinese soybean varieties for somatic embryogenesis and selected 12 varieties based on their embryogenic capacity. The greatest average number of plantlets regenerated per explant (1.35) was observed in N25281. Bonacin et al. [99] demonstrated the influence of genotype on somatic embryogenic capability of five Brazilian cultivars. Droste et al. [100] reported somatic embryo induction, proliferation and transformation of commercially grown Brazilian soybean cultivars for the first time. Soybean somatic embryo conversion is genotype dependent; germination frequency of H7190 was approximately three fold lower than that of PI 417138 [101]. Hiraga et al. [102] examined the capacity for plant regeneration through somatic embryogenesis in Japanese soybean cultivars and identified Yuuzuru and Yumeyutaka as having high potential for somatic embryogenesis. Several cultivars were identified as uniformly embryogenic at the primary induction phase at all locations, among which Jack was the best [103]. Kita et al. [104] evaluated somatic embryogenesis, proliferation of embryogenic tissue, and regeneration of plantlets in backcrossed breeding lines derived from cultivar Jack and a breeding line, QF2. The backcrossed breeding lines exhibited an increased capacity for induction and proliferation of somatic embryos and were used successfully to generate transgenic plants.

3.3. Agrobacterium mediated transformation

Recovery of the first transgenic plant via somatic embryogenesis in soybean was reported by Parrott et al. [105]. Immature cotyledon tissues were inoculated with Agrobacterium strain which contained 15 kD zein gene and the neomycin phosphotransferase gene. The explants were placed on medium containing high auxin for somatic embryo induction. Three transgenic plants containing the introduced 15 kD zein gene were regenerated. Unfortunately, these plants were chimeric and the 15 kD zein gene was not transmitted to the progeny. Sonication-assisted Agrobacterium-mediated transformation (SAAT) of immature cotyledons tremendously improves the efficiency of Agrobacterium infection by introducing large numbers of micro wounds into the target plant tissue [48]. The highest GUS expression was obtained when immature cotyledons were sonicated for 2s in the presence of Agrobacterium followed by co-cultivation for 3 days. Trick and Finer, [108] successfully employed Sonication-assisted Agrobacterium-mediated transformation of embryogenic suspension culture tissue and when SAAT was not used, no transgenic clones were obtained. Yan et al. [109] demonstrated the feasibility of Agrobacterium mediated transformation of cotyledon tissue for the production of fertile transgenic plants by optimising the Agrobacterium concentration, using co-cultivation time and selecting proper explant. Ko and Korban, [110] investigated optimal conditions for induction of transgenic embryos followed by Agrobacterium mediated transformation. Using cotyledon explants from immature embryos of 5-8mm length, a 1:1 (v/v) concentration of bacterial suspension and 4-day co-cultivation period significantly increased the frequency of transgenic somatic embryos. The Agrobacterium tumefaciens strain KYRT1 harboring the virulence helper plasmid pKYRT1 induces transgenic somatic embryos at a high frequency from infected immature soybean cotyledons [111]. Recently, the successful recovery of a high number of soybean transgenic fertile plants was obtained from the combination of DNA- free particle bombardment and Agrobacterium-mediated transformation using proliferating soybean somatic embryos as targets [112].

3.4. Particle bombardment

Particle bombardment is a widely used technique for transformation of embryogenic cultures of soybean; the major advantage of this technique over Agrobacterium is the removal of biological incompatibilities. Particle bombardment in soybean was first reported by Finer and McMullen [64], in which embryogenic suspension culture tissue of soybean was bombarded with particles coated with plasmid DNAs encoding hygromycin resistance and β-glucuronidase. Analysis of DNA from progeny plants showed genetic linkage for multiple copies of introduced DNA. Using particle bombardment, fertile plants could be routinely produced from the proliferating transgenic embryogenic clones. Hazal et al. [113] studied growth characteristics and transformability of embryogenic cultures and found that cultures bombarded between 2-6 days after transfer to fresh medium showed more transient expression of the reporter gene. Histological analysis showed that the most transformable cultures had cytoplasmic-rich cells in the outermost layers of the tissue. Maughan et al. [114] bombarded embryogenic cultures with plasmid containing 630-bp DNA fragment encoding a bovine milk protein, β-casein. Hadi et al. [115] co-transformed 12 different plasmids into embryogenic suspension culture by particle bombardment. Hybridization analysis of hygromycin resistance clones verified the presence of introduced plasmid DNAs. Santarem and Finer [116] investigated the effect of desiccation of target tissue, period of subculture prior to bombardment and number of bombardments per target tissue for enhancement of transient expression of the reporter gene. Desiccation of proliferating tissue for 10 min, subculture on the same day prior to bombardment and three times bombardment on a single day enhanced the transient expression of β-glucuronidase [116]. Dufourmantel et al. [117] successfully transformed chloroplasts from embryogenic tissue of soybean using DNA carrying spectinomycin resistance gene (aadA) by bombardment. All transplastomic T0 plants were fertile and T1 progeny was uniformly spectinomycin resistant, showing the stability of the plastid transgene. Droste et al. [100] successfully transformed embryogenic cultures of soybean cultivars recommended for commercial growing in South Brazil by bombardment, and this opened the field for the improvement of this crop in this country by genetic engineering.

3.5. Genes for trait improvement

Li et al. [6] attempted to transform two antifungal protein genes (chitinase and ribosome-inactivating protein) by co-transformation. Transgenic soybeans expressing the Yeast SLC1 Gene showed higher oil content [118]. They reported that, compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos and also found that a maximum of 3.2% increase in seed oil content was observed in a T3 line. Transfer of Δ6 desaturase, fatty acid elongase and D5 desaturase into soybean under seed specific expression produced arachidonic acid (ARA) in seeds of soybean [119]. In an attempt to enhance soybean resistance to viral diseases, several groups successfully generated transgenic plants by expressing an inverted repeat of soybean dwarf virus SbDV coat protein (CP) genes [120], or soybean mosaic virus (SMV) coat protein gene [121]. The nutritional quality of soybean has been improved for enhanced amino acid, proteins and vitamin production by transgenic technology [114, 122, 123, 124, and 125]. The feasibility of genetically engineering soybean seed coats to divert metabolism towards the production of novel biochemicals was tested by transferring the genes phbA, phbB, phbC from Ralstonia eutropha. Each gene was under the control of the seed coat peroxidase gene promoter [126]. The analysis of seed coats demonstrated that polyhydroxybutyrate (PHB) was produced at an averge of 0.12% seed coat dry weight.

4. Conclusion and future prospects

As demands increase for soybean oil and protein, the improvement of soybean quality and production through genetic transformation and functional genomics becomes an important issue throughout the world. Modern genetic analysis and improvement of soybean heavily depend on an efficient regeneration and transformation process, especially commercially important genotypes. The transformation techniques developed until now till date do not allow high-throughput analyses in soybean functional genomics; though significant improvements have been made in the particle bombardment of embryogenic culture and Agrobacetrium mediated transformation of the cotyledonary node over the past three decades. However, routine recovery of transgenic soybean plants using either of these two transformation systems has been restricted to a few genotypes with no reports of transformation on other locally available commercial genotypes. Therefore, development of an efficient and consistent transformation protocol for other locally available commercial genotypes, will greatly aid soybean functional genomics and transgenic technology.


1 - Sairam RV, Franklin G, Hassel R, Smith B, Meeker K, Kashikar N, Parani M, Abed Dal, Ismail S, Berry K, Goldman SL. A study on effect of genotypes, plant growth regulators and sugars in promoting plant regeneration via organogenesis from soybean cotyledonary nodal callus. Plant Cell, Tissue and Organ Culture 2003, 75:79-85.
2 -
3 - Birt DF, Hendrich S, Anthony M and Alekel DL (2004) Soybeans and the prevention of chronic human disease. Soybeans: Improvement, Production and Uses. J. Specht and R. Boerma, Eds. 3rd ed. pp. 1047–1117 American Society of Agronomy, Madison, WI
4 - Wilson RF. Seed composition. In: H.R. Boerma and J.E. Specht (eds) Soybean: improvement, production and uses. 3rd ed. ASA, CSSA, SSA, Madison, WI 2004. 621-677.
5 - Manavalan LP, Guttikonda SK, Tran LS, Nguyen HT. Physiological and molecular approaches to improve drought resistance in soybean. Plant Cell Physiology 2009, 50:1260-1276.
6 - Li HY, Zhu YM, Chen Q, Conner RL, Ding XD, Li J, Zhang BB. Production of transgenic soybean plants with two anti-fungal protein genes via Agrobacterium and particle bombardment. 2004 48(3):367-374.
7 - Wang G, Xu Y. Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA interference. Plant Cell Reports 2008, 27:1177-1184.
8 - Hiromoto DM, Vello NA. The genetic base of Brazilian soybean (Glycine max (L.) Merrill) cultivars. Brazil Journal of Genetics 1986, 2:295-306.
9 - Cao D, Hou W, Song S, Sun H, Wu C, Gao Y, Han T. Assessment of conditions affecting Agrobacterium rhizogenes-mediated transformation of soybean. Plant Cell Tissue and Organ Culture 2009, 96:45-52.
10 - Hinchee MA, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB. Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Nature Biotechnology 1988, 6:915-922.
11 - Trick HN, Dinkins RD, Santarem, ER, Di R, Samoylov V, Meurer CA, Walker DR, Parrott WA, Finer JJ, Collins GB. Recent advances in soybean transformation. Plant Tissue Culture and Biotechnology 1997, 3(1):9-24.
12 - Kothari SL, Joshi A, Kachhwaha S, Ochoa-Alejo N. Chilli peppers -A review on tissue culture and transgenesis. Biotechnology Advances 2010, 28:35-48.
13 - Kimball SL, Bingham ET. Adventitious bud development of soybean hypocotyl sections in culture. Crop Science 1973, 13:758-760.
14 - Cheng TY, Saka H, Voqui-Dinh TH. Plant regeneration from soybean cotyledonary node segments in culture. Plant Science Letters 1980, 19:91-99.
15 - Paz MM, Shou H, Guo Z, Zhang Z, Banerjee AK, Wang K. Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant. Euphytica 2004, 136:167-179.
16 - Olhoft PM, Somers DA. L.Cysteine increases Agrobacterium- mediated T-DNA delivery into soybean cotyledonary-node cells. Plant Cell Reports 2001, 20:706-711.
17 - Benson EE. Special symposium: in vitro plant recalcitrance, do free radicals have a role in plant tissue culture recalcitrance? In Vitro Cellular and Developmental Biology Plant 2000, 86:163-70.
18 - Barwale UB, Keans HR, Widholm JM. Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis. Planta 1986, 167:473- 481.
19 - Barwale UB, Mayer Jr MM, Widholm JM. Screening of Glycine max and Glycine soja genotypes for multiple shoot formation at the cotyledonary node. Theoretical and Applied Genetics 1986, 72:423-428.
20 - Wright MS, Koehler SM, Hinchee MA, Carnes MG. Plant regeneration by organogenesis in Glycine max. Plant Cell Reports 1986, 5:150-154.
21 - Shetty K, Asano Y, Oosawa K. Stimulation of in vitro shoot organogenesis in Glycine max (Merrill.) by allantoin and amides. Plant Science 1992, 81:245-251.
22 - Kaneda Y, Tabei Y, Nishimura S, Harada K, Akihama T, Kitamura K. Combination of thidiazuron and basal media with low salt concentrations increases the frequency of shoot organogenesis in soybeans [Glycine max (L.) Merr.]. Plant Cell Reports 1997, 17 8-12.
23 - Shan Z, Raemakers K, Tzitzikas EN, Ma Z, Visser RGF. Development of a highly efficient, repetitive system of organogenesis in soybean (Glycine max (L.) Merr). Plant Cell Reports 2005, 24:507-512.
24 - Paz MM, Martinez JC, Kalvig AB, Fonger TM, and Wang K. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium mediated soybean transformation. Plant Cell Reports 2006, 25:206- 213.
25 - McCabe DE, Swain WF, Martinell BJ, Christou P. Stable transformation of soybean (Glycine max) by particle acceleration. Biotechnology 1988, 6:923-926.
26 - Saka H, Voqui Dinh TH, Cheng TY. Stimulation of multiple shoot formation on soybean stem nodes in culture. Plant Science Letters 1980, 19 193-201.
27 - Kim JH, Lamotte E Hack E. Plant regeneration in vitro from primary leaf nodes of soybean (Glycine max) seedling. Journal Plant Physiology 1990, 136:664-669.
28 - Wright MS, Williams MH, Pierson PE, Carnes MG. Initiation and propagation of Glycine max L. Merrill; Plants from tissue-cultured epicotyls. Plant Cell Tissue Organ Culture 1987, 8:83-90.
29 - Wright MS, Ward DV, Hinchee MA, Carnes MG, Kaufman RJ. Regeneration of soybean (Glycine max L. Merr.) from cultured primary leaf tissue. Plant Cell Reports 1987, 6:83-89.
30 - Mante S, Scorza R, Cordts J. A simple, rapid protocol for adventitious shoot development from mature cotyledons of Glycine max cv.Bragg. In Vitro Cellular and Developmental Biology Plant 1989, 25(4):385-388.
31 - Franklin G, Carpenter L, Davis E, Reddy CS, Al-Abed D, Alaiwi WA, Parani M, Smith B, Goldman SL, Sairam RV. Factors influencing regeneration of soybean from mature and immature cotyledons. Plant Growth Regulation 2004, 43:73-79.
32 - Yang N and Christou P. Cell type specific expression of a CaMV 35s-GUS gene in transgenic soybean plants. Developmental Genetics 1990, 11:289-293.
33 - Dan, Y, Reichert NA. Organogenic regeneration of soybean from hypocotyl explants. In Vitro Cellular and Developmental Biology Plant 1998, 34 14-21.
34 - Yoshida T. Adventitious shoot formation from hypocotyl sections of mature soybean seeds. Breeding Science 2002, 52:1-8.
35 - Liu HK, Yang C, Wie ZM. Efficient Agrobacterium tumefaciens mediated transformation of soybeans using an embryonic Tip regeneration system. Planta 2004, 219:1042-1049.
36 - Ma XH, Wu TL. Rapid and efficient regeneration in soybean [Glycine max (L.) Merrill] from whole cotyledonary node explants. Acta Physiologiae Plantarum 2008, 30:209-216.
37 - Murashige T. and Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 1962, 15:473-479.
38 - Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research 1968, 50:151-158.
39 - Joyner EY, Boykin LS, Lodhi MA. Callus Induction and Organogenesis in Soybean [Glycine max (L.) Merr.] cv. Pyramid from Mature Cotyledons and Embryos. The Open Plant Science Journal 2010, 4:18-21.
40 - Kim J, Hack E, LaMotte CE. Synergistic effects of proline and inorganic micronutrients and effects of individual micronutrients on soybean (Glycine max) shoot regeneration in vitro. Journal of Plant Physiology 1994, 144:726-734.
41 - Reichert NA, Young MM, Woods AL. Adventitious organogenic regeneration from soybean genotypes representing nine maturity groups. Plant Cell, Tissue and Organ Culture 2003, 75:273-277.
42 - Hu CY, Wang L. In planta soybean trasnformation technologies developed in China: procedure, confirmation and field performance. In Vitro Cellular and Developmental Biology Plant 1999, 35:417-420.
43 - Hong HP, Zhang H, Olhoft P, Hill S, Wiley H, Toren E, Hillebrand H, Jones T, Cheng M. Organogenic callus as the target for plant regeneration and transformation via Agrobacterium in soybean (Glycine max (L) Merr). In Vitro Cellular and Developmental Biology Plant, 2007, 43(6):558-568.
44 - Delzer BW, Somers DA, Orf JH. Agrobacterium tumefaciens susceptibility and plant regeneration of 10 soybean genotypes in maturity groups 00 to II. Crop Science 1990, 30:320-322.
45 - Chee PP, Fober KA, Slightom JL. Transformation of soybean (Glycine max) by infecting germinating seeds with Agrobacterium tumefaciens. Plant Physiology 1989, 91:1212-1218.
46 - Stewart CN Jr, Adang MJ, All JN, Barmy HR, Cardineau G, Tucker D, Parrot WA. Genetic transformation, recovery and characterization of fertile soybean [Glycine max (L.) Merr.] transgenic for a synthetic Bacillus thuringensis cryIAc gene. Plant Physiology 1995, 112:121-129.
47 - Aragao FJL, Sarokin L, Vianna GR, Rech, EL. Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean [Glycine max (L) Merrill] plants at a high frequency. Theoretical and Applied Genetics 2000, 101 (1&2):1-6.
48 - Santarem ER Trick HN Essig JS Finer JJ. Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression. Plant Cell Reports 1998, 17:752-759.
49 - Zhang Z, Guo Z, Shou H, Pegg SE, Clemente TE, Staswick PE, Wang K. Assessment of conditions affecting Agrobacterium-mediated soybean transformation and routine recovery of transgenic soybean. In: A.D. Arencibia (Ed.), Plant Genetic Engineering: Towards the Third Millennium: Proceedings of the International Symposium on Plant Genetic Engineering–10 December 1999, 88-94. Havana, Cuba, Elsevier, Amsterdam / New York 2000.
50 - Meurer CA, Dinkins RD, Collins GB. Factors affecting soybean cotyledonary node transformation. Plant Cell Reports 1998, 18:180-186.
51 - Donaldson PA, Simmonds DH. Susceptibility of Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean. Plant Cell Reports 2000, 19:478-484.
52 - Maruyama EK, Ishi A, Migita S, Migita K. Screening of suitable sterilization of explants and proper media for tissue culture of eleven tree species of Peru-Amazon forest. Journal of Agricultural Science 1989, 33:252-261.
53 - Perl A, Lotan O, Abu-Abied M, Holland D. Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): the role of antioxidants during grape-Agrobacterium interactions. Nature Biotechnology 1996, 14(5):624-628.
54 - Enriquez-Obregon GA, Prieto-Samsonov DL, de la Riva GA, Perez M, Selman-Housein G, Vasquez-Padron RI. Agrobacterium-mediated Japonica rice transformation: a procedure assisted by antinecrotic treatment. Plant Cell, Tissue and Organ Culture 1999, 59:159-168.
55 - Frame BR, Shou V Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg SEK, Li B, Nettleton DS, Pei D, Wang K. Agrobacterium tumefaciens-mediated transformation of maize embryos using standard binary vector system. Plant Physiology 2002, 129:13-22.
56 - Olhoft PM, Lin K, Galbraith J, Nielsen NC, Somers DA. The role of thiol compounds increasing Agrobacterium-mediated transformation of soybean cotyledonary-node cells. Plant Cell Reports 2001, 20:731-737.
57 - Olhoft PM, Flagel LE, Donovan CM, Somers DA. Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 2003, 216:723-735.
58 - Yun CS. High-efficiency Agrobacterium-mediated Transformation of Soybean. Acta Botanica Sinica 2004, 46 (5):610-617.
59 - Olhoft PM, Bernal LM, Grist LB, Hill DS, Mankin SL, Shen YW, Kalogerakis M, Wiley H, Toren E, Song HS, Hillebrand H, Jones T. A novel Agrobacterium rhizogenes-mediated transformation method of soybean [Glycine max (L.) Merrill] using primary-node explants from seedlings. In Vitro Cellular and Developmental Biology Plant 2007, 43:536-549.
60 - Clemente TE, LaVallee BJ, Howe AR, Conner Ward D, Rozman R, Hunter P, Broyles DL, Kasten D, Hinchee MA. Progeny analysis of glyphosate selected transgenic soybeans derived from Agrobacterium-mediated transformation. Crop Science 2000, 40:797-803.
61 - Zhang Z, Xing A, Staswick PE, Clemente TE. The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean. Plant Cell Tissue Organ Culture 1999, 56:37-46.
62 - Christou P, Swain WF, Yang NS, McCabe DE. Inheritance and expression of foreign genes in transgenic soybean plants. Proceedings of the National Academy of Sciences of the United States of America 1989, 86:7500-7504.
63 - Christou P, McCabe DE, Martinell BJ, Swain WF. Soybean genetic engineering-commercial products of transgenic plants. Trends in Biotechnology 1990, 8:145-15.
64 - Finer JJ, McMullen MD. Transformation of soybean via particle bombardment of embryogenic suspension culture tissue. In Vitro Cellular and Developmental Biology Plant 1991. 27:175-182.
65 - Donaldson PA, Anderson T, Lane BG, Davidson AL, Simmonds DH. Soybean plants expressing an active oligomeric oxalate oxidase from the wheat gf-2.8 (germin) gene are resistant to the oxalate-secreting pathogen Sclerotina sclerotiorum. Physiol. Mol. Plant Path 2001, 59:297-307.
66 - Piller KJ, Clemente TE, Jun SM, Petty CC, Sato S, Pascual DW, Bost KL. Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean. Planta 2005, 222:6-18.
67 - Xue RG, Zhang B, Xie HF. Overexpression of a NTR1 in transgenic soybean confers tolerance to water stress. Plant Cell Tissue Organ Culture 2007, 89:177-183.
68 - Chen DF, Zhang M, Wang YQ, Chen XW. Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed. Biologia Plantarum 2012, 56(1):131-134.
69 - Jiang N, Jeon EH, Pak JH, Ha TJ, Baek IY, Jung WS, Lee JH, Kim DH, Choi HK, Cui Z, Chung YS. Increase of isoflavones in soybean callus by Agrobacterium-mediated transformation. Plant Biotechnology Reporter 2010, 4 253-260.
70 - Yang S, Li G, Li M, Wang J. Transgenic soybean with low phytate content constructed by Agrobacterium transformation and pollen-tube pathway. Euphytica 2011, 177:375-382.
71 - Merkle SA, Parrott WA and Flinn BS. Morphogenic aspects of somatic embryogenesis. In: Thorpe TA (ed.): In vitro Embryogenesis in Plants. Kluwer Academic Publishers 1995, 155-203.
72 - McKersie BD and Brown DCW. Somatic embryogenesis and artificial seeds in Forage legumes. Seed Science Research 1996, 6:109-126.
73 - Reinert J. Morphogenese und ihre kontrolle an gewebekulturen aus carotten. Naturwissenchaften 1958, 45:344-345.
74 - Ammirato PV. The regulation of somatic embryo development in plant cell culture: suspension culture, techniques and hormone requirements. Nature Biotechnology 1983, 1:68-74.
75 - Mathews HR, Litz H, Wilde S, Merkle H and Wetzstein HY. Stable gene expression of β-glcuronidase and npt II genes in mango somatic embryos. In vitro Cellular and Developmental Biology Plant 1992, 28:172-178.
76 - Beversdorf WD, Bingham ET. Degrees of differentiation obtained in tissue cultures of Glycine species. Crop Science 1977, 17(2):307-311.
77 - Christianson ML, Warick DA, Carlson PS. A morphogenetically competent soybean suspension culture. Science 1983, 222:632-634.
78 - Von AS, Sabala I, Bozhkov P, Dyachok J, Filonova L. Developmental pathways of somatic embryogenesis. Plant Cell Tissue Organ Culture 2002, 69:233-249
79 - Finer JJ. Apical proliferation of embryogenic tissue of soybean (Glycine max (L.) Merrill). Plant Cell Reports 1988, 7(4):238-241.
80 - Hoffmann N, Nelson RL, Korban SS. Influence of media components and pH on somatic embryo induction in three genotypes of soybean. Plant Cell, Tissue and Organ Culture 2004, 77(2):157-163.
81 - Santarem ER, Pelissier B, Finer JJ. Effect of explant orientation, pH, solidifying agent and wounding on initiation of soybean somatic embryos. In Vitro Cellular and Developmental Biology-Plant 1997, 33 (1):13-19.
82 - Finer JJ, Nagasawa A. Development of an embryogenic suspension culture of soybean (Glycine max Merrill). Plant Cell, Tissue and Organ Culture 1988, 15:125-136.
83 - Liu W, Moore PJ, Collins GB. Somatic embryogenesis in soybean via somatic embryo cycling. In Vitro Cellular and Developmental Biology Plant 1992, 28:153-160.
84 - Lippmann B, Lippmann G. Induction of somatic embryos in cotyledonary tissue of soybean, Glycine max L. Merr. Plant Cell Reports 1984, 185(3):215-218.
85 - Lazzeri PA, Hildebrand DF, Collins GB. Soybean somatic embryogenesis: effects of hormones and culture manipulations. Plant Cell, Tissue and Organ Culture 1987, 10:197-208.
86 - Lazzeri PA, Hildebrand DF, Sunega J, Williams EG, Collins GB. Soybean somatic embryogenesis: interactions between sucrose and auxin. Plant Cell Reports 1988, 7:517-520.
87 - Parrott WA, Dryden G, Vogt S, Hildebrand DF, Collins GB, Williams EG. Optimization of somatic embryogenesis and embryo germination in soybean. In Vitro Cellular and Development Biology Plant 1988, 24:817-820.
88 - Moon H, Hildebrand DF. Effects of proliferation, maturation, and desiccation methods on conversion of soybean somatic embryos. In Vitro Cellular and Developmental Biology Plant 2003, 39:623-628
89 - Samoylov VM, Tucker DM, Parrott WA. Soybean [Glycine max (L.) Merrill] embryogenic cultures: the role of sucrose and total nitrogen content on proliferation. In Vitro Cellular and Developmental Biology Plant 1998, 34:8-13.
90 - Walker DR, Parrott WA. Effect of polyethylene glycol and sugar alcohols on soybean somatic embryo germination and conversion. Plant Cell, Tissue and Organ Culture 2001, 64(1):55-62.
91 - Korbes AP, Droste A. Carbon sources and polyethylene glycol on soybean somatic embryo conversion. Pesquisa Agropecuária Brasileira 2005, 40(3):211-216.
92 - Santos KGB, Mundstock E, Bodanese Zanettini MH. Genotype-specific normalization of soybean somatic embryogenesis through the use of an ethylene inhibitor. Plant Cell Reports 1997, 16 (12):859-864.
93 - Jang GW, Park RD, Kim KS. Plant regeneration from embryogenic suspension cultures of soybean (Glycine max L. Merrill). Journal of Plant Biotechnology 2001, 3:101-106.
94 - Buchheim JA, Colburn SM, Ranch JP. Maturation of soybean somatic embryos and the transition to plantlet grown. Plant Physiology 1989, 89:768-77.
95 - Durham RE and Parrott WA. Repetitive somatic embryogenesis from peanut cultures in liquid medium. Plant Cell Reports 1992, 11:122-125.
96 - Bailey MA, Boerma HR, Parrott WA. Genotype effects on proliferative embryogenesis and plant regeneration of soybean. In vitro Cellular and Developmental Biology Plant 1993, 29:102-108.
97 - Simmonds DH, Donaldson PA. Genotype screening for proliferative embryogenesis and biolistic transformation of short-season soybean genotypes. Plant Cell Reports 2000, 19:485-490.
98 - Yang C, Zhao T, Yu D, Gai J. Somatic embryogenesis and plant regeneration in Chinese soybean (Glycine max (L.) Merr.) Impacts of mannitol, abscisic acid, and explants age. In Vitro Cellular and Developmental Biology-Plant 2009, 45 (2):180-181.
99 - Bonacin GA, DiMauro AO, Oliveira RC, Perecin D. Induction of somatic embryogenesis in soybean: physicochemical factors influencing the development of somatic embryos. Genetics and Molecular Biology 2000, 23(4):865-868.
100 - Droste A, Pasquali G, Bodanese-Zanettini, MH. Transgenic fertile plants of soybean [Glycine max (L) Merrill] obtained from bombarded embryogenic tissue. Euphytica 2002, 127(3):367-376.
101 - Bailey MA, Boerma HR, Parrott WA. Genotype-specific optimization of plant regeneration from somatic embryos of soybean. Plant Science 1993, 93:117-120.
102 - Hiraga S, Minakawa H, Takahashi K, Takahashi R, Hajika M, Harada K, Ohtsubo N. Evaluation of somatic embryogenesis from immature cotyledons of Japanese soybean cultivars. Plant Biotechnology 2007, 24 (4):435-440.
103 - Meurer CA, Dinkins RD, Redmond CT, Mcallister KP, Tucker DT, Walker DR, Parrott WA, Trick HN Essig JS, Frantz HM, Finer JJ, Collins GB. Embryogenic response of multiple soybean [Glycine max (L.) Merr.] cultivars across three locations. In Vitro Cellular and Developmental Biology Plant 2001, 37(67):62-67.
104 - Kita Y, Nishizawa K, Takahashi M, Kitayama M, Ishimoto M. Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines. Plant Cell Reports 2007, 26(4):439-447.
105 - Parrott WA, Hoffman LM, Hildebrand DF, Williams EG, Collins GB. Recovery of primary transformants of soybean. Plant Cell Reports 1989, 7:615-61.
106 - Hood EE, Gelvin SB, Melchers LS and Hoekema A. New Agrobacterium helper strains for gene transfer to plants. Transgenic Research 1993, 2:208-218.
107 - Torisky RS, Kovacs L, Avdiushko S, Newman JD, Hunt AG and Collins GB. Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens strain Chry5. Plant Cell Reports 1997, 17:102-108.
108 - Trick HN, Finer JJ. Sonication-assisted Agrobacterium-mediated transformation of soybean (Glycine max) embryogenic suspension culture tissue. Plant Cell Reports 1998, 17:482-488.
109 - Yan B, Reddy MSS, Collins GB, Dinkins RD. Agrobacterium tumefaciens mediated transformation of soybean [Glycine max (L) Merrill] using immature zygotic cotyledon explants. Plant Cell Reports 2000, 19(11):1090-1097.
110 - Ko TS, Korban SS. Enhancing the frequency of somatic embryogenesis following Agrobacterium-mediated transformation of immature cotyledons of soybean [Glycine max (L.) Merrill.]. In Vitro Cellular & Developmental BiologyPlant 2004, 40:552-558.
111 - Ko TS, Lee S, Farrand SK, Korban SS. A partially disarmed vir helper plasmid, pKYRT1, in conjunction with 2,4-dichlorophenoxyacetic acid promotes emergence of regenerable transgenic somatic embryos from immature cotyledons of soybean. Planta 2004, 218(4):536-541.
112 - Wiebke Strohm B, Droste A, Pasquali G, Osorio MB, Bucker Neto L, Passaglia LMP, Bencke M, Homrich MS, Margis Pinheiro M, Bodanese Zanettini MH. Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and Agrobacterium system. Euphytica 2011, 177(3):343-354.
113 - Hazel CB, Klein TM, Anis M, Wilde HD, Parrott WA. Growth characteristics and transformability of soybean embryogenic cultures. Plant Cell Reports 1998, 17:765-772.
114 - Maughan PJ, Philip R, Cho MJ, Widholm JM and Vodkin LO. Biolistic transformation, expression, and inheritance of bovine β-casein in soybean (Glycine max). In vitro Cellular and Developmental Biology Plant 1999, 35:334-349.
115 - Hadi MZ, McMullen MD, Finer JJ. Transformation of 12 different plasmids into soybean via particle bombardment. Plant Cell Reports 1996, 15:500-505.
116 - Santarem ER, Finer JJ. Transformation of soybean [Glycine max (L.) Merrill] using proliferative embryogenic tissue maintained on semisolid medium. In Vitro Cellular and Developmental Biology Plant 1999, 35 451-455.
117 - Dufourmantel N, Pelissier B, Garcon F, Peltier G, Jean-Marc F, Tissot G. Generation of fertile transplastomic soybean. Plant Molecular Biology 2004, 55 479-489.
118 - Rao SS, Hildebrand D. Changes in Oil Content of Transgenic Soybeans Expressing the Yeast SLC1 Gene. Lipids 2009, 44:945-951.
119 - Chen R, Matsui K, Ogawa M, Oe M, Ochiai M, Kawashima H, Sakuradani E, Shimizu S, Ishimoto M, Hayashi M, Murooka Y, Tanaka Y. Expression of Delta 6, Delta 5 desaturase and GLELO elongase genes from Mortierella alpina for production of arachidonic acid in soybean [Glycine max (L.) Merrill] seeds. Plant Science 2006, 170:399-406.
120 - Tougou M, Furutani N, Yamagishi N, Shizukawa Y, Takahata Y, Hidaka S. Development of resistant transgenic soybeans with inverted repeat-coat protein genes of soybean dwarf virus. Plant Cell Reports 2006, 25:1213-1218.
121 - Furutani N, Yamagishi N, Hidaka S, Shizukawa Y, Kanematsu S, Kosaka Y. Soybean mosaic virus resistance in transgenic soybean caused by post-transcriptional gene silencing. Breed Science 2007, 57:123-128.
122 - Ishimoto M, Rahman SM, Hanafy MS, Khalafalla MM, El-Shemy HA, Nakamoto Y, Kita Y, Takanashi K, Matsuda F. Murano Y, Funabashi T, Miyagawa H, Wakasa K. Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation. Molecular Breeding 2010, 25:313-326.
123 - Tavva VS, Kim YH, Kagan IA, Dinkins RD, Kim KH and Collins GB. Increased α-tocopherol content in soybean seed over- expressing the Perilla frutescens γ-tocopherol methyltransferase gene. Plant Cell Reports 2007, 26:61-70.
124 - Li Z, Meyer S, Essig JS, Liu Y, Schapaugh MA, Muthukrishnan S, Hainline BE,. Trick HN. High-level expression of maize γ-zein protein in transgenic soybean (Glycine max) Molecular Breeding 2005, 16:11-20.
125 - Dinkins RD, Srinivasa Reddy MS, Meurer CA, Yan B, Trick HN, Finer JJ, Thibaud-Nissen F, Parrott WA and Collins GB. Increased sulfur amino acids in soybean plants overexpressing the maize 15 kDa zein protein. In vitro Cellular and Developmental Biology Plant 2001, 37:742-747.
126 - Schnell JA, Treyvaud-Amiguet V, Arnason JT, Johnson DA. Expression of polyhydroxybutyric acid as a model for metabolic engineering of soybean seed coats. Transgenic Research 2012, 21(4):895-899.
127 - Luo XM, Zhao GL, Jian YY. Plant regeneration from protoplasts of soybean (Glycine max L.). Acta Botanica Sinica 1990, 32:616-621.
128 - Yeh MS. In vitro culture of immature soybean embryos II. The abilities of organogenesis and plantlet regeneration from different aged immature embryo in Glycine species. Journal of the Agricultural Association of China 1990, 39:73-87.
129 - Dhir SK, Dhir S, Widholm JM. Plantlet regeneration from immature cotyledon protoplasts of soybean (Glycine max L.). Plant Cell Reports 1991, 10:39-43.
130 - Sato S, Newell C, Kolacz K, Tredo L, Finer JJ, Hinchee M. Stable transformation via particle bombardment in two different soybean regeneration systems. Plant Cell Reports 1993, 12(7-8):408- 413.
131 - Di R, Purcell V, Collins GB, Ghabrial SA. Production of transgenic soybean lines expressing the bean pod mottle virus coat protein precursor gene. Plant Cell Reports 1996, 15:746-750.
132 - Xing A, Zhang Z, Sato S, Staswick PE, Clemente TE. The use of the two T-DNA binary system to derive marker-free transgenic soybeans. In Vitro Cellular and Developmental Biology Plant 2000, 36:456-463.
133 - Wang X, Eggenberger AL, Nutter Jr FW, Hill JH. Pathogen-derived transgenic resistance to soybean mosaic virus in soybean. Molecular Breeding 2001, 8:119-127.
134 - Zeng P, Vadnais DA, Zhang Z, Polacco JC. Refined glufosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill]. Plant Cell Reports 2004. 22:478-482.
135 - Miklos JA, Alibhai MF, Bledig SA, Connor Ward DC, Gao AG, Holmes BA, Kolacz KH, Kabuye VT, MacRae TC, Paradise MS, Toedebusch AS, Harrison LA. Characterization of soybean exhibiting high expression of a synthetic Bacillus thuringiensis cry1A transgene that confers a high degree of resistance to Lepidopteran pests. Crop Science 2007, 47:148-157.
136 - Liu SJ Wei ZM, Huang JQ. The effect of co-cultivation and selection parameters on Agrobacterium-mediated transformation of Chinese soybean varieties. Plant Cell Reports 2008, 27(3):489-498.
137 - Lee K, Yi BY, Kim KH, Kim JB, Suh SC, Woo HJ, Shin KS, Kweon SJ. Development of Efficient Transformation Protocol for Soybean (Glycine max L.) and Characterization of Transgene Expression after Agrobacterium-mediated Gene Transfer. Journal of the Korean Society for Applied Biological Chemistry 2011, 54:37-45.
138 - Lazzeri PA, Hilderbrand DF, Collins GB. A procedure for plant regeneration from immature cotyledon tissue of soybean. Plant Molecular Biology Reporter 1985, 3(4):160-167.
139 - Ranch JP, Ogelsby L, Zielinski AC. Plant regeneration from embryo-derived tissue cultures of soybean. In Vitro Cellular and Developmental Biology Plant 1985, 21:653-658.
140 - Ghazi TD, Cheema HV, Nabors MW. Somatic embryogenesis and plant regeneration from embryonic callus of soybean [Glycine max (L.) Merr.]. Plant Cell Reports 1986, 5(6):452-456.
141 - Kerns HR, Barwale VB, Meyer MM. Correlation of cotyledonary node shoot proliferation and somatic embryoid development in suspension cultures of soybean [Glycine max (L.) Merr.]. Plant Cell Reports 1986, 5(2), 140-143.
142 - Hartweck LM, Lazzeri PA, Cui D, Collins GB, Williams EG. Auxin orientation effects on somatic embryogenesis from immature soybean cotyledons. In Vitro Cellular and Developmental Biology Plant 1988, 24(8):821-828.
143 - Komatsuda T, Ohyama K. Genotype of high competence for somatic embryogenesis and plant regeneration in soybean Glycine max. Theoretical and Applied Genetics 1988, 75(5):695-700.
144 - Parrott WA, Williams EG, Hildebrand DF, Collins GB. Effect of genotype on somatic embryogenesis from immature cotyledons of soybean. Plant Cell, Tissue and Organ Culture 1989, 16(1):15-21.
145 - Christou, P, Yang NS. Developmental aspects of soybean (Glycine max) somatic embryogenesis. Annals of Botany 1989, 64(2):225-234.
146 - Komatsuda T, Ko SW. Screening of soybean (Glycine max (L.) Merrill) genotypes for somatic embryo production from immature embryo. Japanese Journal of Breeding 1990, 40:249-251.
147 - Komatsuda T, Kanebo K, Oka S. Genotype × sucrose interactions for somatic embryogenesis in soybean. Crop Science 1991, 31(2):333-337.
148 - Wright MS, Launis KL, Novitzky R, Duesiing JH, Harms CT. A simple method for the recovery of multiple fertile plants from individual somatic embryos of soybean [Glycine max (L.) Merrill]. In Vitro Cellular and Developmental Biology Plant 1991, 27:153-157.
149 - Lippmann B, Lippmann G. Soybean embryo culture: factors influencing plant recovery from isolated embryos. Plant Cell, Tissue and Organ Culture 1993, 32(1):83-90.
150 - Parrott WA, All JN, Adang MJ, Bailey MA, Boerma HR, Stewart CN Jr. Recovery and evaluation of soybean plants transgenic for a Bacillus thuringiensis var.kurstaki insecticidal gene. In Vitro Cellular and Development Biology Plant 1994, 30:144-149.
151 - Saravitz CH, Raper CDJr. Responses to sucrose and glutamine by soybean embryos grown in vitro. Physiologia Plantarum 1995, 93:799-805.
152 - Liu W, Torisky RS, McAllister KP, Avdiushko S, Hildebrand D, Collins GB. Somatic embryo cycling: evaluation of a novel transformation and assay system for seed-specific gene expression in soybean. Plant Cell, Tissue and Organ Culture 1996, 47:33-42.
153 - Rajasekaran K, Pellow JW. Somatic embryogenesis from cultured epicotyls and primary leaves of soybean [Glycine max (L.) Merrill]. In Vitro Cellular and Developmental Biology Plant 1997, 33:88-91.
154 - Samoylov VM, Tucker DM, Parrott WA. A liquid medium-based rapid regeneration from embryogenic soybean cultures. Plant Cell Reports 1998, 18 49-54.
155 - Chanprame S, Kuo TM, Widholm AM. Soluble carbohydrate content of soybean [Glycine max (L.) Merr.] somatic and zygotic embryos during development. In Vitro Cellular and Developmental Biology Plant 1998, 34:64-68.
156 - Ponappa T, Brzozowski AE, Finer JJ. Transient expression and stable transformation of soybean using jellyfish green fluorescent protein. Plant Cell Reports 1999, 19:6-12.
157 - Tian L, Brown DCW. Improvement of soybean somatic embryo development and maturation by abscisic acid treatment. Canadian Journal of Plant Science 2000. 80:721-276.
158 - Walker DR, All JN, McPherson RM, Boerma HR, Parrott WA. Field Evaluation of Soybean Engineered with a Synthetic cry1Ac Transgene for Resistance to Corn Earworm, Soybean Looper, Velvetbean Caterpillar (Lepidoptera: Noctuidae), and Lesser Cornstalk Borer (Lepidoptera: Pyralidae). Journal of Economic Entomology, 2000, 93(3) 613-622.
159 - Droste A, Pasquali G, Bodanese-Zanettini MH. Integrated bombardment and Agrobacterium transformation system: an alternative method for soybean transformation. Plant Molecular Biology Reporter 2000, 18:51-59.
160 - Droste A, Leite PCP, Pasquali G, Mundstock EC, Bodanese-Zanettini MH. Regeneration of soybean via embryogenic suspension culture. Scientia Agricola 2001, 58(4):753-758.
161 - Fernando JA, Vieira MLC, Geraldi IO, Appezzato-da-Gloria B. Anatomical study of somatic embryogenesis in Glycine max (L.) Merrill. Brazilian Archives of Biology and Technology 2002, 45 (3):277-286.
162 - Tomlin ES, Branch SR, Chamberlain D, Gabe H, Wright MS, Stewart CN Jr. Screening of soybean, Glycine max (L.) Merrill, lines for somatic embryo induction and maturation capability from immature cotyledons. In Vitro Cellular and Developmental Biology Plant 2002, 38:543-548.
163 - Chiera JM, Finer JJ, Grabau EA. Ectopic expression of a soybean phytase in developing seeds of Glycine max to improve phosphorus availability. Plant Molecular Biology 2004, 56:895-904.
164 - Schmidt MA, Tucker DM, Cahoon EB, Parrott WA. Towards normalization of soybean somatic embryo maturation. Plant Cell Reports 2005, 24:383- 391.
165 - Santos KGB, Mariath JEA, Moco MCC, Bodanese Zanettini MH. Somatic Embryogenesis from Immature Cotyledons of Soybean (Glycine max (L.) Merr.): Ontogeny of Somatic Embryos. Brazilian Archives of Biology and Technology 2006, 49 (1):49-55.
166 - Wiebke B, Ferreira F, Pasquali G, Bodanese Zanettini MH, Droste A. Influence of antibiotics on embryogenic tissue and Agrobacterium tumefaciens suppression in soybean genetic transformation. Bragantia 2006, 65(4):543-551.
167 - Chiera JM, Streeter JG, Finer JJ. Ononitol and pinitol production in transgenic soybean containing the inositol methyl transferase gene from Mesembryanthemum crystallinum. Plant Science 2006. 171:647-654.
168 - Tougou M, Yamagishi N, Furutani N, Shizukawa Y, Takahata Y, Hidaka S. Soybean dwarf virus-resistant transgenic soybeans with the sense coat protein gene. Plant Cell Reports 2007, 26:1967-1975.
169 - El-Shemy H A, Khalafalla MM, Fujita K, Ishimoto M. Improvement of protein quality in transgenic soybean plants. Biologia Plantarum 2007, 51:277-284.
170 - Weber RLM, Korber AP, Baldasso DA, Callegari Jacques SM, Bodanese Zanettini MH, Droste A. Beneficial effect of abscisic acid on soybean somatic embryo maturation and conversion into plants. Plant Cell Culture and Micropropagation 2007, 3(1):1-9.
171 - Yemets AI, Radchuk VV, Pakhomov AV, Blume Ya B. Biolistic Transformation of Soybean Using a New Selectable Marker Gene Conferring Resistance to Dinitroanilines. Cytology and Genetics 2008, 42( 6):413-419.
172 - Schmidt MA, LaFayette PR, Artelt BA, Parrott WA. A comparison of strategies for transformation with multiple genes via microprojectile-mediated bombardment. In Vitro Cellular and Developmental Biology Plant 2008, 44(3):162-168.
173 - Loganathan M Maruthasalam S, Shiu LY, Lien WC, Hsu WH, Lee PF, Yu CW, Lin CH. Regeneration of soybean (Glycine max L. Merrill) through direct somatic embryogenesis from the immature embryonic shoot tip. In Vitro Cellular and Developmental Biology Plant 2010, 46:265-273.
174 - Droste A, Silva da AM, Souza de IF, Wiebke‑Strohm, Bucker Neto L, Bencke M, Sauner MV, Bodanese‑Zanettin MH. Screening of Brazilian soybean genotypes with high potential for somatic embryogenesis and plant regeneration. Pesquisa Agropecuaria Brasileira 2010, 45(7):715-720.
175 - He Y, Young TE, Clark KR, Kleppinger-Sparace K F, Bridges WC, Sparace SA. Developmental profile of storage reserve accumulation in soybean somatic embryos. In Vitro Cellular and Developmental Biology Plant 2011, 47:725-733.
176 - Mariashibu TS, Subramanyam K, Arun M, Mayavan S, Rajesh M, Theboral J, Manickavasagam M, Ganapathi A. Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars. Acta Physiologiae Plantarum 2012, (published online) DOI 10.1007/s11738-012-1046-3.