**1. Introduction**

[43] Malatesta, M., Boraldi, F., Annovi, G., Baldelli, B., Battistelli, S., Biggiogera, M., &

[44] Cho, W. C. S. (2007). Proteomics technologies and challenges. *Genomics, Proteomics &*

soybean: effects on liver ageing. *Histochemistry and Cell Biology*, 130-967.

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

*Bioinformatics*, 5-77.

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412

Quaglino, D. (2008). A long-term study on female mice fed on a genetically modified

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 mil‐ lion 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 popu‐ lation 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-conglyci‐ nin (7S globulin) and glycinin (11S globulin). The oil portion of the seed is composed pri‐ marily 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 soy‐ bean 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 re‐ sistance 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 dif‐ ficult through conventional breeding. The technology of plant transformation is only moder‐ ately 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 ge‐ nomes, the transformation efficiency for soybean still remains low [9]. Since the first success‐ ful 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 cul‐ ture period and tends to produce multiple insertion events, while the latter is labour inten‐ sive and requires specially trained personnel to undertake the work [9]

For soybean *in vitro* regeneration, two principal methods have been identified: somatic em‐ bryogenesis 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 molecu‐ lar 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 efficien‐ cy 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.

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

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

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 soy‐ bean 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

The main objectives of soybean improvement include increase in yield, development of re‐ sistance 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 dif‐ ficult through conventional breeding. The technology of plant transformation is only moder‐ ately 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

Although numerous methods have been developed for introducing genes into plant ge‐ nomes, the transformation efficiency for soybean still remains low [9]. Since the first success‐ ful 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 cul‐ ture period and tends to produce multiple insertion events, while the latter is labour inten‐

For soybean *in vitro* regeneration, two principal methods have been identified: somatic em‐ bryogenesis 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 molecu‐ lar 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 efficien‐ cy 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.

sive and requires specially trained personnel to undertake the work [9]

osteoporosis [4].

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414

issue throughout the world [7].

and the wider application of genomics.




**Table 1.** Major landmarks in soybean organogenesis and transformation

**Year Explant tissue Major contribution Reference**

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

node transformation system

meristematic soybean cells

transformation frequency

soybean mosaic virus (SMV)

organogenic regeneration

transformation efficiency

2001 Cotyledonary node Increased *Agrobacterium* infection using L-cystine Olhoft and Somers, [16]

sclerotinia stem rot caused by *Sclerotinia*

regulators and sugars on regeneration from calli

based selection for increased transformation

source, selection agent and antioxidant on *Agrobacterium* mediated transformation efficiency

inactivating protein gene to enhance fungal

mediated transformation system

Shoot regeneration Franklin et al. [31]

in *Agrobacterium*-mediated transformation of

strategy to derive marker free transgenic soybean

agent for *Agrobacterium* mediated cotyledonary

Zhang et al. [61]

Xing et al. [132]

Clemente et al. [60]

Aragao et al. [47]

Olhoft et al. [56]

Wang et al. [133]

Donaldson et al. [65]

Reichert et al. [41]

Sairam et al. [1]

Olhoft et al. [57]

Zeng et al. [134]

Paz et al. [15]

Li et al. [6]

Liu et al. [35]

1999 Cotyledonary node Assessed the use of glufosinate as a selective agent

soybean

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416

2000 Cotyledonary node *Agrobacterium* two T-DNA binary system as a

2000 Cotyledonary node Evaluated the effect of glyphosate as a selective

2000 Embryonic axes Used of Imazapyr as selection agent for selection of

2001 Cotyledonary node Investigated the use of thiol compound to increase

2001 Cotyledonary node Developed transgenic soybean plants resistant to

2001 Cotyledonary node Expressed oxalate oxidase gene for resistant to

2003 Hypocotyl Screened soybean genotype for adventitious

2003 Cotyledonary node Assessed the effect of genotype, plant growth

2003 Cotyledonary node Used mixture of thiol compounds and hygromycin

efficiency

2004 Cotyledonary node Investigated the effect of seed vigor of explant

2004 Cotyledonary node Transferred chitinase gene and the barley ribosome-

resistance

2004 Embryonic tip Established regeneration and *Agrobacterium*

2004 Mature and immature cotyledon

2004 Cotyledonary node Assessed glufosinate selection for increased

*sclerotiorum*


**Explant Tissue Year Major Contribution Reference**

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Immature cotyledon 1984 Somatic embryo Induction Lippmann & Lippmann, [84]

1986 Somatic embryogenesis from callus Ghazi et al. [140]

Immature cotyledon 1985 Plant regeneration *via* somatic embryogenesis Lazzeri et al. [138]

Hypocotyl and cotyledon 1986 Embryoids development in suspension culture Kerns et al. [141]

1987 Investigated the effect of nutritional, physical, and chemical factors on somatic embryogenesis

explant on somatic embryogenesis

concentration of auxin on somatic embryo

sucrose during somatic embryogenesis

embryogenic suspension culture

somatic embryo formation.

conversion into plantlets

*hpt* gene *via* particle bombardment

been improved by reducing the exposure to auxin

gene by agrobacterium mediated transformation

Christianson et al.[77]

Ranch et al. [139]

Lazzeri et al. [85]

Lazzeri et al. [86]

Parrott et al. [87]

Finer, [79]

Parrott et al. [144]

Parrott et al. [105]

Buchheim et al. [94]

Christou and Yang, [145]

Komatsuda et al. [146]

Finer and McMullen., [64]

Finer and Nagasawa, [82]

[143]

Hartweck et al. [142]

Komatsuda and Ohyama,

Embryonic axes 1983 Embryoids development and plant regeneration

Immature embryo 1985 Somatic embryogenesis and assessment of

Immature embryo, cotyledon and, hypocotyl from germinating seedling

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418

Immature embryo and

cotyledon

*via* suspension culture

genotypic variation

Immature cotyledon 1988 Investigated the effect of auxin and orientation of

Immature cotyledon 1988 Analysed genotype dependency and High

induction

Immature cotyledon 1988 Investigated the interaction between auxin and

Immature cotyledon 1988 Germination frequency of somatic embryo has

Immature cotyledon 1988 Histological analysis to investigate secondary

embryogenesis

Immature cotyledon 1989 Developed primary transformants expressing zein

Immature cotyledon 1989 Investigated the developmental aspects of somatic embryogenesis

Immature cotyledon 1990 Screened soybean genotypes for somatic embryo production

Immature cotyledon 1991 Transformed embryogenic cultures with *gus* and

Immature cotyledon 1989 Demonstrated the effect of genotype on

Immature cotyledon 1989 Assayed somatic embryo maturation for

Immature cotyledon 1988 Developed rapid growing maintainable



**Explant Tissue Year Major Contribution Reference**

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

mediated transformation of soybean immature

mediated transformation of embryogenic

cultures by modifying sucrose and nitrogen

histodifferentiation of embryogenic cultures

somatic and zygotic embryo during development.

prolific embryogenic cultures using bombardment

culture with the aim to improve transformation

embryo development from immature cotyledons.

expressing a synthetic cry1Ac gene from *Bacillus thuringiensis* for resistance to variety of insects

soybean somatic embryo germination and

suspension culture tissue

content in medium

Santarem et al.[48]

Trick and Finer, [108]

Samoylov et al. [89]

Samoylov et al. [154]

Chanprame et al. [155]

Santarem and Finer, [116]

Maughan et al. [114]

Ponappa et al. [156]

Tian and Brown, [157]

Bonacin et al. [99]

Yan et al. [109]

Buchheim et al. [94]

Walker et al. [158]

Walker and Parrott, [90]

[97]

Simmonds and Donaldson,

Immature cotyledon 1998 Established sonication-assisted A*grobacterium*

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420

cotyledon

Immature cotyledon 1998 Established sonication-assisted Agrobacterium

Immature cotyledon 1998 Improved proliferation efficiency of embryogenic

Immature cotyledon 1998 Studied soluble carbohydrate content in soybean

Immature cotyledon 1999 Studied the factors influencing transformation of

Immature cotyledons 1999 Developed transgenic plants with bovine milk protein, β-casein

Immature cotyledons 1999 Transformed GFP into embryogenic suspension

Immature cotyledons 2000 Improved somatic embryo development and

Immature cotyledons 2000 Studied physical factors influencing somatic

Immature cotyledon 2000 Investigated the factors affecting *Agrobacterium*

Immature cotyledon 1989 Investigated maturation of somatic embryo for

Immature cotyledon 2000 Developed and evaluated transgenic soybean

Immature cotyledon 2001 Effect of polyethylene glycol and sugar alcohols on

conversion

embryogenesis

Immature cotyledon 2000 Screened genotypes for proliferative

and regeneration strategy

maturation by application of ABA

mediated transformation soybean

efficient conversion into plantlets

Immature cotyledon 1998 Developed liquid medium based system for




**Table 2.** Major landmarks in soybean somatic embryogenesis and transformation

### **2. Organogenesis and transformation**

**Explant Tissue Year Major Contribution Reference**

embryogenic cultures and *Agrobacterium tumefaciens* suppression in soybean

cultivars by back cross with a highly regenerable

transformed with modified Gy1 proglycinin gene with a synthetic DNA encoding four continuous

production of ononitol and pinitol

Santos et al. [165]

Tougou et al. [120]

Wiebke et al. [166]

Chiera et al. [167]

Tougou et al. [168]

Kita et al. [104]

Hiraga et al. [102]

Tavva et al. [123]

EI-Shemy et al. [169]

Weber et al. [170]

Furutani et al. [121]

Yemets et al. [171]

Schmidt et al. [172]

Rao and Hildebrand, [118]

Yang et al. [98]

Immature cotyledon 2006 Improved fatty acid content Chen et al. [119]

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Immature cotyledon 2006 Investigated the ontogeny of somatic

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422

embryogenesis

transformation

Somatic embryo 2006 Developed transgenic soybean resistance to dwarf virus

Immature cotyledon 2006 Investigated the influence of antibiotics on

Immature cotyledon 2006 Developed transgenic soybean for increased

Immature cotyledon 2007 Developed transgenic soybean resistant to dwarf virus

Immature cotyledon 2007 Improved somatic embryogenesis in recalcitrant

cultivar Jack

Immature cotyledon 2007 Soybean seed over expressing the *Perilla frutescens*

methionines.

soybean mosaic virus

resistance to Dinitroanilines

Immature cotyledon 2007 Analysed the effect of Abscisic acid on somatic

Immature cotyledon 2007 Developed transgenic soybean resistance to

Immature cotyledon 2008 Used a new Selectable Marker Gene Conferring

Immature cotyledon 2008 Developed strategy for transfer of multiple genes

Immature cotyledon 2009 Assessed the effect mannitol, abscisic acid and

Somatic embryo 2009 Developed transgenic soybean with increased oil content

soybean cultivars

Immature cotyledon 2007 Improved protein quality in transgenic soybean

somatic embryogenesis

γ -tocopherol methyltransferase gene

embryo maturation and conversion.

*via* micro projectile-mediated bombardment

explant age on somatic embryogenesis in Chinese

Immature cotyledon 2007 Evaluated Japanese soybean genotypes for

Organogenesis is characterized by the production of a unipolar bud primordium with sub‐ sequent development of the primordium into a leafy vegetative shoot. A successful plant re‐ generation 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 regenera‐ tion by organogenesis in soybean was first reported by Kimball and Bingham, [13] from hy‐ pocotyl sections followed by Cheng et al.[14] by culturing seedling cotyledonary node segments. Transfer of T-DNA into cotyledonary node cells by *Agrobacterium* mediated trans‐ formation 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. Soy‐ bean transformation efficiency has been improved by optimizing the selection system, en‐ hancing 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*. Tis‐ sue culture responses are greatly influenced by three main factors viz. whole plant physiolo‐ gy 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] re‐ searchers have used different parts of the soybean plant as explants for successful shoot mor‐ phogenesis 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), hypoco‐ tyls [22,33,34], and embryo axes [25,35]. Plant regeneration *via* organogenesis from cotyledo‐ nary 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 orga‐ nogenesis 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 re‐ ported 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 prolifera‐ tion of meristems. Use of pre-existing shoot meristems in transformation procedures can in‐ crease 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 adventi‐ tious shoots more efficiently than BA. Histological analysis of adventitious shoot regenera‐ tion 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 regula‐ tors and carbohydrates in the medium. Hong et al. [43] reported organogenic callus induc‐ tion 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**

**2.1. Organogenesis**

Relationships

424

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*. Tis‐ sue culture responses are greatly influenced by three main factors viz. whole plant physiolo‐ gy 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] re‐ searchers have used different parts of the soybean plant as explants for successful shoot mor‐ phogenesis 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), hypoco‐ tyls [22,33,34], and embryo axes [25,35]. Plant regeneration *via* organogenesis from cotyledo‐ nary 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

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

growth difficulties, which prevent the plant from being regeneration-competent[36].

[40], and ureide in the form of allantoin and amides [21].

regeneration efficiency was higher than for either cytokinin alone.

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 orga‐ nogenesis 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 re‐ ported 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

Adventitious shoot regeneration from cotyledonary node or leaf node is based on prolifera‐ tion of meristems. Use of pre-existing shoot meristems in transformation procedures can in‐ crease 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 adventi‐ tious shoots more efficiently than BA. Histological analysis of adventitious shoot regenera‐ tion 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

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 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 re‐ generation protocols. Genotype specificity for regeneration in soybean is well documented, although organogenesis is less genotype dependent and has become routine in several labo‐ ratories [18,20,28,29&33]. Reichert et al. [41] tested organogenic adventitious regeneration from hypocotyl explants excised from 18 genotypes. Plant formation from hypocotyl ex‐ plants showed that all genotypes were capable of producing elongated shoots that could be successfully rooted. This study confirmed the genotype independent nature of this organo‐ genic regeneration from the hypocotyl explant. Sairam et al. [1] developed an efficient geno‐ type 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 dif‐ ference in shoot bud formation among different genotypes, but there was significant differ‐ ence 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 cotyledonarynode. 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 sys‐ tem 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 re‐ mains low, apparently because of infrequent T-DNA delivery to cells in the cotyledonarynode axillary meristem, inefficient selection of transgenic cells that give rise to shoot

meristems, and low rates of transgenic shoot regeneration and plant establishment. The de‐ velopment of an effective Agrobacterium transformation method for soybean depends on several factors including plant genotype, explant vigor, Agrobacterium strain, vector, selec‐ tion 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 trans‐ formed plants. It has been reported that soybean genotype contributed to variation in sus‐ ceptibility 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 steriliza‐ tion 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 effi‐ ciency [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 soy‐ bean 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 soy‐ bean 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 tumefa‐ ciens* strain AGL1. Clemente et al. [60] successfully used and evaluated the effect of glypho‐ sate as a selective agent within the *Agrobacterium mediated* cotyledonary transformation system. Imazapyr is a herbicidal molecule that inhibits the enzymatic activity of acetohy‐ droxyacid 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 meristemat‐ ic 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 af‐ ter *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. Trans‐ forming meristems of soybean bu DNA coated gold particles followed by shoot regenera‐ tion 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 se‐ lection of plants that contained transgenic germ-line cells [32,62&63]. Shoot apex transfor‐ mation 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**

meristems, and low rates of transgenic shoot regeneration and plant establishment. The de‐ velopment of an effective Agrobacterium transformation method for soybean depends on several factors including plant genotype, explant vigor, Agrobacterium strain, vector, selec‐ tion 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 trans‐ formed plants. It has been reported that soybean genotype contributed to variation in sus‐ ceptibility 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 steriliza‐ tion 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 effi‐ ciency [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 soy‐ bean 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 soy‐ bean 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 tumefa‐ ciens* strain AGL1. Clemente et al. [60] successfully used and evaluated the effect of glypho‐ sate as a selective agent within the *Agrobacterium mediated* cotyledonary transformation system. Imazapyr is a herbicidal molecule that inhibits the enzymatic activity of acetohy‐ droxyacid 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 meristemat‐ ic 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 af‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

ter *Agrobacterium* transformation of cotyledonary node cells.

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. Trans‐ forming meristems of soybean bu DNA coated gold particles followed by shoot regenera‐ tion 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 se‐ lection of plants that contained transgenic germ-line cells [32,62&63]. Shoot apex transfor‐

**2.4. Particle bombardment**

Relationships

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Soybean has been improved by *Agrobacterium* mediated transformation followed by shoot regeneration. Wheat germin gene (gf-2.8) encoding an oligomeric protein and oxalate oxi‐ dase (*oxo*) genes were introduced into soybean to improve resistance to the oxalate-secret‐ ing pathogen *Sclerotina sclerotiorum* [65]. Li et al.[6] successfully utilized *Agrobacterium*mediated transformation to transfer chitinase gene (*chi*) and the barley ribosomeinactivating 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 *Brassi‐ ca campestris* into soybean cv.Jungery that produces methyl jasmonate and showed toler‐ ance 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 αand β-tocopherols by overexpressing γ-tocopherol methyltransferase from *Brassica napus* (BnTMT) [68]. Jiang et al. [69] transferred isoflavone synthase (IFS) gene into soybean cal‐ lus using *Agrobacterium*-mediated transformation and the transgenic plants produced in‐ creased levels of the secondary metabolite, isoflavone. Transgenic soybean plant containing PhyA gene of *Aspergillus ficuum* exhibited a lower amount of phytate in differ‐ ent 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 degrada‐ tion of phytate and potentially in turn mobilize more inorganic phosphate from phytate and thus reduce phosphate load on agricultural ecosystems [70].
