**3***. Agrobacterium-***mediated transformation of soybean**

#### **3.1.** *Agrobacterium-***mediated transformation mechanism**

*Agrobacterium* is a unique organism to generate transgenic plants and in natural conditions [41]. It allows introduction of a single stranded copy of the bacterial transferred DNA (T-DNA) into a host cell and integration of the genomic DNA of interest, resulting in genetic manipula‐ tion of the host. Since the development of disarmed tumour-inducing (Ti) plasmid [42-43], *Agrobacterium* has been used to transform various major crops for genetic modification [44-46].

*Agrobacterium* recognizes wounded host plant cells which produce penolic compounds such as acetosyringone as inducers of *vir* gene expression [47], and attach to the plant cells to ex‐ port the T-DNA after virulence (Vir) protein activation. Acetosyringone is now routinely used for improving transformation efficiency. After *vir* gene activation, a single stranded T-DNA copy (T-strand) is transferred into the plant by type IV secretion system (T4SS) which is related to VirB complex [48]. The VirB complex is composed of at least 12 proteins (VirB1-11 and VirD4) which form a multisubunit envelope-spanning structure [49]. Various

*Agrobacterium* proteins, such as VirD2-T-DNA, VirE2, VirE3, VirF, and VirD5, pass though VirB complex to transfer into plant cells [50-51]. VirE2 and VirD2 interact with cytosolic T-DNA in the plant cells and form a complex which is later imported into the nucleus when it is bound to VIP1 plant protein [52-55]. Recently, Gelvin et al., hypothesized that T-complex (T-DNA, VirE2, VirD2 and VIP1) is imported into the nucleus through actin cytoskeleton and thus myosin may be involved in *Agrobacterium*-mediated transformation [56]. However, the specific mechanism of T-DNA movement through myosin is still unknown.

The T-complex is imported into the nucleus by the phosphorylation of VirE2 Interacting Protein 1 (VIP1), induced by mitogen-activated protein kinase (MAPK), such as MPK3 [55]. After T-complex is imported into the host nucleus, VirE2 and VIP1 need to be degraded be‐ fore T-DNA integration by a subunit of the SCF (SKP-CUL1-F-box protein) ubiquitin E3 li‐ gase complex. Not only *Agrobacterium* protein VirF but also protein VBF can mark VIP1 protein for the degradation. Furthermore, binding of VIP1-binding F-box (VBF) to T-com‐ plex can induce the degradation of VIP1 and VirE2 by the 26S proteosome, and at the end Tstrand is integrated into plant genomic DNA and expressed in the host plants [57-58].

### **3.2. History of** *Agrobacterium-***mediated soybean transformation research**

Among various transformation technologies, *Agrobacterium*-mediated transformation meth‐ od has shown to be effective for the production of transgenic soybeans because of straight‐ forward methodology, familiarity to researchers, minimal equipment cost, and reliable insertion of a single transgene or a low copy number [13]. Till now, a number of reports have been published related to the optimum condition to achieve a high yield of soybean transformation; such as *Agrobacterium* inoculation conditions, regeneration media compo‐ nents, etc. For *Agrobacterium*-mediated transformation methods, the susceptibility of soy‐ bean to *Agrobacterium* and various *Agrobacterium* strains have been tested to improve the transformation efficiency (Table 1). Also, *Agrobacterium* strains and growth conditions which affect the soybean transformation efficiency have been published [8, 59-62]. After Pederson et al., [46] and Owens et al., [59] showed the susceptibility of certain soybean genotypes against tumor induction, *Agrobacterium* biology study has been advanced to enhance trans‐ formation efficiency. In addition to *Agrobacterium* biology study, chemical contents for inoc‐ ulation have been studied such as varying acetosyringone and syringaldehyde concentrations [63]. For high inoculation efficiency, Mauro et al., [64] tested various *Agrobac‐ terium* biotypes (nopaline, agropine and octopine) to identify the most effective *Agrobacteri‐ um* biotype for soybean transformation.

After Hinchee et al., [8] developed *Agrobacterium*-mediated soybean transformation meth‐ ods, many *Agrobacterium* strains have been tested and employed, such as EHA101, EHA105, LBA4404 and AGL1. Parrott et al., [33] showed that EHA101 was highly potent to transform immature soybean cotyledons, especially PI283332, and had higher recovery of transformed plants over LBA4404. Dang and Wei [65] tested transformation efficiency using embryonic tips instead of cotyledonary explants and somatic embryos, and when embryogenic tips were infected for 20 hours, hypervirulent strain KYRT1 showed increased efficiency over EHA105 and LBA4404.

Recently, *A. tumefaciens* KAT23 (AT96-6) which has an ability to efficiently transfer the T-DNA into soybean, was isolated from peach root. After 20 stains were confirmed by com‐ mon bean and soybean transformation, Yukawa et al. [66] tested their potential availability as legume super virulent *A. tumefaciens* in various soybean cultivars (Peking, Suzuyutaka, Fayette, Enrei, Mikawashima, WaseMidori, Jack, Leculus, Morocco, Serena, Kentucky Won‐ der and Minidoka). Without modifying vectors or *vir* function, they showed that KAT23 (AT96-6) has a high potential to function as a common strain to increase soybean transfor‐ mation efficiency. Therefore, this study identified a novel soybean super virulent *A. tumefa‐ ciens* strain which transferred not only the T-DNA of the Ti-plasmid but also introduced T-DNA of the binary vector efficiently. These results indicate that KAT23 (AT96-6) has the ability to transform soybeans at high efficiency.

There has been a significant improvement in soybean transformation over the past two deca‐ des. However, the efficiency of soybean transformation is not great enough for practical needs and shows high variation. Thus, considering the potential application of soybean transforma‐ tion, the importance of *Agrobacterium* can't be over-emphasized.


**Table 1.** Summary of cotyledonary-node transformation system.

*Agrobacterium* proteins, such as VirD2-T-DNA, VirE2, VirE3, VirF, and VirD5, pass though VirB complex to transfer into plant cells [50-51]. VirE2 and VirD2 interact with cytosolic T-DNA in the plant cells and form a complex which is later imported into the nucleus when it is bound to VIP1 plant protein [52-55]. Recently, Gelvin et al., hypothesized that T-complex (T-DNA, VirE2, VirD2 and VIP1) is imported into the nucleus through actin cytoskeleton and thus myosin may be involved in *Agrobacterium*-mediated transformation [56]. However,

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

The T-complex is imported into the nucleus by the phosphorylation of VirE2 Interacting Protein 1 (VIP1), induced by mitogen-activated protein kinase (MAPK), such as MPK3 [55]. After T-complex is imported into the host nucleus, VirE2 and VIP1 need to be degraded be‐ fore T-DNA integration by a subunit of the SCF (SKP-CUL1-F-box protein) ubiquitin E3 li‐ gase complex. Not only *Agrobacterium* protein VirF but also protein VBF can mark VIP1 protein for the degradation. Furthermore, binding of VIP1-binding F-box (VBF) to T-com‐ plex can induce the degradation of VIP1 and VirE2 by the 26S proteosome, and at the end Tstrand is integrated into plant genomic DNA and expressed in the host plants [57-58].

Among various transformation technologies, *Agrobacterium*-mediated transformation meth‐ od has shown to be effective for the production of transgenic soybeans because of straight‐ forward methodology, familiarity to researchers, minimal equipment cost, and reliable insertion of a single transgene or a low copy number [13]. Till now, a number of reports have been published related to the optimum condition to achieve a high yield of soybean transformation; such as *Agrobacterium* inoculation conditions, regeneration media compo‐ nents, etc. For *Agrobacterium*-mediated transformation methods, the susceptibility of soy‐ bean to *Agrobacterium* and various *Agrobacterium* strains have been tested to improve the transformation efficiency (Table 1). Also, *Agrobacterium* strains and growth conditions which affect the soybean transformation efficiency have been published [8, 59-62]. After Pederson et al., [46] and Owens et al., [59] showed the susceptibility of certain soybean genotypes against tumor induction, *Agrobacterium* biology study has been advanced to enhance trans‐ formation efficiency. In addition to *Agrobacterium* biology study, chemical contents for inoc‐ ulation have been studied such as varying acetosyringone and syringaldehyde concentrations [63]. For high inoculation efficiency, Mauro et al., [64] tested various *Agrobac‐ terium* biotypes (nopaline, agropine and octopine) to identify the most effective *Agrobacteri‐*

After Hinchee et al., [8] developed *Agrobacterium*-mediated soybean transformation meth‐ ods, many *Agrobacterium* strains have been tested and employed, such as EHA101, EHA105, LBA4404 and AGL1. Parrott et al., [33] showed that EHA101 was highly potent to transform immature soybean cotyledons, especially PI283332, and had higher recovery of transformed plants over LBA4404. Dang and Wei [65] tested transformation efficiency using embryonic tips instead of cotyledonary explants and somatic embryos, and when embryogenic tips were infected for 20 hours, hypervirulent strain KYRT1 showed increased efficiency over

the specific mechanism of T-DNA movement through myosin is still unknown.

**3.2. History of** *Agrobacterium-***mediated soybean transformation research**

*um* biotype for soybean transformation.

EHA105 and LBA4404.

Relationships

494

### **4. New directions of soybean genetic engineering, skills and vectors**

To date, the *Agrobacterium*- and biolistic-mediated transformation methods remain the very successful methods in soybean transformation, whereas other available transformation tech‐ nologies have not been practical in soybean, which include electroporation-mediated trans‐ formation [72], PEG/liposome-mediated transformation [73], silicon carbide-mediated

transformation [74], microinjection [75] and chloroplast-mediated transformation [76]. Of these two, *Agrobacterium*-mediated transformation has become more adapted in public labo‐ ratories worldwide. On the other hand, there are unintended insertions such as unwanted antibiotic markers and promoters, which can be inserted during transformation. This prob‐ lem has raised potential biosafety issues related to environmental concerns and human health risks. To overcome these potential risks, methods of developing marker free trans‐ genic plants have been developed, such as cotransformation [77], transposon-mediated transformation [78] and site-specific recombination [79].

Among the various methods, co-transformation system is one of the most commonly used methods to produce marker free transgenic plants. In co-transformation systems, a marker gene and genes of interest are placed on separate DNA molecules and introduced into plant genomes. Then, the non-selectable genes segregate from the marker gene in the progeny generations. Most strains of *A. tumefaciens* have the ability to contain more than one T-DNA, and crown gall tumors were often co-transformed with multiple T-DNAs [42]. As a result, there are two possibilities; Multiple T-DNAs were delivered into plant cells either from a mixture of strains ('mixture methods') or from a single strain ('single-strain methods'). De‐ picker et al. [80] described that a single strain method was higher in efficiency than a mix‐ ture method. For a single-strain method of co-transformation, Kamori et al., [77] tested cotransformation method to develop a suitable superbinary vector system. Using the unique plasmids which carried two T-DNA segments marker free rice and tobacco were produced and evaluated. LBA4404, a derivative of an octopine strain, were used for these co-transfor‐ mation methods and they hypothesized that LBA4404 may be an important factor contribu‐ ting to the high frequency of unlinked loci.

To improve plant genetic traits, many soybean research labs have developed tools for soy‐ bean functional genomics, such as several libraries containing large inserts of bacterial artifi‐ cial chromosome (BAC) and plant transformation competent binary plasmids clone (BIBAC) (81). In functional genomic research, bacterial artificial chromosome (BAC) is a single copy artificial chromosome vector and is based on the *E. coli* fertility (F-factor) plasmid. They are not only stable in host cell, but also are used for large scale gene cloning and discovery [82]. However, some BAC libraries that are desirable for functional genomics are often not ame‐ nable for transformation directly into plants because of their large subclones. Therefore, bi‐ nary bacterial artificial chromosome (BIBAC) libraries have been developed for *Agrobacterium*-mediated plant transformation and gene functional complementation. The BI‐ BAC library is based on BAC vector and has both an F-factor plasmid for replication origin of *E.coli* and an Ri plasmid for replication origin of *Agrobacterium rhizogenes*. The vector also has a *sacB* gene as a positive selection for *E. coli* and a selectable marker gene for plant. Since BIBAC vectors were reported, these vectors have been used for plant transformation in some model plant species including tobacco, canola, tomato, and rice [83-86]. Although the transformation efficiency was very low, the BIBAC vectors have been successfully employed to transfer large inserts into those crops as a single locus via *Agrobacterium*-mediated trans‐ formation. The introduced T-DNA was stably maintained and inherited through several generations and no gene silencing was observed [85]. However, soybean transformation us‐ ing a BIBAC vector has not been achieved to date. Moreover, plant transformation with DNA fragments below 20 kb is routine whereas the stable plant transformation with DNA fragments larger than 50 kb is challenging.

transformation [74], microinjection [75] and chloroplast-mediated transformation [76]. Of these two, *Agrobacterium*-mediated transformation has become more adapted in public labo‐ ratories worldwide. On the other hand, there are unintended insertions such as unwanted antibiotic markers and promoters, which can be inserted during transformation. This prob‐ lem has raised potential biosafety issues related to environmental concerns and human health risks. To overcome these potential risks, methods of developing marker free trans‐ genic plants have been developed, such as cotransformation [77], transposon-mediated

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

Among the various methods, co-transformation system is one of the most commonly used methods to produce marker free transgenic plants. In co-transformation systems, a marker gene and genes of interest are placed on separate DNA molecules and introduced into plant genomes. Then, the non-selectable genes segregate from the marker gene in the progeny generations. Most strains of *A. tumefaciens* have the ability to contain more than one T-DNA, and crown gall tumors were often co-transformed with multiple T-DNAs [42]. As a result, there are two possibilities; Multiple T-DNAs were delivered into plant cells either from a mixture of strains ('mixture methods') or from a single strain ('single-strain methods'). De‐ picker et al. [80] described that a single strain method was higher in efficiency than a mix‐ ture method. For a single-strain method of co-transformation, Kamori et al., [77] tested cotransformation method to develop a suitable superbinary vector system. Using the unique plasmids which carried two T-DNA segments marker free rice and tobacco were produced and evaluated. LBA4404, a derivative of an octopine strain, were used for these co-transfor‐ mation methods and they hypothesized that LBA4404 may be an important factor contribu‐

To improve plant genetic traits, many soybean research labs have developed tools for soy‐ bean functional genomics, such as several libraries containing large inserts of bacterial artifi‐ cial chromosome (BAC) and plant transformation competent binary plasmids clone (BIBAC) (81). In functional genomic research, bacterial artificial chromosome (BAC) is a single copy artificial chromosome vector and is based on the *E. coli* fertility (F-factor) plasmid. They are not only stable in host cell, but also are used for large scale gene cloning and discovery [82]. However, some BAC libraries that are desirable for functional genomics are often not ame‐ nable for transformation directly into plants because of their large subclones. Therefore, bi‐ nary bacterial artificial chromosome (BIBAC) libraries have been developed for *Agrobacterium*-mediated plant transformation and gene functional complementation. The BI‐ BAC library is based on BAC vector and has both an F-factor plasmid for replication origin of *E.coli* and an Ri plasmid for replication origin of *Agrobacterium rhizogenes*. The vector also has a *sacB* gene as a positive selection for *E. coli* and a selectable marker gene for plant. Since BIBAC vectors were reported, these vectors have been used for plant transformation in some model plant species including tobacco, canola, tomato, and rice [83-86]. Although the transformation efficiency was very low, the BIBAC vectors have been successfully employed to transfer large inserts into those crops as a single locus via *Agrobacterium*-mediated trans‐ formation. The introduced T-DNA was stably maintained and inherited through several generations and no gene silencing was observed [85]. However, soybean transformation us‐

transformation [78] and site-specific recombination [79].

Relationships

496

ting to the high frequency of unlinked loci.
