**2. Different approaches for soybean transformation**

In soybean transformation, two major methods are now widely utilized: *Agrobacterium-medi‐ ated* transformation of different explant tissues and particle bombardment. The *Agrobacteri‐ um*-mediated method, as a simple protocol, does not require any specific or expensive equipment. Moreover, this method usually produces single or low copy numbers of inser‐ tions with relatively rare rearrangement [13]. On the other hand, bombardment technique directly introduces desired genes into the target plant cell with small tungsten or gold parti‐ cles [9]. The success of this approach critically depends upon the ability of the target tissue to proliferate as well as proper pre-cultures to make a target plant.

### **2.1. Cotyledonary-node-based transformation**

The routine regeneration system was first reported by using the mature cotyledonary-node [14]. The multiple adventitious buds and shoots from explant tissues were proliferated and re‐ generated on culture media containing cytokinin by organogenesis. The transgenic soybean plants have been successfully and reproducibly produced using mature or immature cotyle‐ don explants via *Agrobacterium*-mediated transformation. Hinchee et al. [8] for the first time re‐ ported the production of fertile transgenic soybean plants using mature cotyledonary-node by *Agrobacterium*-mediated transformation, but transformation efficiency was very low. The sys‐ tem employed the neomycin phosphotransferase II (*NPT* II) gene as a selectable marker and combined kanamycin as a selective agent. However, this selection was addressed with a prob‐ lem of regeneration of non-transgenic or chimeric shoots at the shoot formation stage. More‐ over, the system was highly genotype-dependent. To overcome the high genotypedependency and high chimerism problems by the *NPT* II selection and develop a new selection system for soybean transformation, Zhang et al. [10] developed the selection system employ‐ ing herbicide bialaphos resistance (*bar*) gene as a selectable marker coupled with glufosinate as a selective agent. This system enabled to transform many soybean genotypes with stable trans‐ gene inheritance, albeit transformation efficiency remained to be improved. Meanwhile, to solve the escape problem caused by kanamycin selection, Clemete et al. [15] deployed the her‐ bicide glyphosate as a selective agent, leading to high stringent selection and good transgene inheritance. It was discovered later that addition of various thiol compounds in the co-cultiva‐ tion medium significantly increased the transformation efficiency [11, 16-17]. These thiol com‐ pounds, as antioxidants, reduce the oxidative burst that caused tissue browning or necrosis and also promote organogenesis and shoot growth from buds [18].

Recently, an alternative cotyledonary explant derived from mature soybean seed for *Agro‐ bacterium* transformation has been reported by Paz et al. [19]. The term half-seed explants were used as an experiment material and fertile transgenic plants were attained.

preferred and reproducible transformation is the use of the cotyledonary node as a plant material, which is based on *Agrobacterium*-mediated gene transfer [10-12]. Nevertheless, new methods have been developed for more efficient soybean transformation. There still remain, however, many challenges for genotype- and tissue- specific independent transformation of soybean. This review provides an overview of historical efforts in developing and advanc‐ ing soybean regeneration and transformation systems. In addition, recent advances and

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

In soybean transformation, two major methods are now widely utilized: *Agrobacterium-medi‐ ated* transformation of different explant tissues and particle bombardment. The *Agrobacteri‐ um*-mediated method, as a simple protocol, does not require any specific or expensive equipment. Moreover, this method usually produces single or low copy numbers of inser‐ tions with relatively rare rearrangement [13]. On the other hand, bombardment technique directly introduces desired genes into the target plant cell with small tungsten or gold parti‐ cles [9]. The success of this approach critically depends upon the ability of the target tissue

The routine regeneration system was first reported by using the mature cotyledonary-node [14]. The multiple adventitious buds and shoots from explant tissues were proliferated and re‐ generated on culture media containing cytokinin by organogenesis. The transgenic soybean plants have been successfully and reproducibly produced using mature or immature cotyle‐ don explants via *Agrobacterium*-mediated transformation. Hinchee et al. [8] for the first time re‐ ported the production of fertile transgenic soybean plants using mature cotyledonary-node by *Agrobacterium*-mediated transformation, but transformation efficiency was very low. The sys‐ tem employed the neomycin phosphotransferase II (*NPT* II) gene as a selectable marker and combined kanamycin as a selective agent. However, this selection was addressed with a prob‐ lem of regeneration of non-transgenic or chimeric shoots at the shoot formation stage. More‐ over, the system was highly genotype-dependent. To overcome the high genotypedependency and high chimerism problems by the *NPT* II selection and develop a new selection system for soybean transformation, Zhang et al. [10] developed the selection system employ‐ ing herbicide bialaphos resistance (*bar*) gene as a selectable marker coupled with glufosinate as a selective agent. This system enabled to transform many soybean genotypes with stable trans‐ gene inheritance, albeit transformation efficiency remained to be improved. Meanwhile, to solve the escape problem caused by kanamycin selection, Clemete et al. [15] deployed the her‐ bicide glyphosate as a selective agent, leading to high stringent selection and good transgene inheritance. It was discovered later that addition of various thiol compounds in the co-cultiva‐ tion medium significantly increased the transformation efficiency [11, 16-17]. These thiol com‐ pounds, as antioxidants, reduce the oxidative burst that caused tissue browning or necrosis

challenges in soybean transformation are discussed.

Relationships

490

**2. Different approaches for soybean transformation**

to proliferate as well as proper pre-cultures to make a target plant.

and also promote organogenesis and shoot growth from buds [18].

**2.1. Cotyledonary-node-based transformation**

In fact, several laboratories have contributed to enhanced soybean transformation using a cotyledonary-node explant. To overcome the low transfer of *Agrobacterium* into plant cell, the infection media were first amended with the phenolic compound, 4'-Hydroxy-3',5'-dime‐ thoxyacetophenone (acetosyringone), to induce expression of the virulence (*Vir*) genes [20-21]. To increase the infection sites, Trick and Finer [22] evaluated cotyledonary node transformation efficiency using a developed sonication assisted *Agrobacterium*-mediated transformation (SAAT) protocol. Although this treatment was not able to obtain fertile transgenic plants, the increase of *Agrobacterium* transfer was shown. Olhoft et al. [16-17] dis‐ covered that thiol compounds enhanced *Agrobacterium* infection in soybean. At the same time, however, these compounds caused counter-selection effect when glufosinate was used as a selective agent under previously published selection conditions. To solve this problem, Olhoft et al. [11] developed Hygromycin phosphotransferase (*HPT* II) selection system using hygromycin B as a selective agent. This has led to a substantial increase in transformation frequency. Transformation efficiency with thiol compounds was increased 5-fold by using refined glufosinate selection [12].

Since the transformation process by use of kanamycin or hygromycin B as selection agent has been proven to be genotype-dependent, the most widely used selection system has been the combination of *bar* gene with the herbicide phosphinothricin (glufosinate) [10, 12]. In this selection system, the concentration of agent glufosinate greatly affects the transforma‐ tion frequency [12], so the appropriate selection schemes can be varied among genotypes, seed vigor and other *in vitro* culture conditions.

**Figure 1.** Scheme for genetic transformation of soybean (*Glycine max* (L.) Merrill) cotyledonary nodes.

### **2.2. Immature embryos-based transformation**

The regeneration using immature embryos via somatic embryogenesis was first reported by Christianson et al. [23]. The immature embryos excised from soybean pods were suspended on semi-solid media or liquid media containing high concentration of auxin, 2,4-Dichlorophe‐ noxyacetic acid (2,4-D), and the whole plantlets were recovered [24-25]. After immature em‐ bryos were developed as an alternative plant material, transgenic plants were first obtained from this explant tissue via particle bombardment [26]. This system has been exclusively used to produce transgenic soybean such as glyphosate tolerant, hygromycin resistance, and *Bacil‐ lus thuringiensis* (BT) transgenic soybean [27-29]. As the formation of proliferative embryogen‐ ic tissue depends on genotype, the use of immature embryos for transformation has been limited to few genotypes cultivars including "Jack" and "Williams 82."

The use of particle bombardment with immature embryos tends to be highly variable, and multiple copies of the introduced DNAs are commons. Moreover, this problem has com‐ pounded with aged embryogenic suspension cultures from which a high percentage of re‐ generated plants lost their fertility [29]. In spite of this limitation, the embryogenic cultures have several advantages, one of which is its relatively high transformation efficiency and less chimeric plants recovered.

### **2.3. Embryogenic shoot tips-based transformation**

The embryonic shoot tip explant is another source of explant which has been used for soy‐ bean transformation. McCabe et al., [30] first reported the stable transformation using meris‐ temic cell, shoot apex, by particle acceleration. The shoot derived from these meristems via organogenesis has been produced to form multiple shoots prior to mature plants. However, all of the primary transgenic plants were chimeric. Martinell et al., [31] described the suc‐ cessful method using meristemic shoot tip from germinated seedling by *Agrobacterium*mediated transformation. This system has provided rapid and efficient soybean transformation. Liu et al. [32] also reported the regeneration system using embryonic shoot tips by shoot organogenesis. The explants have been shown the high regeneration and the transformation efficiencies using *Agrobacterium*-mediated with up to 15.8%.

#### **2.4. Immature cotyledonary-nodes**

The regeneration capacity of immature cotyledonary-node was found by Parrott et al [33]. Based on this regeneration system, first transgenic soybean plants have been developed by *Agrobacterium tumefaciens* [34]. This system was tested using two different *Agrobacterium* strains, LBA4404 and EHA101 and deploying kanamycin selection. The system utilized aux‐ in 1-Naphthaleneacetic acid (NAA) for plant regeneration. Although these systems allowed development of transgenic plants from the explants, no fertile transgenic plants were recov‐ ered. Recently, Ko et al [35] described the efficient transformation system using immature cotyledonary-nodes by *Agrobacterium*-mediated transformation, but transformation efficien‐ cy was still very low.

### **2.5. Hypocotyl based transformation**

**2.2. Immature embryos-based transformation**

Relationships

492

less chimeric plants recovered.

**2.4. Immature cotyledonary-nodes**

cy was still very low.

**2.3. Embryogenic shoot tips-based transformation**

The regeneration using immature embryos via somatic embryogenesis was first reported by Christianson et al. [23]. The immature embryos excised from soybean pods were suspended on semi-solid media or liquid media containing high concentration of auxin, 2,4-Dichlorophe‐ noxyacetic acid (2,4-D), and the whole plantlets were recovered [24-25]. After immature em‐ bryos were developed as an alternative plant material, transgenic plants were first obtained from this explant tissue via particle bombardment [26]. This system has been exclusively used to produce transgenic soybean such as glyphosate tolerant, hygromycin resistance, and *Bacil‐ lus thuringiensis* (BT) transgenic soybean [27-29]. As the formation of proliferative embryogen‐ ic tissue depends on genotype, the use of immature embryos for transformation has been

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

The use of particle bombardment with immature embryos tends to be highly variable, and multiple copies of the introduced DNAs are commons. Moreover, this problem has com‐ pounded with aged embryogenic suspension cultures from which a high percentage of re‐ generated plants lost their fertility [29]. In spite of this limitation, the embryogenic cultures have several advantages, one of which is its relatively high transformation efficiency and

The embryonic shoot tip explant is another source of explant which has been used for soy‐ bean transformation. McCabe et al., [30] first reported the stable transformation using meris‐ temic cell, shoot apex, by particle acceleration. The shoot derived from these meristems via organogenesis has been produced to form multiple shoots prior to mature plants. However, all of the primary transgenic plants were chimeric. Martinell et al., [31] described the suc‐ cessful method using meristemic shoot tip from germinated seedling by *Agrobacterium*mediated transformation. This system has provided rapid and efficient soybean transformation. Liu et al. [32] also reported the regeneration system using embryonic shoot tips by shoot organogenesis. The explants have been shown the high regeneration and the

The regeneration capacity of immature cotyledonary-node was found by Parrott et al [33]. Based on this regeneration system, first transgenic soybean plants have been developed by *Agrobacterium tumefaciens* [34]. This system was tested using two different *Agrobacterium* strains, LBA4404 and EHA101 and deploying kanamycin selection. The system utilized aux‐ in 1-Naphthaleneacetic acid (NAA) for plant regeneration. Although these systems allowed development of transgenic plants from the explants, no fertile transgenic plants were recov‐ ered. Recently, Ko et al [35] described the efficient transformation system using immature cotyledonary-nodes by *Agrobacterium*-mediated transformation, but transformation efficien‐

transformation efficiencies using *Agrobacterium*-mediated with up to 15.8%.

limited to few genotypes cultivars including "Jack" and "Williams 82."

Another type of explant tissue, hypocotyl, was also investigated with 13 different soybean genotypes. Most of the genotypes initiated shoots from this type of explant [36]. This meth‐ od was reported to be genotype-independent regeneration protocol via organogenesis and utilized the acropetal end of a hypocotyl section from a 7-day old seedling. Despite inducing adventitious shoots from the explant, most recovered shoot did not matured in the soil. Wang et al [37] reported successful production of fertile transgenic plants using hypocotylbased *Agrobacterium*-mediated transformation. To improve the transformation system, two different chemicals, cytokinin hormone 6-Benzylaminopurine (BAP) and silver nitrate, were added to the shoot formation media. In spite of the term "hypocotyl" used in the above transformation system, the true tissues responsible for regeneration are actually the preexist‐ ing meristem tissues located at the nodal area of the cotyledon, essentially the same source of tissue as cotyledonary-nodes [17] except that cotyledons were removed [45, 46].

### **2.6. Leaf tissue-based transformation**

The reproducible regeneration methods for whole plants from primary leaf tissue or epico‐ tyls were first reported by Wright et al [38]. The multiple shoots from those explants were continually initiated and proliferated with cytokinin BAP hormone. Rajasckaren et al [39] described regeneration of several varieties of soybean by embryogenesis from epicotyls and primary leaf tissues, thereby inducing fertile plants from those explants. Kan et al [40] first tested transformation efficiency using epicotyls and leaf tissues by *Agrobacterium tumefaciens*. To find out proper transformation condition for those explants, they investigated different *Agrobacterium* strains, EHA101 and LBA4404, but also different treatments on inoculation stage, sucrose and mannose.
