**3.3 Soybean** *Agrobacterium* **mediated transformation**

Hinchee and his colleagues first time reported soybean transformation with *Agrobacterium* strain pTiT37-SE harboring pMON9749 (GUS + nptII) and pMON894 (nptII + glyphosate tolerance). They successfully regenerated plants on media containing kanamycin and

Engineering technologies and molecular mindsets expiated asset of *Agrobacterium* to transfer the genes of interest into plant cell. This revolution lighted the pathway to break inert kingdom genetic exchange restrictions. They terminated the property of *Agrobacterium* to cause tumor but did not change the belongings that are involved in T-DNA transfer mechanism. Finally, plant biotechnological era came to revolution to produce transgenic plant species with desired characters. However, all the barriers could not be departed productively. Factors responsible for production of transformants have been studies worldwide and are found more or less same for all genotypes even plant or Agrobacterium. These factors, at *Agrobacterium* flank, include genotype, plasmid constrains, T-DNA length and signaling mechanism. While at plant cell side, the factors include type, age, genetic makeup and welcome address to T-DNA. The welcome discourse also depends upon physical and chemical conditions that finally lead to produce whole plant from a single

Although initially dicots were considered host for *Agrobacterium* but advancement in procedures commanded *Agrobacterium* to display same role in monocots as in dicots. The process of plant transformation is a routine matter in most of the labs but some plant species

Soybean genotype susceptibility for tumor induction was studied by Pedersen et al., 1983 and Owens & Cress in 1984 on infection with *Agrobacterium*. According to their reports, crown gall formation is dependent upon soybean genotype and *Agrobacterium* strain used as well as on environmental conditions. Physiological age of soybean cotyledons also exert great influence on tumor initiation and tumor morphology. Owens and Smigocki (1988) indicated that transformed soybean cells could be recovered by co-infecting with supervirulent strain and addition of phenolic compounds (Acetosyringone or Syringaldehyde) in inoculation medium increase transformation efficiency. It is also possible to produce tumorigenic genotype by crossing non-tumorigenic with highly tumorigenic genotype in soybean so conventional crossing may help to transform non-susceptible genotypes. Luo et al. (1994) observed production of transformed calli from mature seed cotyledons working on transformation friendly genotype "Peking" with *Agrobacterium* strain A281 harboring pZA-7 (UidA + nptII). They mentioned that production of transformed calli is a simple tool to test constructs designed for soybean transformation. Genotype of *Agrobacterium* (nopaline, agropine, octopine) also plays an important role in infection and T-DNA inheritance (Mauro et al., 1995). Acetosyringone may facilitate tumor formation significantly but not for all *Agrobacterium* strains. However, strain/genotype difference was observed significant while older plant parts showed less susceptibility to tumor formation. Transformation event occurs in number of cells but poor selection and non-regenerable callus formation attribute to poor recovery of transformed plants (Donaldson & Simmond, 2000). A new *Agrobacterium tumefaciens* strain KAT23 isolated from peach root also found effective to induce callus at soybean tissues (Yukawa et al., 2007). This nopaline type strain can transform T-DNA of Ti

plasmid and of binary vector efficiently to many legumes including soybean.

Hinchee and his colleagues first time reported soybean transformation with *Agrobacterium* strain pTiT37-SE harboring pMON9749 (GUS + nptII) and pMON894 (nptII + glyphosate tolerance). They successfully regenerated plants on media containing kanamycin and

**3.3 Soybean** *Agrobacterium* **mediated transformation** 

transformed cell.

are still considered recalcitrant to transformation.

**3.2 Susceptibility of soybean to** *Agrobacterium* 

glyphosate (Hinchee et al., 1988). Modification to regeneration protocol is essential to get high level of transformants. Greater number of mitotic cycles are required before embryo initiation and production of plants with transformed germ lines cells. EHA101 was found more potent to transform soybean immature cotyledons and recovery of transformed plants over LBA4404 (Parrott et al., 1989). However, McKenzie & Cress (1992) were able to get transformed plants from cotyledon and hypocotyl explants from 10 days old seedlings working with LBA4404 harboring pBI121. Transformation efficiency is not dependent only on Agrobacterium genotype but soybean cultivar, age of explant and other conditions also influence. Trick & Finer (1997) introduced sonication assisted *Agrobacterium* mediated transformation (SAAT) system. SAAT permits efficient delivery of T-DNA to large number of plant cell in a variety of different plant tissues. In soybean, GUS expressing surface area increased upto 79.9% by SAAT treatment for 10 sec. Other tissues that are considered difficult to transform can be subjected to SAAT that permit Agrobacterium to infect deep within the plant tissue. While SAAT treatment was not found effective at post co-cultivation period with decreased shoot proliferation from cotyledonary node of some soybean genotypes (Meurer et al., 1998). They also reported that inoculum OD600 1.0 gave better transient expression but no interaction was found between SAAT, *Agrobacterium* strain and soybean genotype. Micro-wound in plant tissues due to SAAT treatment release compounds that facilitate growth and accumulation of bacteria under aerobic conditions so facilitate transformation efficiency (Finer & Finer, 2000). However, longer sonication time may damage plant tissue (Santarem et al. 1998). Way of placement of explant (adaxial side incontact with medium) on medium (Ko et al., 2003); exposure of soybean explants to AgNO3 throughout shoot induction and shoot elongation (Olhoft et al., 2004); explant preparation in the presence of *Agrobacterium* culture and varying level of kanamycin during selection and regeneration (Zia et al., 2010b) are important for better recovery of transformants.

Instead of kanamycin resistant plant, glufosinate resistant (bar gene) plants were produced by Zhang et al. (1999) and Clemente et al. (2000) using cotyledonary node explants of 5 days old seedlings. Glyfosinate selection regime is important to get rid of non-transformed plants and to minimize chimerism. Yan et al. (2000) analyzed that immature zygotic size 8-10mm and co-cultivation for short period increase transient expression while selection by direct replacement at low concentration of hygromycin also increase somatic embryo development and plant regeneration. Cystine present in co-cultivation medium increase transformation efficiency due to presence of thiol group and polyphenol oxidase and peroxidase inhibition (Olhoft & Somers, 2001; Olhoft et al., 2001). Copper and iron chelators were also found effective for better expression. Olhoft and his colleagues successfully transformed soybean by cot node method (Olhoft et al., 2003). High frequency upto 16.4% was observed due to presence of cystine, Dithiothreitol (DTT) and thiol compound in infection and co-cultivation medium. Beside this, addition of Silwet-77 as surfactant; co-cultivation at 22C also played significant role in transformation (Liu et al., 2007). Donaldson & Simmonds (2000) demonstrated that competent cells, in the case of cotyledonary node transformation, are few so has low transformation competency therefore using cotyledonary nodes as explants present low transformation efficiency. Tight selection procedure (selection of explants on selective agent before infection) increases transformation efficiency and occurrence of less escape (Chen, 2004).

Xing and his colleagues produced marker free plants by introducing two T-DNA binary systems (Xing et al., 2000). Integration of two T-DNA followed by their independent

Soybean: Plant Manipulation to *Agrobacterium* Mediated Transformation 305

number of genes, different biological processes and symbiotic relation etc. Klink et al. (2008) introduced a new soybean variety MiniMax with a rapid and short life cycle that produced hairy roots under non-axenic conditions when infected with *A. rhizogens* strains K599 harboring disarmed vector pKSF3. These transgenic roots were capable of compatible

The development of the in planta transformation system (Floral-dip method and Vacuum infiltration) radically accelerated research in basic plant molecular biology. These methods have been targeted mostly for meristems or other tissues that ultimately give rise to

Soybean transformation also has been subjected by infecting partially germinated seeds with *Agrobacterium* to vacuum infiltration with high frequency (de Ronde et al., 2001). In planta soybean transformation has also been carried out by Lei et al, (1991); Liu et al., (1996) and Hu & Wang (1999). They introduced foreign DNA by pollen tube pathway and by ovarian injection. Such procedures pass tissue culture steps but for routine transformation physiological conditions of recipient plant, type and concentration of DNA, location of ovary etc are critical factors. By such methods, they produced new varieties that yield batter protein and oil contents. But Li et al. (2002) were not able to produce positive results by pollen tube pathway. They reported that DNA was inside the cell but not integrated into soybean genome. Shou et al., 2002 also performed pollen tube pathway transformation procedure using different soybean cultivars. They observed that only 2% progenies were partially resistant to herbicide. However, no plant was confirmed by Southern blotting carrying transformed T-DNA as well as by histochemical GUS assay. They concluded that

pollen tube pathway transformation technique is not reproducible for soybean.

Plant tissue culture has attained a lot of attention in recent years because it is a gateway to modern plant biotechnology including plant genetic transformation. Although soybean in vitro manipulation and transformation has passed more then thirty years but still establishment of acceptable protocol is far behind that could be used for all cultivars all over the word. The work is going on to overcome the limitations but soybean genotype could not be overlooked in all methodologies. Now destiny is near where new genetically modified varieties of soybean like Roundup ready will be produced globally by following the

Bailey, M.A.; Boerma, H.R. & Parrott, W.A. (1993). Genotype-specific optimization of plant –

Beversdrof, W.D. & Bingham, E.T. (1977). Degree of differentiation obtained in tissue cultures of *Glycine* species. *Crop Science*, Vol,17, pp. 307-311, ISSN 0011-183X Buising, C.M.; Shoemaker, R.C. & Benbow, R.M. (1994). Early events of multiple bud

regeneration from somatic embryos of soybean. *Plant Science*, Vol.93, No.1-2, pp.

formation and shoot development in soybean embryonic axes treated with the cytokine. *American Journal of Botany*, Vol.81, No.11, pp. 1435-1448, ISSN 0002-9122

reactions with several *Heterodera glycines* races.

gametes.

**4. Conclusions** 

established protocols.

117-120, ISSN 0168-9452

**5. References** 

**3.5 In planta** *Agrobacterium* **mediated transformation** 

segregation in progeny is a viable mean to produce maker free soybean transgenic plants. Transformation efficiency was observed upto 15.8% using embryonic tips of soybean pre grown on MS medium containing BAP (Liu et al., 2004). They also observed that shoot regeneration and transformation efficiency increased using embryonic tips over hypocotyls and cotyledons. Embryonic tips were also found sensitive against kanamycin treatment at level higher then 10 mg/l. Addition of antioxidant in co-cultivation medium result in significant decrease in browning and necrosis of hypocotyls and increased GUS expression (Wang & Xu, 2008). Embryogenic tips showed better response for hypervirulent strain KYRT1 than EHA105 and LBA4404 when infected for 20 hours (Dang & Wei, 2007). While co-cultivation for 5 days in dark at 22C in acidic medium (pH 5.4) also enhanced transformation efficiency. Paz et al. (2004) concluded that use of high vigor seed and minimum seed sterilization also raise transformation efficiency from cotyledonary node of 5-6 days seedling plants. Cystine and DDT during co-cultivation increase T-DNA delivery while glyfosinate selection over bialaphos during shoot induction and shoot elongation also increase transformation efficiency. Ko & Korban (2004) reported that size of immature cotyledon (5-8 mm in length), concentration of bacterial culture and co-cultivation for 4 days significantly increase transformation efficiency. However, they failed to get transformants in the presence of kanamycin during selection. Paz et al. (2006) used cotyledonary node of half seeds as an explant. Use of half seed explants ranged transformation efficiency 1.4 to 8.7% and this system is simple and does not require deliberate wounding of explants. Use of thin 30 fibers needle to wound cotyledonary node cells of half seeds also increased transformation efficiency up to 12% confirmed by gfp activity and L- Phosphinothricin (PPT) selection (Xue et al. 2006). Organogenic callus induced from axillary nodal tissue of soybean was also subjected for *Agrobacterium* mediated transformation (Hong et al., 2007). Moderate concentration of TDZ was required for induction of organogenic calli while low concentration of BAP proved best for organogenic response from callus. They also observed that young callus was more competent to T-DNA delivery and multiple shoot regeneration. Olhoft et al. (2007) tested two disarmed *Agrobacterium* strains for soybean transformation. Regeneration frequency was not significantly different when inoculated with *A rhizogenes* strain SHA17 and *A tumefaciens* strain AGL1 while infection with SHA17 increased transformation efficiency upto 3.5 folds.

### **3.4 Soybean transformation with** *Agrobacterium rhizogenes*

Instead of *Agrobacterium tumefaciens*, soybean transformation also been studied by *Agrobacterium rhizogenes* to study efficiency of strain, properties of roots and resistance against nematodes. Cho et al. (2000) got transformed hairy roots by *A. rhizogenes* strain K599 harboring pBI121 (gus + nptII) and pBINm-gfp5-ER (nptII and gfp). They observed that cyst nematode may complete their life cycle in transformed hairy root cultures containing these genes but concluded that such system can be ideal for testing genes that might impart resistance to soybean against nematodes. RNAi silencing was also studied by *A. rhizogenes* mediated transformation to cotyledon explants of soybean (Subramanian et al., 2005). More than 50% roots were transformed with RNAi construct that exhibited more then 95% silencing. Kereszt et al. (2007) reported that infection of *A. rhizogenes* at cotyledonary node of few days seedling might produce 5-7 roots at infection site with 70-100% efficiency. These roots fully support the plants, are capable of nodulation, have phenotype as determined by genotype of shoot. This can further be used for high throughput transformation, to test high number of genes, different biological processes and symbiotic relation etc. Klink et al. (2008) introduced a new soybean variety MiniMax with a rapid and short life cycle that produced hairy roots under non-axenic conditions when infected with *A. rhizogens* strains K599 harboring disarmed vector pKSF3. These transgenic roots were capable of compatible reactions with several *Heterodera glycines* races.
