**4. Conclusions**

304 Soybean – Genetics and Novel Techniques for Yield Enhancement

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 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 established protocols.
