Transformation Of Soybean Using Giyphosate As A Selecive Agent

Genetic engineering of soybean to improve agronomic performance and pest or herbicide resistance has played an important role in promoting soybean production worldwide. The Agrobacterium-mediated transformation system is the most commonly used method in soybean transformation. In this study, twenty two soybean genotypes that originated from different soybean production regions were screened by transient infection, regeneration capacity and stable transgenic efficiency. Six soybean genotypes showed comparable stable transgenic efficiencies and regeneration rates in our experimental system. For the Tianlong1, Yuechun03-3, Yuechun04-5, Jack, and Williams82, the average stable transformation efficiencies were all higher than2.4%. Moreover, quick and effective procedures to identify the To plants and the separation statistics on T1offspring were discussed.In this study, we also modified two glyphosate-resistance genes G6-EPSPS and G10-EPSPS. With appropriate soybean genotypes and efficient new genes, we have successfully established a transformation system using glyphosate as a selective agent. In this system, the transformation efficiency ranged between0.41%to3.06%. The concentration of glyphosate was the most important factor for the success of the transformation. When the glyfosate concentration increased from5mg/L to100mg/L, the shoot induction rate decreased2to3times. However, the total transformation efficiency remained the same. In the experiments using high selective concentration, the transformed cells showed a significant advantage on growth compared with the nontransformed ones. Thus, the stringent selection scheme, which eliminates non-transgenic cells at earlier stage, will greatly reduce the lab cost and the false-positive rate. In addition, the resulted transformants are expected to be more tolerance to the herbicide. Other than the selection scheme, the transformation system using embryonic meristem explants was optimized to obtain transgenic soybean.In this study, a total of thirty seven independent transgenic lines with stacked traits of herbicide tolerance with pest resistance, or high-yield were obtained. Through systematic screening of herbicide and insect resistance, transgene expression, Western blot, and Southern blot, S7and other4events with outstanding herbicide and insect resistance were selected. In summary, we have established a new soybean transformation system using glyphosate as a direct selective agent. Due to the avoidance of selective marker, the system is safe for generation of new transgenic soybean germplasm.