Optimization Of Agrobacterium Tumerfaciencs-mediated Transformation Systems And Transformation Of GmGT-2A Into Soybean

Soybean (Glycine max L. Merr) is a primary source of protein and vegetable oil throughout the world. It is also the most valuable grain legume crop. Soybean growth and yield would be significant effected when that suffered external environmental stress (such as drought, salt, frost, cryogenic). Therefore, breeding of stress-tolerant soybean germplasms is a key method to manage crisis of soybean industry in China. Currently, transgenic technologies is one of the most important and effective methods which were utilized to breed new stress-tolerant soybean germplasms, and establishing an appropriate transformation system is the premise of that.In this study, A transcription factor, GmGT-2A, which is related to enhancing the ability of resistanting stress such as salt tress, was introduced into soybean genome 'zhechun No.3' and 'zhonghuang 13'produced by Hangzhou Sub-center of National Soybean. And Some important factors including sterilizing method, explant size, angle of the explant in the medium,6-BA concentration in the germination culture or the inducing culture and the hormone in elongation culture medium were studied to establish a transformation system. The main results were as follows:When chlorine gas was used in the seeds sterilization, the contamination frequency and germination frequency of seeds were 1.67% and 98.33% respectively. They were significant higher than those of using NaCIO with 70% ethanol in the sterilization, which were 35.33% and 64.67% respectively. The optimal seeds sterilizing time is 4-6 h with the highest regeneration rate of 88-89% and 0% pollution rate. When cotylendonary was embedded in the inducing medium, we found that 2/3 of total size and 45 angle between cotylendonary and medium face could get best regeneration rate and the quality of the shoot. The concentration of 6-BA in the germination medium and inducing medium were also analyzed in this study, the result showed that regeneration rate of explant and the number of cluster buds with the obviously stem were highest, when concentration of 6-BA were 0.5 mg/L and 2 mg/L in the germination medium and inducing medium respectively. We concluded that 6-BA plays an important role in both germination culture stage and inducing culture stage. The accummulation of 6-BA in the early culture stage also have effects on the shoot inducing and root inducing. The key factors on shoot enlongation was also discussed in this paper. The elongation frequency of shoot reached highest as 78.33% when 0.15 mg/L of ZT,0.6 mg/L of GA3,0 mg/L of TDZ,1 mg/L of 6-BA were added in the enlongation medium.Using above transgenic system, we introduced transcription factor GmGT-2A, constructed in the binary vector pBin438, into cultivar'zhonghuang 13' successfully, and 19 transgenic lines with target gene that had been confirmed using target gene-specific markers, selective marker gene-specific, construction-specific PCR marker analysis and stress torlerance tests were gained.In this paper, another transgenic system Agrobacterium-mediated soybean mature embryonic tip was also studied. Three main factors including pre-culture time of embryonic tips,6-BA concentration in the co-culture medium, and the infection time on the transformation efficiency were disscused. Results of the single factor optimizing test showed that transformation efficiency of embryonic tip was significant affected by the three factors respectively. The optimal levels for each factor were 1.5 d for pre-culture time of embryonic tip,10 h for infection time of embryonic tip and 8 mg/L for 6-BA concentration in the co-culture medium. Results of further orthogonal test showed that these three factors could be ranked as infection time, pre-culture tim, and 6-BA concentration, according to their effectiveness on transformation efficiency. The optimal transformation system included the levels for each factor as mentioned above. A highest transformation efficiency of 5.67% of embryonic tip was achieved in this study. A preliminary test also showed that rooting medium with no additional IBA was better than that with 0.2 mg/L IBA, in terms of root emerging time and root qualities. The transcription factor GmGT-2A also constructed in the binary vector pCambia3300 was introduced into soybean variety 'zhechun NO.3' via above transformation systems successfully, the target gene in these lines was confirmed using target gene-specific, selective marker gene -specific and construction-specific PCR analysis.