Establishment Of Large-Scale Technique For Agrobacterium-Mediated Transformation In Soybean

Soybean is one of the most important crops, which is rich in protein, vegetable oil and a variety of useful physiological chemical compositions of human. Soybean is not only a major human food source of vegetable protein, is also an important materials for industry and animal feed. With the technology and people's living standards improve, soybean production began to not content the needs of people's lives. Traditional breeding methods and can not effectively solve the problem of soybean's gap between supply and demand. So that, more and more researchers have tried to overcome these problems use genetic engineering technology since 1988, and has achieved some success. But, compare with other plants, the transformation frequency of soybean is still low relative, and the repeatability is not good. Actually a few of cases of transgenic soybeans were successful due to the low transformation rate.Establish an efficient and stable system for soybean transformation technology is still one of the hot topic for the scientists.In this study,we use Agrobacterium-mediated transformation method for transforming 7 soybeans, the cotyledon-node explant was uesd for the transgenic receptor. In order to improve transformation efficiency, this experiment was conducted to compare the efficiency of several factors during transformation. This will lay a good foundation for the gene function research and molecular breeding of soybean.The main results of this research as follows1. 7 soybean genotypes were used for transformation.The Agrobacterium tumefaciens strain was EHAl05 and GV3101,which carried binary vector PBI121.The result in this research had shown that regeneration frequency was higher of"zhonghuang10"and"wenfeng7".2. To reduce the browning effect,we added L-cy in the co-cultivation medium.The result indicate that though the experiments for concentration gradient of L-cy,the suitable L-cy cencentration is 400mg/L for the zhonghuang13 soybean.The browning rate has dropped by more than 90%。3. Reduce the level of residual Agrobacterium contamination.We design of three methods to wash the cotyledon-node explant,after the co-cultivation,and finally chose one methods that the lever of residual Agrobacterium was lowest.4. Improve the efficiency of shoot induction.We chose some of cotyledon-node explant that just shooting after the co-cultivation,cut off the new shoot,and then transfer them to a new medium.The result indicate that the efficiency of shoot induction was most increased.5. We add Thidiazuron(TDZ) in the shoot induction medium.And found TDZ is more effective than 6-BA at activated cell development.6. Duing the root regeneration stage,We had determined the optimal concentration of IBA,and found that the genotypes has some infulence on the rooting ability.7. We also did some research on transplanting, and improve the efficiency of the survival of transplanted.It is made some good provide for the molecular detection.8. Through the soybean transformation experiments,we finally obtained 43 transgenic plants,increases in transformation efficiency to an average of 1.7%, which the most is 4.61%. Conversion efficiency is higher than the current average level of soybean transformation.