Study On Plantlets Regeneration And Genetic Transformation System For Vegetable-type Soybean

The vegetable-type soybean has high nutritional and medicinal properties, while the study of regeneration and genetic engineering of vegetable-type soybean lagged. To study the effects of sterilization, pre-culture days, different concentrations of6-BA and medium formulations on induction, elongation of adventitious buds, and different concentration of IBA on rooting induction, embryonic tips from nine vegetable-type soybean were used as the initial explants. To improve Agrobacterium tumefaciens mediated transformation of embryonic tips of vegetable-type soybean, the effect of several factors on transformation efficiency were examined by measuring transient expression levels of β-glucuronidase and the number of resistant explants. Test results showed that:1. The study results of sterilizing methods showed that the optimal sterilization was using0.1%HgCl2for10min and5%NaCIO for5min, the lowest pollution rate was0.3%.2. The study results of duds induction, elongation and rooting induction in embryonic tips of vegetable-type soybean showed that the optimal adventitious buds induction formulation was MSB5base medium with3.0mg·L-16-BA, and the optimal early-culture days of embryonic tips was2d. The combination of1.0mg-L-16-BA and0.1mg-L-1NAA benefited to produce many adventitious buds. The combination of0.05mg-L-16-BA and0.1mg·L-1IBA benefited to the elongation of adventitious buds. The medium of1/2MSB5is the best medium for root induction. The average root frequency was97%. The survival rate was100%when transferred to soil. The regenerated plantlets with healthy roots grew successfully and matured.3. Among the nine varieties, the embryonic tips from K06-82are the best one for regeneration, with92%of adventitious buds induction rate,4.5of mean number of adventitious buds and2.47cm of mean length of adventitious buds after culture for20d. These simple, reproducible and high frequency regeneration systems provide a basis for genetic transformation of common bean with Agrobacterium tumefaciens.4. The study results of effects of different factors on transformation efficiency of embryonic tips of vegetable-type soybean showed that co-cultivation with200 umol-L-1AS for3d is advantage to improve transformation frequency.5. Combination300mg·L-1Tic with100mg·L-1Cef is good for bacteriostasis; medium with antioxidants can reduce the browning of embryonic tips. And the best elongation medium for resistant shoots from embryonic tips in vegetable-type soybean is1/2MSB5＋KT0.2mg·L-1.6. GUS transient expression and PCR analyses confirmed the stable integration of the binary insect resistance genes in the primary transgenic plants. The transformation frequency ranged from6.4to17.6%.