Optimization Of High-efficiency Agrobacterium-mediated BADH Gene Transformation System Of Alfalfa And Detection Of Transgenic Plants

Saline soil is a very important nonbiological force factor influence agricultural production and ecological environment. How to use the great area saline soil and improve the tolerance of salt is an important research subject in bioscience. The alfalfa is a kind of perennial high-quality legume family herb that is praised as "king of herb". It has agreeable taste and costly nutrition, and has a good adaptability. It is able to bear the drought and resist cold, and has a good ecological benefit to ecological environment protection and prevents soil erosion. Development and application of genetic engineering opened up a new way for the alfalfa in resisting salt breeding. In this research, the BADH gene was transformed through Agrobacterium-mediated, and all kinds of factors that affect transformation had been studied and optimized. This experiment was on the basis of high-frequency regeneration system of alfalfa that had already been set up, and many transgenic plants were obtained. The main results were as following:1. The high-efficiency Agrobacterium-mediated BADH gene transformation system of alfalfa had been established. In the Agrobacterium-mediated gene transformation, frequency of GUS+ positive explants, frequency of resistant callus were used to optimize the parameters affecting transformation. In the optimization of every factor of the system, we had confirmed Cef rightest density as 400mg/L and Kan as 50mg/L. Results showed the efficiency of transformation were varied greatly with the genetype of alfalfa, the explant type and its physiological state. At the same time, the transformation frequency was affected greatly by bacterial concentration and infection time. The highest transformation efficiency was obtained when the bacterial cell density was OD600 0.3-0.5 with 5-10 minutes of infection. It was also higher to transformation efficiency under no pre-culture and co-culture for 3-4 days and AS concentration with10mg/L.2. PCR screen with transformed plants. In a series of experiments, a great of kanmycin resistant lines were obtained. DNA of transformed plants was extracted for PCR and PCR-Southern analysis. The result indicated BADH genes were integrated into genomes of 34 out of 145 lines. It revealed that the transformation system was efficiency and stability, and certified BADH gene was transformed successfully.3. The salt test of the transgenic plant. Through tissue culture, the transgenic alfalfa was investigated with variant NaCl salt gradient. The results showed transgenic alfalfa differentiated normally on the medium with 0.7% NaCl, and Just present the obvious injured symptom when the salt concentration at 1.2%, while exceed 0.7%, most the control palnts take the death in vain already. These results indicated that the transgenic alfalfa had better tolerance ability than the control plants, Therefore it proved that BADH gene had been expressed well in transgenic plants.