Transformation Of BdDREB2 Gene To Alfalfa By Agrobacterium Tumefaciens

As"the king of forages",Medicago sativa is the earliest cultivation,the most extensive distribution and the most important perennial quality legumes which is high yield, good palatability and protein-rich. Recently, Medicago sativa is cultivated in north China area widely and the cultivation scale has the trendency of expanding gradually year. As the environment of China turn worse, the water resourse turn less. The soil salinization of part areas'land is serious. These greatly restrict the planting of alfalfa and the development of animal husbandry. Improving the soil has many shortcomings, for example, tremendous cost of soil and little effect. But these years,improveing plant cultivar by genetic engineering technology becomes a hot spot of biological engineering research.In this study, the gene transformation acceptor selected is the alfalfa cultivar Medicago sativa L."zhongmu NO.1", which comes from the conventional big field breeding technology by the CASI. I selected the radicle, the hypocotyls and the cotylecdons as the explant, conducted the tissue culture systematic research, seeked a high differentiation rate explant and the basic culture medium which suit for this variety, and determined the best concentration of bacilli, the best infection time and the co-culture time. Addition to these, the BdDREB2 (Buchloe dactyloides Dehydration Responsive Element Binding protein 2)gene which was cloned from Buchloe dactyloides (Nutt.) Engelm was introduced into the genome of the alfalfa (Zhongmu No.1) by using Agrobacterium tumefaciens-mediated transformation. The main results were as following:1. The radicle, the hypocotyls and the cotylecdons callus rate are all higher, but the radicle and the hypocotyls are hard to differentiated embryo. The callus rate of cotylecdon is lower than the hypocotyl's callus rate, but its differentiation rate is higher. It is indicated that cotylecdon is suitable as explant. UM medium is the best embryogenic callus induction medium and the best embryo induction medium and 1/2MS medium is the best root development medium.2. The best concentration of bacilli is OD600≈0.6; the best infection time is 10 minutes; and the co-culture time is 3 days.3. The best transformation system is as following.Embryogenic callus induction medium: UM+2,4-D 2mg/L+KT 0.25mg/L+Cef 500mg/L+Kan 75mg/L The best embryo induction medium: UM+KT 2mg/L+Cef 500mg/L+Kan 75mg/L The best root development medium: 1/2MS4. Finally eight transformant was achieved. PCR analysis indicated that four of the eight transformant were transgenic plants. The positive rate is 50%. RT-PCR results showed that the geneof BdDREB2 is transcribed into mRNA in positive transgenic alfalfa. Stable integration of the BdDREB2 gene into the genome of transgenic plants was confirmed by Southern blot analyses.