Establishment Of Tissue Culture Regeneration System And Transgene For Quality Improvement In Medicago Falcata L.

Alfalfa was important legume forages, and its resources was very rich in our country, but the digestion rate was low, so how to improve the digestion rate and quality, enhance the nutritional value were the main tasks of workers for the alfalfa breeding. With the development of genetic engineering, molecular method had been used widely of alfalfa breeding, it made breeding process speed up after clearing the breeding targets. In this study, it was as materials that optimization for aseptic culture, tissue culture regeneration system and Agrobacterium-mediated genetic transformation system had been established, and aimed to obtain higher sulfur amino acids and lower lignin of transgenic plants of Alfalfa by transgenic means. The results as followed:1. The best growth conditions for Alfalfa was seed pretreatment about70s by75%alcohol,0.1%HgCl2and concussion disinfection8min in add30g-L-1sucrose in MS culture. The germination rate could reached91.32%;0.1%HgCl2sterilization time was key factors affecting the growth of seedlings, so did sugar concentration.2. The results that regeneration system of native Alfalfa, showed that the callus induction rate could be up to100%of cotyledons and hypocotyls were cultivated for lOd on the optimal medium for callus induction that MS+1.5mg-L"12,4-D+0.4mg-L"16-BA+3%sucrose+0.7%agar. The optimal medium for embryogenic callus was MS+0.5mg-L-1KT+0.1mg·L-1NAA+2%sucrose+0.7%agar, and the rate of embryogenic callus was81.5%.The optimal medium for differentiated buds was MS+0.25mg-L-KT+0.05mg·L-1NAA+2%sucrose+0.7%agar. the rate of differentiated buds was36.5%. The optimal rooting medium was1/2MS+0.5mg·L-1NAA+1.5%sucrose+0.7%agar. and the rooting rate was93.3%. A third of the plant’s callus was better than other plants’ on the same hormone level. There were quaint differences between individuals of Alfalfa material in the tissue culture regeneration.3. genetic transformation system of Native Alfalfa had been Established which transformed Agrobacterium tumefaciens LBA4404with over-expression vector pDESAK-APR and interference vector p449hct-c3h. The results included that the two bacteria of Agrobacterium tumefaciens with vectors were mixed that equal volume and concentration, the OD600of both the two bacteria were0.4-0.5. and infecting time was 6～8min, the cotyledons and hypocotyls of Alfalfa were explants which were infected. co-cultured time was3d, inhibitory concentration of Cef was500mg·L-1, screening concentration of selecting pressure Kan was60mg-L-1. transgenic seedlings of expression level of target gene had been Obtained.