The Pilot Study For The Improvement Of Alfalfa Frost Resistance Using Transgenic Technology

The alfalfa is a kind of perennial high-quality legume family herb that is praised as "king of herb".It has agreeable taste,costly nutrition and has 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 cold breeding.In this research,the CBF1 gene was transferred to alfalfa by Agrobacterium-mediated transformation to improve cold-resistant of alfalfa WL-323.The study in this paper is aimed at establishing and optimizing the regeneration and transformation system fromZhongmuNO.1,Hunter River,Gongnong NO.1 and WL-323.Preliminary study on the impact of alfalfa callus induction rate and the differentiation factors of renewable to sets up and optimizes tissue culture regeneration systems of alfalfa..It turned out that hypocotyl is the most effective explant and the best acceptor is WL-323.The resisting cold breeding gene CBF1 was transferred to alfalfa by Agrobacterium-mediated transformation in this paper.The main achievements of our research are following:1.Regeneration tissiue culture system establishment。(1)definite the optimizing culture medium embryogenic callus induction:improveSH+2,4-D(4.0mg/L)+6-BA(0.5mg/L) subculture media:MSO+NAA(0.5mg/L) + 6-BA(1.0mg/L) + AgNO₃(1.0 mg/L) differential medium:MS(2)Any explant of the four alfalfa is bale to callus induction and differentiation a new one.It turned out that hypocotyl is the most effect explant and the best acceptor is WL-323.2.Regeneration high-efficiency Agrobacterium-mediated CBF1 gene transformation system of alfalfa.(1) The best concentration of Kan for screening of resistance seedlings is 60mg/L.(2) Carb is better than Cef in inhibiting the growth of Agrobacterium.the experimental selection of 350mg/L Carb to inhibit the growth of Agrobacterium.(3) The proper concentration of Agrobaeterium to infection explants is 0.4—0.6。(4) The best infection period is 10min。(5) It was also higher to transformation efficiency under AS concentration with 10mg/L.3.PCR screen with transformed plants.In a series of experiments,a great of kanmycin esistant lines were obtained.DNA of transformed plants was extracted for PCR,and Consistent with the positive control access to specific bands. Preliminary results show that the resisting cold breeding gene CBF1 had been successfully transferred to the alfalfa genome.