Study On Transformation Of DesC Gene Of "Zhongmu No.2" By Agrobacterium Tumefaciens

High-frequency regeneration system and optimized genetic transformation system of alfalfa had been established by using Medicago sativa L."zhongmuNO.2"as plant material,which implied a stable basis for alfalfa transformation. Based on that,gene desC,which related cold-tolerance,was transformed into Medicago sativa L."zhong mu NO.2".by Agrobacterium-mediated,which aimed to get the transgenic alfalfa plants overexpressing desC and the expression elements could be integrated into the genome of alfalfa by identification. The main results were as following:1. Regeneration tissue culture system establishment of"zhong mu NO.2"Effects of various factors on the form of callus were investigated using orthogonal test design method. The results showed that the most helpful explants for callus induction was hypocotyls. 2,4-D is the most effect factor to the callus induction. The most important is that the optimal medium for callus induction of"Zhong mu NO.2"was MS+2,4-D(0.2mg/L)+ NAA(0.01mg/L)+6-BA(1mg/L)+KT(0.2mg/L).2. Establishment and optimization of genetic transformation system by Agrobacterium-mediated. The results showed that the effects of the three factors to Kan-resistance callus induction is that OD value of stock solution>inoculation time>co-culture time.The optimized parameters of callus transformation by desC are: OD value of stock solution was adjusted to 0.4 at A600; inoculation time is 10min; co-culture time is 5d.3. The results of PCR showed that the gene of desC had been transcribed into callus of positive transgenic alfalfa.More experiments also be needed to see whether the desC gene has transcribed into the genomic DNA.