Preliminary Study On The Transformation Of Perennial Ryegrass With ZxNHX And ZxVP1 Bivalent Genes

This study selected three varieties of Perennial ryegrass (Lolium perenne L.) for gene transformation acceptor, and the mature seeds and bud clumps as explants, the influences of varied factors on callus and shoot induction,subculture were studied through the orthogonal experimental.In the process of transferring gene ZxNHX and ZxVP binary vector by the Agrobacterium-mediated of Perennial ryegrass,we established a high-frequency callus regeneration system and the efficient proliferation system of multiple shoots, Based on that, the gene ZxNHX and ZxVP binary vector was transformed into perennial ryegrass, and we also want a better performance and drought tolerant varieties of it. Through preliminary testing, we have found the expression gene in the transformed plants, the main results were as follows:(1)Optimize and improve the regeneration system of perennial ryegrass. Learning from the experience of previous work, and now we selected the mature seeds of vertical cutting as explants, the gradient through the design of handling different showed that:when the 2,4-D concentration of 5mg/L,6-BA concentration of 0.05 mg/L, the callus induction rate was higher; subculture was induced with the same medium with the Induction medium; when the 6-BA concentration of lmg/ L, we gained the high rate of callus differentiation; best rooting medium for:1/2MS medium+0.2 mg/L KT+0.5 mg/L IAA.(2) we established an efficient propagation system of multiple shoots of Perennial ryegrass. As the aseptic shoot tips for explants, we studied the shoot induction and proliferation of perennial ryegrass. By analyzing the different treatment of the results showed that:when the 2,4-D and 6-BA concentrations were 0.5 mg/L and 2.0 mg/L, it was helpful to induced multiple shoots; The suitable subculture concentration of 6-BA is 2.0 mg/L; the long subculture days is not conducive to the growth of multiple shoots; the genotypes also have a great impact on the induction and proliferation of shoot clumps;the induction and proliferation rates of shoot clumps is different with different varieties of perennial;the suitable rooting medium was 1/2 MS+NAA 0.1 mg/L.(3), Establishment and optimization of Perennial ryegrass genetic transformation system by Agrobacterium-mediated method.. Through comparative analysis of the experimental results show that:Callus and shoots were used as recipient material for transformation, the concentration of Agrobacterium tumefaciens suspension was OD600=0.5-0.6 added with AS of 150μmol/L, then the infected Explants were incubated for 3-4d. After that cultivated in the medium supplemented with 75mg/L hygromycin and 250mg/L Carb.(4) The transformation of ZxNHX and ZxVP gene and the preliminary identification of transgenic plants. According to transformation procedure showed above, as embryogenic callus and multiple shoots for the transformation acceptor respectively, gene ZxNHX and ZxVP was transformed into Perennial ryegrass by Agrobacterium-mediated,9 and 11 transgenic plants were gained respectively. By PCR analysis,the results showed that there were the target gene in the transformed seedlings, parts of the plants were tested positive.