The Tissue Culture And Construction Of Agrobacterium-mediated Transformation System For Oat

BaiYan10naked oat is selected by means of using pedigree method breeding on F3generations materials of Canada introduced by Academy of Agricultural Sciences inBaiCheng city of JiLin province. Oat is a kind of crop which has strong resistance, and if weconstruct an efficient system of the tissue culture and transformation for oat, it will playimportant significance for researching the resistance gene of oat and improving oat varieties.We have developed procedures of stable and effective regeneration andagrobacterium-mediated transformation of BaiYan10Oat, which will lay the foundation forthe researching the transformation on BaiYan10Oat and other series of BaiYan Oat. Themain research results are presented as follows:1. Construction of efficient system of the tissue culture and regeneration for BaiYan10Oat”(1) Mature embryo is the most suitable explant which was ascertained by comparingcallus ratio and differentiation rate of seeds, mature embryos, leaf base segments and apicalmeristem.(2) We have screened out the optimum callus induction medium of BaiYan10Oat whichis MS+2，4-D0.5mg/L+NAA0.1mg/L.(3) Optimize the regeneration medium which is MS+6-BA1.5mg/L+NAA0.4mg/L+IAA0.4mg/L, through orthogonal experimental design.2. Construction of genetic transformation system for BaiYan10Oat(1) For the transformation, the screener is homomycin，and20-30mg/L is the optimizedconcentration, by sensitivity test of callus to homomycin; The test of agrobacteriumEHA105for cephalosporins and carbenicillin with sensitivity shows that carbenicillin is more suitablefor bacteriostat, and200-400mg/L is the optimized concentration.(2) We have ascertained that OD600=0.5-0.8is the optimized concentration and10min isthe optimized time for callus differentiation, by employing agrobacterium-mediatedbacterium fluid with different concentration to inflect the callus.(3) Genetic transformation: the agrobacteriumEHAl05, which was infected by plasmidpCAMBIA1304-GFP, and the A. tumefaciens suspension whose concentration is OD600=0.5was used to infect an old callus which was induced during one month for10min, and theywere co-cultured for the next3days on the cocultivation medium which was add into200uMacetosyringone. Next, they were transferred to that screening culture medium supplemented with25mg/L homomycin and350mg/L carbenicillin. The subsequent culture was replacingthe same media with the same selection agent added every10days. After the medium wasreplaced two times, the callus were transferred to the differentiation medium, when theregrowth long to3-4cm, transferred them to the rooting medium, at last, tried seedlings andtransplanting.(4) Identification of genetically modified oats: as the plasmid contains GFP genes, wecould observe the callus slice with fluorescence microscope, and then the green fluorescenthas been observed, which preliminary evidence that the plasmid has been integrated into thegenome of oats. There are78PCR positive plants involved in132regeneration plants, and thePCR positive rate is59.1%. Then total RNA is extracted from some of the PCR positiveplants, which is used for RT-PCR analysis, and all the plants assayed were positive.