Study On Somatic Embryos Induction Of Vitis Vinifera（L.）and Transformation Of Chinese Wild Vitis Stilbene Synthase Gene

In this study, the stamens, pistils and flowers from3kinds of European grape varieties"Thompson Seedless","Carignane","Red Globe" were tested as explants. And the somaticembryos were induced form these floral organs. The stilbene synthase gene and its specificpromoter were previously isolated from Chinese wild Vitis rseudoreticulata accession"Baihe-35-1". We transfered this gene to somatic embryo cells of those grape cultivars aboveby Agrobacterium-mediated transformation. The main results of this study are as follows:1. The embryonic callus were induced form stamens, pistils and flowers of these3kindsof different European grape varieties in5kinds of different medium. The inducing rateindicated that the MC medium (NN69inorganic elements+B5organic elements+0.55mg·L^-12,4-D+0.505mg·L^-1NOA+1.24mg·L^-14-CPPU+500mg·L^-1glutamine+3g·L^-1Phytogel+3%sucrose, pH5.8) is optimal for Thompson Seedless, Carignane and Red Globe stamensembryogenic callus induction, the induction rate is1.7%,0.3%and11.8%; PIV medium(NN69inorganic elements+B5organic elements+1.0mg·L^-12,4-D+2.0mg·L^-1BA+3g·L^-1Phytogel+6%sucrose, pH5.7) is best for Thompson Seedless and Carignane pistilsembryogenic callus induction, the induction rate is4.0%and3.2%; only flowers from RedGlobe in MS1medium (MS inorganic elements+B5organic elements+0.55mg·L^-12,4-D+0.505mg·L^-1NOA+1.24mg·L^-14-CPPU+500mg·L^-1glutamine+3g·L^-1Phytogel+3%sucrose,pH5.8)could induce embryogenic callus and the induction rate is0.5%.2. Somatic embryos can be induced from all kinds of explants in hormone-free somaticembryo induction medium (X6medium+3g·L^-1Phytogel+0.5g·L^-1AC+2%sucrose, pH6.0).The improved X6liquid medium (X6inorganic elements+MS organic elements+1.0mg·L^-1IAA+1.0mg·L^-1BA+6%sucrose+400mg·L^-1glutamine,pH5.8) is the best medium forsecondary somatic embryo induction.3. Somatic embryo germination medium (1/2MS medium+1.75mg·L^-1IAA+0.35mg· L^-1GA+3%sucrose+0.25g·L^-1AC+3g·L^-1Phytogel) have the best effect for somatic embryosgermination in the3kinds of germination medium tested.4. The enhanced expression vector contained Chinese wild grape stilbene synthase geneand its specific promoter and other functional elements was transformed into the Europeangrape somatic embryos with the Agrobacterium-mediated transformation method. Two "Thompson Seedless" transgenic plants were obtained after antibiotics resistance gene screening,PCR and PCR-Southern blot analysis.