Genetic Transformation Of The VpCN Gene From Chinese Wild Vitis Pseudoreticulata In Thompson Seedless

Grapevine(Vitis spp.)is one of the most important economic fruit all over the world.Cultivated grapevine(Vitis vinifera L.),particularly,occupies the significant position in viticulture,owing to its good quality,genuine flavor and high output.However,a fungus diseases powdery mildew causes enormous economic loss to grapevine industry.An effective approach is through genetically modified technology to obtain disease resistant cultivated grapevine in oriented direction.Chinese wild grapevine genotype Vitis pseudoreticulata Baihe-35-1 contains a high resistance to powdery mildew,and identification and function analysis of relative disease resistance gene to powdery mildew from Chines wild Vitis have excellent significance to grapevine breeding.Our research group previously cloned a VpCN gene from Chinese wild grapevine Vitis pseudoreticulata Baihe-35-1,which showed the resistance to powdery mildew in transgenic Arabidopsis thaliana.In order to further verify this gene function,five grapevines were established the somatic embryogenesis in this study,and used pro-embryogenic mass as the acceptor materials,this gene was transformed into susceptible powdery mildew European grape Thompson Seedless by co-cultivation with Agrobacterium tumefaciens.This provides a basis for improvement of European grapevine diseases resistance and the main results of this study are described as follows:1.Induced embryonic callus and somatic embryos of Vitis vinifera cv.Thompson Seedless,Chardonnay,Red Globe,Vitis quinquangularis Shang-24 and Vitis davidii Tang Wei.Anthers,ovaries,and flower buds of this five grapevines were used as the explants and inoculated on MC、MS1 and DM medium.Anthers of Vitis vinifera cv.Thompson Seedless on MC medium which contains 30 g/L sucrose,2.5 uM 2,4-D,2.5 uM NOA,5.0 uM 4-CPPU,0.1 g/L inositol and 7 g/L TC agar were the highest efficiency of embryonic callus induction(24%).X6 medium(MS+60 g/L sucrose+7 g/L TC agar)was more suitable for somatic embryos induction than NB medium.2.The plant regeneration of Thompson Seedless was obtained via somatic embryogenesis.DM medium(DKW+30 g/L sucrose+2.5 uM 2,4-D+5.0 uM 6-BA+2.5uM NOA+1.0 g/L inositol+7 g/L TC agar)was more suitable for secondary embryonic callus induction.3.Based on the Agrobacterium-mediated transformation system,VpCN gene was transformed into Thompson Seedless grapevine to obtain disease-resistance grapevines.But now the kanamycin resistance somatic embryos were just formed.Moreover,pro-embryogenic mass as the acceptor materials were more suitable for genetic transformation than somatic embryos in Thompson Seedless.4.The subcellular localization vector of pBI221-VpCN-GFP was constructed,and introduced into onion epidermal cells by Agrobacterium tumefaciens.The finding is that VpCN located in the nucleus.