Transformation With An Arabidopsis Broad-Spetrum Resistance Gene RPW8.2 In Garpevine (Vitis Vinifera) Cv.Thompson Seedless

The most economically important disease of cultivated grapevines worldwide is powdery mildew caused by the biotrophic fungal pathogen Erysiphe necator.The main of the grapevines widely cultivated around the world are European grapevines and they are susceptible to powdery mildew.Therefore,to improve grape resistance via integrating effective genetic resistance genes into European grapevines becomes obviously important in grape breeding.Using somatic embryos as genetically modified material,an Arabidopsis broad-spetrum resistance genes RPW8.2 was successfully transformed for over-expression.Expression of the RPW8.2 and resistance of transgenic European grapevine lines were further analyzed.The main results are listed as follows:1.Induced embryogenic callus of Vitis vinifera L.cv.Thompson Seedless.Anthers and flower buds were used as the explants and inoculated on medium PIV(which contain 60 g/L Sucrose,1 mg/L 2,4-D,2 mg/L 6-BA,0.1 g/L Inositol and 1 g/L Active chlorite),then subcultured until embryonic callus were induced.Gradually embryogenic callus were germinated to mature somatic embryos,somatic embryos induced in the secondary medium KBN(which contain 30 g/L Sucrose,1 g/L Inositol,0.3 g/L KNO3,1.126 mg/L 6-BA,0.552mg/L 2,4-D,0.5505 mg/L NOA,and 3 g/L Phytagel)obtained on embryogenic callus.With the modified phenol fuchsin,observing for cell morphology,the results show that embryonic callus cells were stained darker and densely,whereas non-embryonic callus cells were lighter and loose.2.Vectors with fusion gene RPW8.2-YFP were transferred into Thompson Seedless via Agrobacterium tumefaciens.After cocultivation,embryonic callus were cultured on the selection medium to produce the resistance lines.The resistance lines were detected bymolecular techniques,which showed that gene RPW8.2-YFP was integrated into Thompson Seedless.The rate of the liquid co-cultivation medium KBN cultured was 93.7 %,1/2MS(which contain MS and 30 g/L Sucrose)was 4.7 %.80 transgenic lines with gene RPW8.2 of native promoter were obtained.22 transgenic lines with 35 S promoter were obtained.3.Resistant embryos culture on seedling medium(which contain MS and 0.2 mg/L6-BA),lines of native promoter to grow weakly and slowly,rate of survival was only 8.75 %,while the survival rate of 35 S promoter was zero;rooting medium(which contain MS and IBA)culture,when the IBA concentration was 1.0 mg/l,rate of survival native promoter lines was 100%,while 35 S promoter plants was 54.17 %.4.Transgenic lines of Thompson Seedless enhanced the resistance to powdery mildew.RPW8.2 transgenic plants positively response to the induction of powdery mildew,mainly inhibited mycelial growth while making powdery mildew haustorium deformity,H2O2 got accumulation.In 5 mM SA process,the expression of RPW8.2 gene also had been induced.