Transformation Of Vitis Vinifera L.cvs Thompson Seedless With The VpPR10.1 And Study Of Resistance To Downy Mildew

Grapevine（Vitis L.）is an ancient fruit crop which is also one of the most important in the modern world,based on its high economic value.Grapes can be consumed fresh as a popular table fruit,eaten dried as raisins or its expressed juice processed to wine-a drink of huge cultural significance with a large added-value component.However,the European grapevine（Vitis vinifera L.）has coevolved with some significant fungal pathogens.Hence,most V.vinifera cultivars are susceptible to a number of fungal pathogens,with downy mildew being one of the most important,especially in warmer and more humid areas.A more efficient method is described for facilitating Agrobacterium-mediated transformation of Vitis vinifera L.cv.Thompson Seedless somatic embryogenesis using polyamines（PAs）.The efficacies of putrescine,spermidine and spermine are identified at a range of concentrations（10,100 and 1000 μM）added to the culture medium during somatic embryo growth.Putrescine（PUT）and spermidine（SPD）promote the recovery of proembryonic masses（PEM）and the development of somatic embryos（SE）after co-cultivation.Judging from the importance of the time-frame in genetic transformation,PAs added at the co-cultivation stage have a stronger effect than delayed selection treatments,which are superior to antibiotic treatments in the selection stage.Best embryogenic responses are with 1000 μM PUT and 100 μM SPD added to the co-culture medium.Using the above method,a pathogenesis-related gene（VpPR10.1）from Chinese wild Vitispseudoreticulata was transferred into Thompson Seedless for functional evaluation.The transgenic line,confirmed by western blot analysis,was inoculated with Plasmopara viticola to test for downy mildew resistance.Based on observed restrictions of hyphal growth and increases in H₂O₂ accumulation in the transgenic plants,the accumulation of VpPR10.1 likely enhanced the transgenic plant’s resistance to downy mildew.Results were obtained as follow:1.Exogenous free polyamines can increase transformation efficiency of Vitis vinifera grapevine.Addition of spermidine（SPD）100μM or putrescine（PUT）1000μM in the co-culture medium which also contains 1/2MS,20g/L sucrose and 150μL acetosyringone during the transformation give the best response.2.Successful genetic transformation of VpPR10.1 were conducted through Agrobacterium-mediated method of Vitis vinifera L.cv.Thompson Seedlesssomatic embryogenesis.11 transgenic plants were identified out of 70 resistant plants by RT-PCR and western blot,which indicates the transgenic rate is 15.7%.3.Number of haustoria per hypae at 2dpi and sporangia density at 5dpi in the leaves of transgenic line 6904 demonstrated that the transgenic line 6904 has the stronger resistance to Plasmopara viticola.The accumulation of H₂O₂ and inhibition of hypha also verified the high resistance of transgenic line.