Small Rna Based Genetic Engineering For Virus-resistant Potato Plants

Potato is currently the third-largest crop in terms of human consumption after wheat and rice.China is the largest potato producer worldwide,which produces 90 million tones accounts for almost one-quarter of global potato production,and about 28% of total cultivated areas.Meanwhile,potato is vegetatively propagated crop,the virus can accumulate in the plant from generation to generation and cause 50% or more of the potential total yield being lost in single virus,whereas mixed viruses can reach more than 80% lost and affecting the quality as well by inducing internal and surface tuber necrosis.Thus,viruses are among the most economically important biotic constraints in potato production.Much research in recent years has published about 40 viral species to infect potato worldwide,only 11 were found in China.Among those 11 six viruses have been recognized as major potato viruses: PLRV,PVA,PVM,PVS,PVX,and PVY.However,the lack of germplasm resistant for almost of virus diseases,viruses have developed various mechanisms to overcome the plant resistance.Thus,in this study we delivered double-stranded RNA to initiate the RNA interference to target different conserved regions within the six major potato virus genomes: PLRV,PVA,PVM,PVX,PVY,PVS.We used microtubers and internodes as transformation explants in comparative manners.We experienced high transformation efficiency compare with microtubers and high transformation frequency as well from 10-days-old internodes.Using internodes can reduce the cost of carbon source and incubation of microtuberization cultures.Using internodes keeps the transformation in a closed cycle with the micropropagation which reduces the time and efforts.Ten-days-old internodes are much recommended than threeor four-weeks-old.The number of generated transformed shoots varies from vector to vector as a result of foreign gene effect on the Agrobacterium cells;e.g.S17,S51,S34 strains form multicellular clumps that eventually settle at the bottom of culture tubes or flasks and cause bacterial overgrowth to lead to low transformation efficiency.A total of 81 Transformed AC142-01 plants were regenerated.Thirteen engineered with different artificial s RNAs precursor to target several conserved regions within PLRV,another 16 for PVA,4 for PVS,1 for PVY,3 for PVX,8 for PVM based on ami RNA enclosed in intron “GUS” strategy.Eight GUS and 28 ami RNA enclosed in exon “PDS engineered with different artificial s RNAs precursor to target different conserved regions within multiple viruses(PLRV,PVA,PVY,PVX,PVS,PVM).The screening of transformants was done at the genomic DNA level by PCR.Transgenic lines AC142-01 modified with ami RNA-GUS precursor to target several conserved regions within the PLRV genome shows specific resistance to the virus.Thus,PLRV accumulation and disease symptoms were decreased in the transgenic lines I12 and I25 based on ELISA assay and phenotype characterization.