Study On Construction Of RNAi Vectors And Resistance To TYLCV In Transgenic Tomato

Tomato is an important cultivated breed of fruits and vegetables. Tomato yellow leafcurl virus (TYLCV), which belongs to the genus Begomovirus in the family Geminiviridae,is transmitted by whitefly Bemisia tabaci and cause serious losses to tomato production.The best way to control TYLCV is to cultivate vrius-resistant cultivated breed. RNAinterference (RNAi), one of the useful technologies to breed virus-resistant tomato isconcerned by its high efficiency, specificity, heredity and biological safety.In this work, three regions from open reading frames (ORFs) of the viral genome C1,C2, and the overlap region C1C2were used for studying their efficiency to target viralDNA accumulation in plant cells. Each of the three regions had been cloned andconnected into pFGC5941in sense/antisese directions to construct three RNAi vectorspFGC5941-FC1-RC1, pFGC5941-FC2-RC2and pFGC5941-FC1C2-RC1C2.The pFGC5941-FC1-RC1, pFGC5941-FC2-RC2and pFGC5941-FC1C2-RC1C2vectors were transfered into Agrobacterium tumefaciens GV3101, which were introducedinto tomato plant tissues via Agro-infiltration technique. Tomato plants at two-three leavesstage were infiltrated with Agrobacterium harboring the RNAi constructs. All plants werechallenged with virus using the TYLCV infectious clone20days post infiltration. Toidentify the resistance, TYLCV symptoms of tomato plants were investgated and virusDNA was detected by PCR reaction. The result showed that all the three fragments caninhibit or reduce accumulation of viral genome due to the induction of siRNA mechanism.The infiltered plants with the construct containing the as C2region proved to be the bestregion for inducing the resistance to TYLCV with87.5%inhibtion rate.The results of the study showed that the induction rate of callus and adventitious budof cotyledon were higher than that of hypocotyl. The best medium for callus and buddifferentiation of cotyledon was MS+0.2mg/L IAA+1.0mg/L6-BA, for hypocotyl wasMS+0.5mg/L IAA+1.0mg/L6-BA, and the root induction rate was the highest on MSmedium supplemented with0.1mg/L IAA. The optimised experimental conditions oftomato transformation by agrobacterium-mediated transduction were agrobacterium liquidconcentration OD600=0.2, infecting time20min(cotyledon, budding rate reached33.3%)or30min (hypocotyl, budding rate reached23.3%) and coculturing for2days.