Development of Tomato Leaf Curl Virus Resistant Tomato Plants by Post Transcriptional Gene Silencing

Abstract

Tomato (Solanum lycopersicum (Mill.) is one of the most widely grown newlinevegetable crops of India. Tomato leaf curl virus (ToLCV) a member of the genus newlineBegomovirus under the family Geminiviridae is transmitted by whitefly (Bemisia newlinetabaci). Roving survey conducted during rabi 2015-2016 in northern Karnataka newlineindicated that tomato leaf curl disease (ToLCD) was present in almost all fields newlinesurveyed. ToLCD incidence and severity ranged from 5.0 to86.6 per cent and 2.0to 45.2 newlineper cent respectively. Molecular characterization of ToLCV was done by using replicase newline(rep) gene specific primers. Cloning and sequencing of nine isolates were confirmed as newlinestrain of ToLCV. Based on highest sequence similarities with ToLCV sequence newlineavailable in NCBI, ToLCV isolates collected from northern Karnataka was considered newlineas an isolates of Tomato leaf curl Karnataka virus. Replicase gene of ToLCV Dharwad newlineisolate was cloned in ihp generic vector and further subcloned into plant transformation newlinevector (pCAMBIA1305.1). Clones were confirmed for presence of rep gene in generic newlineihp and pCAMBIA1305.1 vector through PCR, it produced an amplicon of ~500 bp rep newlinegenes and further confirmed by restriction analysis, which released ~500 bp band. newlineRecombinant pCAMBIA construct mobilized into the Agrobacteruim tumefaciens by newlinetriparental mating method. The transformed A. tumefaciens clones were confirmed newlinethrough colony PCR and further confirmed through restriction digestion in all test newlineobtained gene specific amplicon. Recombinant Agrobacteruim tumefacien used for the newlinein planta method of transformation of DMT 2 tomato plants. PCR and GUS assay of T0 newlineand T1 transgenic plants indicated the integration of ToLCV rep gene in the genome of newlinetransgenic tomato plants. Resistance of T0 and T1 transgenic tomato plants was newlineconfirmed by challenged inoculation of viruliferous whitefly. No symptoms were newlineobserved in seven T0 and 22 T1 transgenic plants, whereas symptoms were observed in newlinenon-transgenic control plants. newline