Genetic engineering of sunflower (Helianthus annuus L.) for resistance to necrosis disease through deployment of the TSV coat protein gene

Necrosis disease incited by tobacco streak virus (TSV) causes significant yield losses in sunflower in the tropics and sub-tropics. Genetic engineering of sunflower through deployment of the coat protein gene of TSV (TSV-CP) was attempted along with neomycin phosphotransferase gene (nptII) for selection of putative transformants on kanamycin. Split cotyledons from decoated mature seeds were used as target tissues for Agrobacterium tumefaciens-mediated transformation and putative transformed shoots were obtained with an average frequency of 3.3%. Presence of the introduced transgene was identified by PCR; expression was determined through RT-PCR, northern blot analysis and quantitative Real-time PCR (qRT-PCR); stable integration was ascertained through Southern blot analysis of plants in T-0 to T-4 generations. Out of 102 positive T-0 events, 20 events were carried to T-1 generation from which five events (CP-S-237, CP-S-247, CP-S-481, CP-S-648 and CP-S-753) were advanced till T-4 generation. Challenge inoculations of plants of event No 481 showed resistance to necrosis disease and plants grew to maturity in TSV-CP transgenics while control plants (untransformed) showed mortality within 1-2 weeks following inoculation. Expression analysis of the TSV-CP and nptII genes in different tissues at flowering and seed setting stages revealed constitutive expression of the transgene till seed maturation. One event (No 481) was selected for transfer of the TSV-CP gene into agronomically superior genotypes.