Enhanced somatic embryogenesis and agrobacterium-mediated transformation of three cultivars of tomato by exogenous application of putrescine

Efficiency of Agrobacterium mediated transformation of tomato with coat protein (CP) of Tomato yellow leaf curl virus (TYLCV-OM) was optimized using different media. Cotyledons and epicotyls parts from susceptible tomato cvs. Moneymaker, Pusa Ruby and Jinan were used as explants and co-cultivated with Agrobacterium tumefaciens strain AGL1 harbouring CP gene. The effects of putrescine, vitamins, explants and growth regulators on plant transformation and regeneration were studied. Optimal shoot regeneration and callus induction was obtained with 1 μM putrescine, 0.5 μg/mL zeatin riboside and 0.1 μg/mL indole acetic acid. Maximum transformation efficiency was obtained with bacterial concentration of 1x10⁸ cells incubated for 48 h. The above conditions increased transformation efficiency up to 49.2% using cotyledons as explant. Gene specific primers were used to confirm the presence of transgene in the primary transformants selected on kanamycin. Transformation efficiency using cotyledons and epicotyl was found to be 49.2%, 33% in Moneymaker, 32%, 27% Jinan and 48.1%, 32% in Pusa Ruby respectively. All the values were found significantly different (p< 0.05) using LSD. The study shows that exogenous application of putrescine combined with zeatin riboside can enhance regeneration ability of cotyledon as well as epicotyl and decrease rhizogenesis and expansion of necrotic lesions.