Optimization of regeneration and parameters affecting Agrobacterium-mediated transformation of commercial cultivar of cotton (Gossypium hirsutum L.)

Optimization of a plant regeneration protocol for revitalize recalcitrant species like cotton (Gossypium hirsutum L.) from meristem apex culture with higher efficiency will put spurt on applications of functional genomics and selective breeding programs using transformation technologies. Despite of many gene transformation studies in cotton, a limited success has been achieved because of inherent limitation like low regeneration rate, genotype dependency and, variable response in selected group of cultivated varieties. In present study, we illustrated an efficient protocol for genetic transformation in six commercial cultivars of cotton, using a vector containing nptII gene. Several parameters were investigated to optimize genetic transformation of these cultivars. The results showed that, the highest regeneration percentage (98 %) was observed with MS, 0.1 mg/L NAA, 0.1 mg/L 6-BA, 2.5 g/l phytage. The high transformation rate was achieved using short pre-incubation in medium contaning cytokine for 4-5 h, inoculating for 20 min in MS medium containing 100 lM acetosyringone, 1mg/l kin with agrobacterium 0.3 OD600 and co-culturing for two days with media containing acetosyringone. PCR and kan-painting identification of T0 and T1 regenerated plants revealed that the nptII gene is successfully integrated into cotton genome with successful expression in transgenic plants.