Development of high frequency in vitro shoot regeneration system from leaves of apple cultivar ‘Oregon Spur’ and optimization of antibiotics concentration

Abstract

Optimization of efficient shoot regeneration protocol and selection of transformed plants at optimum level of antibiotics are pre-requisite in genetic engineering. Here, we explored standardization of in vitro shoot regeneration protocol from leaf explants of apple cv. Oregon Spur for the first time using different concentrations of plant growth regulators. Further, we determined the effect of two antibiotics (kanamycin and cefotaxime) on shoot regeneration efficiency. About 95% of leaves induced adventitious shoots on MS medium supplemented with 4 mg/L BA and 0.2 mg/L NAA. The frequency of shoot regeneration increased to 100% when leaf explants were cultured on 2 mg/L TDZ along with 0.5 mg/L NAA. The number of shoots regenerated per leaf was enhanced but abnormal and vitrified shoots were obtained in case of TDZ supplemented medium. Dark incubation of initial one week decreased the shoot regeneration frequencies at all the combinations of plant growth regulators. Leaf explants were found highly sensitive to kanamycin and inhibited shoot induction above 5 mg/L. This concentration was found suitable for selection of transgenic shoots. Cefotaxime at 100-300 mg/L promoted organogenesis, whereas 500 mg/L was found optimal to eliminate Agrobacterial overgrowth after co-cultivation with leaf explants. Our results have demonstrated that the above protocol can successfully be applied for transgenic development in ‘Oregon Spur’ cultivar of apple in future trials.