Impact of cefotaxime and kanamycin on in vitro regeneration via Agrobacterium mediated transformation in apple cv. Red Chief

During gene transfer in apple, optimization of antibiotics concentration in post co-cultivation process is critical for the establishment of an efficient transformation system. Keeping in view, the doses of kanamycin (5–25 mg/l) and cefotaxime (100–500 mg/l) alone or in combinations were evaluated for their effects on callus induction and shoot regeneration. Cefotaxime at 100–500 mg/l was also tested for the inhibition of Agrobacterium tumefaciens strain EHA105 following co-cultivation with leaf explants of apple cv. Red Chief. In this study, leaf explants were found highly sensitive to kanamycin, a widely used antibiotic in transformed plant selection and recovery of transgenic plants. Low kanamycin doses of 5–8 mg/l resulted in callus induction and shoot regeneration. Maximum shoot regeneration frequency (60.56%) was obtained on 5 mg/l kanamycin and gradually reduced (12.84%) upto 8 mg/l. It reduced callus induction, completely inhibited the regeneration at 9–25 mg/l and developed chlorosis of leaves. Cefotaxime at 100–200 mg/l enhanced the regeneration rate (95–100%) from leaves comparable to control while higher concentrations gradually reduced the frequency of regeneration with decreased number of shoots per explant. However, no callus and regeneration were observed when kanamycin was combined with cefotaxime at various concentrations. Omission of kanamycin from initial regeneration phase for few days resulted in shoot induction. To eliminate the Agrobacterium overgrowth from cultured leaves, 500 mg/l cefotaxime was found effective. As this concentration inhibited the shoot induction, it was gradually reduced to 200 mg/l over a period of 7 weeks, which stimulated morphogenesis and later formed shoots. Thus, the optimized doses established in the present study could be applicable to suppress the excess Agrobacterium growth and to generate transformed shoots in further genetic transformation work on ‘Red Chief’.