Optimization of critical determinants for efficient transformation in M7 apple root stock

Optimizing the critical determinants for achieving efficient genetic transformation in apple has been considered as the first and foremost step. Therefore, in the current study, effect of different cytokinins-auxins, antibiotics, pre-culturing, co-cultivation duration and infection time were investigated in an effort to increase the efficiency of producing putative transformants in M7 apple rootstock. The findings demonstrated that MS medium supplemented with 4 mg/l BA and 0.5 mg/l NAA resulted in 20 per cent shoot regeneration, whereas, 0.6 mg/l TDZ with 0.5 mg/l NAA in 41.46 per cent regeneration. Cefotaxime at low concentration (200-300 mg/l) was not only non-toxic but also stimulated shoot regeneration. Agrobacterium tumefaciens strain LBA4404 containing pCAMBIA 2300 plasmid encoding neomycin phosphotransferase II and rolB gene were used for leaf transformation. It was found that pre-culturing for 48 hours, an infection time of 7 min and co-cultivation for 48 hours resulted in induction of callus/shoots. To overcome bacterial overgrowth after co-cultivation, 500 mg/l cefotaxime was found appropriate.