A reproducible protocol for adventitious shoot regeneration from leaves of apple rootstock Merton793 and assessment of genetic stability

Abstract

The present studies were aimed to develop an efficient and reproducible method for shoot regeneration from leaf explants of apple rootstock Merton793. The influence of two cytokinins BA and TDZ with three auxins IAA, IBA and NAA in different combinations were evaluated for shoot induction using light and dark incubation. Direct and healthy shoots were obtained from leaves on MS medium with BA-NAA and BA-IAA combinations in light incubation and 100% regeneration was achieved on medium with 4 mg/l BA and 1 mg/l NAA. Both direct and indirect shoot regeneration was obtained on TDZ-NAA and TDZ-IBA. Maximum shoot regeneration was promoted on 0.8 & 0.6 mg/l TDZ with 1 mg/l NAA, however, it showed abnormal, vitrified, small and rosette type of shoots. Dark treated leaf explants showed low rates of shoot induction with TDZ combinations. Healthy adventitious shoots regenerated directly from leaves were multiplied, rooted and hardened successfully. RAPD analysis of regenerants revealed that all the banding profiles from directly originated shoots were monomorphic and similar to those of the mother plant, while shoots originated through intervening callus revealed 61.2% polymorphism. The results show that genetic fidelity of directly produced adventitious shoots was maintained, and thus, our regeneration protocol can be an useful method in transformation research in Merton793 without the induction of variation.