Rapid in vitro cloning of a 40-year-old tree of Azadirachta indica A. Juss. (Neem) employing nodal stem segments

An efficient in vitro process for rapid clonal propagation of a 40-year-old tree of Azadirachta indica employing nodal stem segments was developed. Season of collection and maturity of explants showed direct influence on bud-break. Nodal stem segments collected during the month of April gave best response. Maximum bud-break (78.6-81%) was obtained when middle order nodes (3rd or 4th node from apex) were taken. Amongst different cytokinins used, 6-benzylaminopurine (BAP) at the concentration of 1.11 mu M was found most effective in inducing multiple shoots, whereas inorganic and organic constituents of the medium influenced growth and general condition of proliferating shoots. On an average 3.1 shoots per explant were regenerated in modified Murashige and Skoog medium supplemented with 1.11 mu M BAP, 1.43 mu M indole-3-acetic acid (IAA) and 81.43 mu M adenine hemisulphate. Isolated shoots were rooted in presence of 2.46 mu M indole-3-butryic acid (IBA). Root induction took place in 8-10 days with 100% rooting. The in vitro-raised plantlets were successfully transplanted in potted soil and finally grown under field conditions with 100% survival. The genetic fidelity of such in vitro-raised field-grown plants was ascertained by random amplified polymorphic DNA (RAPD) markers. Furthermore, the azadirachtin content of in vitro-cloned plants was found comparable to the mother tree proving their chemical stability also. The protocol developed holds good for in vitro cloning of mature elite neem trees.