A process with controlled pattern of regenerant differentiation from leaflet segments leading to production of cloned plants of a 40-year-old tree of Azadirachta indica was developed.Atwo-step procedure was adopted for containing intervening callusing during regenerant differentiation using modified Murashige and Skoog (MS) medium, where in the first step the explants were subjected to pulse treatments having higher concentration of 6-benzylaminopurine (BAP), while in the second step they were cultured in one-tenth of the initial concentrations of BAP. In the present case, simultaneous differentiation of two types of morphogenetic structures, that is, shoot buds and the meristematic nodules was observed. However, differentiation of higher number of desirable regenerants—the shoot buds and a few meristematic nodules, rather than vice-versa could be controlled by increasing both, the concentration of BAP in pulse treatment and the duration of pulse treatment. In the optimum treatment, where the explants were exposed to 8.88 µM BAP and 81.43 µM adenine hemisulphate for 5 days followed by their transfer to 0.88 µM BAP and 81.43 µM adenine hemisulphate, on an average, 17.4 shoot buds and only 1.6 meristematic nodules were formed from a leaflet. On subculturing, the shoot buds developed into shoots, whereas the meristematic nodules produced three kinds of organized structures that too in varied proportions. Multiplication of shoots was sustained in proliferation medium supplemented with 1.11 µMBAP, 1.43 µMindole-3-acetic acid (IAA) and 135.72 µM adenine hemisulphate. The isolated shoots were rooted and complete plantlets were transferred to potted soil with 100% survival.
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