ORIGINAL PAPER Control of pattern of regenerant differentiation and plantlet production from leaflet segments of Azadirachta indica A. Juss. (neem) Kavita Arora Æ Meena Sharma Æ A. K. Sharma Received: 12 June 2008 / Revised: 12 October 2008 / Accepted: 30 October 2008 / Published online: 21 November 2008 Ó Franciszek Go ´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako ´w 2008 Abstract 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. A two-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 meriste- matic 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 treat- ment, where the explants were exposed to 8.88 lM BAP and 81.43 lM adenine hemisulphate for 5 days followed by their transfer to 0.88 lM BAP and 81.43 lM adenine hemisul- phate, on an average, 17.4 shoot buds and only 1.6 meristematic nodules were formed from a leaflet. On sub- culturing, 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 lM BAP, 1.43 lM indole-3-acetic acid (IAA) and 135.72 lM adenine hemisulphate. The isolated shoots were rooted and complete plantlets were transferred to potted soil with 100% survival. Keywords Azadirachta indica Á Cloning Á Leaflet segments Á Meristematic nodules Á Shoot buds Abbreviations BAP 6-Benzylaminopurine IAA Indole-3-acetic acid IBA Indole-3-butyric acid 2iP N 6 -(2-isopentenyl) adenine MS medium Murashige and Skoog medium TDZ Thidiazuron or (N-phenyl-N 0 - (1,2,3-thiadiazol-yl) urea Z Zeatin Introduction Azadirachta indica A. Juss., commonly known as Neem, is an indigenous multipurpose tree and highly valued for its medicinal and pesticidal properties. Due to environmental and genetic factors, the content of azadirachtin in different accessions varies considerably (Wewetzer 1998). Due to ever-increasing global demand of neem trees for com- mercial use and the limitations in conventional methods of vegetative propagation, the biotechnological approach is warranted to develop methods for rapid clonal multiplica- tion of elite trees, since the trees produced through seeds exhibit genetic variation. In a number of higher plants the explants respond differently to external morphogenetic stimuli and differentiate a variety of regenerants such as, a shoot bud, unaccompanied and accompanied by root Communicated by E. Lojkowska. K. Arora (&) Á M. Sharma Á A. K. Sharma Tissue Culture Laboratory, National Botanical Research Institute, Lucknow 226 001, India e-mail: aks_nbri@rediffmail.com 123 Acta Physiol Plant (2009) 31:371–378 DOI 10.1007/s11738-008-0244-5