In the present study regeneration efficiency and genetic clonality are the two major aspects for in vitro propagation of candidate plus tree of Pongamia pinnata, a versatile biodiesel legume. Woody Plant Medium (WP) and Murashige and Skoog's medium (MS) supplemented with different concentrations and combinations of plant growth regulators were screened for high frequency regeneration using nodal segment culture and axenically grown seedlings of elite genotype of P. pinnata. Percentage response from field-grown mature nodal segments of P. pinnata were highly dependant on the season, with greater than 68% of culture developing adventitious shoots during spring. Woody Plant Medium supplemented with benzyladenine (5.0 mg L (1)) and kinetin (0.5 mg L (1)) gave the greatest response to initiation and multiplication. The multiplication rate of 11 shoots per explants with an average shoot length of 3.0 cm was observed. Multiplied shoots started to produce roots in the multiplication medium itself containing BA and NAA but subsequent establishment was poor. The rooting response was enhanced in half-strength MS media with indole-3-butyric acid (0.5 mg L (1)). Rooted plants were hardened successfully in glass house with 70% survivability. RAPD and ISSR markers were employed to determine the genetic fidelity of in vitro raised plantlets. (C) 2012 Elsevier Ltd. All rights reserved.