Abstract An in vitro regeneration system was developed using organogenic callus derived from in vitro grown cotyledonary explants of Gleditsia caspica Desf., an important leguminous tree. Murashige and Skoog (MS) basal medium augmented with 0.2 g L-1 myo-inositol and various concentrations of either 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid, or indole-3-butyric acid (IBA) alone as well as combined with cytokinins was used for callus induction. The highest frequency of organogenic yellowishwhite and nodular callus (93 %) was obtained from explants grown on medium supplemented with 13.5 lM 2,4-D and 4.4 lM benzyladenine (BA). The yellowish-white and nodular callus when transferred to MS medium supplemented with BA (2.2–17.7 lM) or kinetin (KT; 2.3–18.8 lM) solely or in combination with 2.3 lM 2,4-D produced several microshoots after 5 weeks culture. The calli cultured on MS medium with 4.4 lM BA singly showed superior growth response and produced both maximum shoot regeneration (94 %) and the highest mean number (4.3) of microshoots per callus. Transfer of regenerated microshoots onto modified MS basal medium fortified with 5.8 lM gibberellic acid and 4.4 lM BA resulted in the maximum number of internodes per shoot and the highest shoot elongation after a period of 6 weeks. Optimum rooting of 90 %, an average 6.1 roots per shoot, and a mean root length of 3.6 cm was observed when half-strength MS medium was supplemented with 9.8 lM IBA and 0.92 lM KT. The regenerated healthy plants with well-developed shoots and roots showed a survival rate of 77 % after acclimatization and transplanting to garden soil for a 10-week hardening period under ex vitro conditions.
