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An efficient protocol for in vitro multiple shoot bud induction and plant regeneration from mature green cotyledon derived callus tissues of Acacia sinuata has been developed. Callus formation occurs at all the concentrations of thidiazuron (TDZ) in Murashige and Skoog’s (MS) medium, but 0.6 μM proved to be the best with maximum callus formation frequency. Supplementation of TDZ in combination with indole-acetic acid (IAA) in MS media accelerates shoot bud organogenesis in differentiating callus tissues with 60-70% conversion of shoot buds into shoot. Most efficient shoot organogenesis was recorded when TDZ induced calli were subcultured at different concentrations of 6-benzyladenine (BA). Optimum shoot bud induction and plant regeneration from callus was achieved when 0.6 μM (TDZ) induced calli were subcultured at 3.0 μM (BA) where 16.6 ± 0.74 shoots/unit callus were obtained. Rooting in in vitro differentiated shoots was achieved when transferred to medium containing different concentration of indole-3-butyric acid (IBA) in full & half strength MS medium. The well rooted plantlets were hardened and transferred to net house with 90% survival rate.