Protoplasts were isolated from cotyledons of Sesbania bispinosa (Jacq.) W.F. Wight. In a liquid-over-agar culture system with Murashige and Skoog (MS) medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D, 2 mg l(-1) benzyladenine (BA), 1 mg l(-1) glutamine and 0.5 M mannitol, 84% of these protoplasts divided and formed callus. The first division occurred after 3-4 days. Callus formed from the protoplasts differentiated shoots by organogenesis on MS medium with 1 mg l(-1) indolebutyric acid (IBA) and 1 mg l(-1) BA. These shoots developed into complete plantlets when excised and cultured on MS medium with 0.5 mg l(-1) IBA.