A simple and efficient procedure to improve plant regeneration from protoplasts isolated from long-term cell-suspension cultures of Indica rice

A simple and efficient method has been developed to improve plant regenerat ion from protoplasts isolated from >1-yr-old cell-suspension culture of Ind ica rice (Oryza sativa) cv. Pusa Basmati I. A two-step regeneration procedu re, involving the transfer of calluses to shoot-regeneration medium contain ing 1% (w/v) agarose prior tc, culture on a medium containing 0.4% (w/v) ag arose, was found to improve plant regeneration. high concentrations of kine tin in the regeneration medium were also found to be beneficial. The two-st ep regeneration procedure, combined with a high concentration of kinetin (1 0.0 mg l(-1)) in the medium, significantly increased plant regeneration. By this method, even though protoplasts were isolated from over 1-yr-old cell -suspension cultures, protoplast-derived plant regeneration frequency reach ed 16.1%, compared with <4% regeneration frequency without such treatment. Use of a similar protocol might improve plant regeneration from other plant species, especially recalcitrant species.