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 regeneration from protoplasts isolated from >1-yr-old cell-suspension culture of Indica rice (Oryza sativa) cv. Pusa Basmati 1. A two-step regeneration procedure, involving the transfer of calluses to shoot-regeneration medium containing 1% (w/v) agarose prior to culture on a medium containing 0.4% (w/v) agarose, was found to improve plant regeneration. High concentrations of kinetin in the regeneration medium were also found to be beneficial. The two-step regeneration procedure, combined with a high concentration of kinetin (10.0 mg 1^-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 reached 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.