Plant regeneration from mesophyll protoplasts of rice (Oryza sativa L.)

We report induction of sustained divisions, colony formation and plant regeneration from mesophyll protoplasts isolated from the leaf base and sheath of rice seedlings. Protoplasts were purified on sucrose density gradients and cultured in modified liquid N6 medium in the presence of a feeder layer prepared from young rice-cell suspension cultures. Protoplasts formed cell walls and divided after 6 to 7 days giving rise to microcolonies which became macroscopic after 25 to 30 days. Nine to ten week old protocalli, on transfer to regeneration medium, formed somatic embryos that developed into shoots which were rooted on MS medium. Plantlets were recovered from four varieties, which on subsequent transfer to Yoshida's culture solution developed into healthy plants that were transferred to pots for further growth. Our results show that contrary to popular belief, mesophyll protoplasts of rice and perhaps other gramineous plants are capable of dedifferentiation and re-entry into the cell cycle, and are totipotent.