Callus formation and plant regeneration from rice protoplasts purified by density gradient centrifugation

Protoplasts were isolated from germinating embryo-derived cell suspensions of rice ( Oryza sativa L.). The protoplast yield from cultured cells was sufficient, but heterogeneity in size and the degree of vacuolation of freshly isolated protoplasts were commonly observed for all preparations from four varieties tested. The use of density gradient centrifugation with media based on a mixture of sucrose and mannitol was effective for separation of a relatively uniform protoplast preparation from the heterogeneous population. The fraction with a high specific gravity from density gradients contained a high percentage of cytoplasmic-rich protoplasts. The plating efficiency (percentage of protoplasts that formed colonies) was improved markedly by the incorporation of this purification step. The purified fraction could be cultured in a liquid medium and a reproducible plating efficiency of up to 0.7% was obtained depending on the variety and the plating density. Otherwise the eliminated fraction with low specific gravity, which contained mainly transparent protoplasts with large vacuoles, rarely underwent sustained divisions. Protoplast-derived calli of tested rice varieties produced plantlets on regeneration media with a frequency of 10–22%. Regenerated plants were transfered to soil and grown to set seeds in a greenhouse.