55. Isolation of protoplasts from Oryza sativa L. and its wild relatives

Plant Breeding Institute, Faculty of Agriculture, Hokkaido University, Sapporo, 060 Japan

The isolation of rice protoplasts and their subsequent regeneration has been achieved by several workers. However, wild Oryza species have not been tested. With the hope of incorporating into rice cultivars some favorable genes of wild species through cell fusion and gene manipulation in the future, we have examined protoplast isolation from seed calli using a number of strains belonging to four species of Oryza.

Calli induced from dehulled seeds were cultured on N6 medium containing 4.4 mg/1 2,4-D and 50 mg/1 sucrose. For protoplast isolation, the seed calli were immersed in enzyme solution containing 1% driselase, 2% cellulase "Onozuka" RS, 2% cellulase "Onozuka" R-10, 2% macerozyme R-10, 1% hemicellulase, 0.1% pectoryase Y-23, 0.2% macerozyme R-10, 1% hemicullulase, 0.1% pectoryase Y-23, 0.2% potassium dextran sulfate, 0.05% albumin, 0.35 mM mannitol, 20 mM MES, and 5 mM MgCl, and were incubated in dark on a reciprocal shaker for 6 hours at 28 degrees C. Protoplast yields were calculated with a hemacytometer.

The protoplast yields indicated a considerable range of variation among strains of O. sativa (Table 1). Many of Japonica and Indica strains showed isolation of intact protoplasts, while the rate of isolation was lower in wild species. A glutinous strain from Thailand gave the best yield (6.1x10 p/gfc), and there was a tendency for the Japonica type to be superior to the Indica type in protoplast yield. The variation range of O. rufipogon was similar to that of Indica varieties.

The isolated protoplasts were cultured and some of them formed callus colonies from which shoots were developed. These shoots continue to grow.