38. Isolation and culture of protoplasts from the common wild rice

Studies of rice protoplasts are still at the callus formation stage. Regeneration of plantlets has not been reported except for root differentiation in the callus derived from leaf-sheath protoplasts of rice (Daka and Sen 1976). Probably, the calli which do not show rapid senescence and have a high differentiation ability during sub-culturing for the isolation of protoplasts would be useful for achieving organogenesis and plantlet regeneration.

Material we used for isolating protoplasts were particle-like embryoids produced by cultured calluses from young panicles of a wild rice (Oryza sativa f. spontanea) found in Caling county, Hunan Province. Aseptic calli weighting 4.5 g were dropped in 8 ml mixed digestion solution containing 2% cellulase, 1% pectinase, 0.2% potassium dextran sulphate, 1 mM caCl\2\.2H\2\O, and 0.6 M mannitol, at pH 5.6.The digestion process lasted 4.5 hours at 30 degrees C. The difestion solution was filtered with 400 mesh stainless-steel sieve so that fibre was excluded. The protoplasts were washed two times with solution containing 1 mM CaCl\2\.2H\2\O and 0.6 M mannitol and then washed once with the protoplast culture medium (Tsai et al. 1978). Density of protoplasts for culture was 3x10⁵ to 4x10⁵/ml.

In the culture of young panicles, the maximum induction and differentiation frequency was obtained at the stage of differentiation of the secondary branchlets and spikelet primordia. At this stage, the frequency of callus induction was 100%, and differentiation frequency was 65.8%. The calli maintained differentiation ability during subcultures. Milk-white and dense calluses produced particle-like embryoids. If the callus had a prolonged culture time, it would proliferate continuously and form a group of embryonic cells capable of developing into embryoids. The developmental stage of the embryoids was relatively synchronous. The enzyme degradation was relatively uniform and thorough. After enzyme treatment intact protoplasts were isolated. During the protoplast culture, clusters of more than 30 cells were formed. This experiment is still under way.

Daka, P.C. and S.K. Sen, 1976. Differentiation in calli originated from isolation protoplasts of rice (Oryza sativa L.) through plating technique. Molecular & General Genetics 145: 239-243.

Tsai, Shikuei, Yingchien Chien, Yunlo Chou and Suhsuen Wu, 1978. A further study on the isolation and culture of rice (Oryza sativa L.) protoplasts. Acta Bot. Sinica 20(2): 97-102.