39. Regeneration of rice plants from protoplasts

Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Sakyoku, Kyoto, 606 Japan

The regeneration of rice plants from callus has been reported (Guha-Mukherjee 1973; Nishi et al. 1968; Tamura 1981; Wakasa 1981), and callus formation from rice protoplasts has also been reported (Cai et al. 1978; Daka and Sen 1976). But, no one has succeeded in regenerating rice plants from protoplasts. This note reports successful plant regeneration from protoplasts of Oryza sativa cv. A-58 MS and Fujiminori.

We induced calluses from husked seeds of 26 varieties of rice, one of which was a cytoplasmic male sterile line, A-58 MS. Calluses from all the varieties were cultured in LS liquid medium with 2x10⁵M 2,4-D on a gyratory shaker at 100 rpm in dark. From a cultured A-58 MS cells, we selected actively dividing cells (T\3\) which showed very high growth and opaque-yellow cytoplasms with small vacuoles. A large number of protoplasts were isolated from the T\3\ cells by treatment with 2% Cellulase Onozuka RS, 2% Macerozyme R-10, 0.1% Pectolyase Y-23, 1% driselase, 0.8% calf serum, 80 mM CaCl\2\.2H\2O, 0.125 mM MgCl\2\, and 0.5 mM MES in hormone-free LS medium containing 0.3 M glucose. Our isolated protoplasts had a high frequency of division and formed colonies at about 10% plating efficiency in a new, RY-2 medium which we established. Two of the other 25 varieties, Fujiminori and Toyotama, also showed good protoplast division and formed callus colonies at plating efficiencies of 8.4% and 5.6% respectively. None of the other varieties showed protoplast division or colony formation.

To regenerate plantlets from protoplast-derived cells of A-58 MS, Fujiminori and Toyotama, we cultured samples for 17 days in N\6\ liquid medium with 8% sucrose but without hormones. Then, we transferred the cell aggregates to flasks containing LS agar basal medium supplemented with 8% sucrose and 4x10^-
6 M 6-benzyladenine. After about 30 days of culture, green spots appeared on some Fujiminori and A-58 MS calluses. No green spotes were formed on Toyotama callus. Some of the green spots became enlarged, and eventually formed shoots. After 40 days, 1-cm long coleoptiles and first leaves emerged from the shoots. After 60 days, leaves of the regenerated plants reached the top of the flask and their roots expanded in the agar. One plant was obtained from the protoplast-derived A-58 MS callus and eight from the Fujiminori callus. These plants were potted and placed in a greenhouse, where they continue to grow.

Cai, Q.G., Y.Q. Qian, Y.L. Zhou, and S.X. Wu, 1978. A further study on the isolation and culture of rice protoplasts. Acta Bot. Sinica 20: 97-103.

Daka, P.C. and S.K. Sen, 1976. Differentiation in calli originated from isolated protoplasts of rice through plating technique. Molec. Gen. Genet. 145: 239-244.

Guha-Mukherjee, S., 1973. Genotypic differences in the in vitro formation of embryoids from rice pollen. J. Exp. Bot. 24: 139-144.

Nishi, T., Y. Yamada, and E. Takahasi, 1968. Organ redifferentiation and plant regeneration in rice callus. Nature 219: 508-509.

Tamura, S., 1981. Studies in the aseptic culture of monocyotledonous cultivated plants and its application. Mem. Fac. Agric. Niigata Univ. 18: 1- 52.

Wakasa, K., 1981. Application of tissue culture to plant breeding: Method improvement and mutant production. Bull. Nat. Inst. Agric. Sci. D33: 122-200.