56. Somatic embryogenesis and plant regeneration from rice protoplasts

Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, nottingham, NG7 2RD U.K.

Several percent reports have claimed plant regeneration from rice protoplasts (Fujimura et al., 1985; Coulibaly and Demarly 1989; Yamada et al., 1986). Relatively few plants were regenerated in these studies and the mode of plant regeneration was not fully documented. Yamada et al. (1986) suggested that enhanced embryogenesis might improve the frequency of plant regeneration.

In this note we describe rapid efficient plant regeneration from protoplast- derived callus of Oryza sativa (cv. Taipei 309 and Fujisaka 5) through somatic embryogenesis. Full details of the procedures used will be published shortly (Abdullah et. al. 1986).

Calli were initiated from seedling leaves, roots and the scutellum of seeds of Taipei 309 on Linsmaier and Sooog (1965) medium with 2.5 mg/1 2, 4-D (LS 2.5). Calli were also initiated from the scutellum of seeds of Fujisaka 5 on the same medium. Embryogenic calli were subsequently placed in liquid LS 2.5 medium to initiate cell suspension cultures, and then transferred to AA liquid medium (Muller and Grafe 1978) with 2 mg/1 2,4-D (AA2) and subcultured weekly. The cultures were 4-9 months old at the time of protoplast isolation.

Protoplasts were isolated from cell suspension cultures 4-5 days after subculture by 4 hours incubation in 1% cellulase RS and 0.1% pectolyase Y23, with 13% mannitol. A modified K8p medium (Kao 1977) with 10% glucose, 0.5 mg / 1 2, 4-D (KpR medium) solidified 1.2% Sea Plaque agarose was used for protoplast culture as described previously (Thompson et al. 1986). Plating efficiencies of 10-30%, based on the percentage of plated protoplasts that had divided at a density of 3.0X10⁵/ml, were obtained after two weeks. After 4-5 weeks of culture, plating efficiencies of 0.5-1.2%, based on macrocolony formation from plated protoplasts were recorded.

Plant regeneration was achieved by direct transfer of calli 1-2 mm in diameter to the surface of hormone-free N6 medium (Chu et al. 1985) with 8% sucrose (N60). Up to 20% of the colonies rapidly produced green plants on this medium. Globular and scutellar notch-stage embryoids were observed after 7 days, fol,lowed by the development of a well-defined scutellum and coleoptile. The embryoids germinated as rapidly as 15 days after colony transfer to N60 medium, i.e. 6-7 weeks after protoplast isolation. Plants have been regenerated from colonies of embryo, leaf and root origin and from embryo-derived suspension protoplasts of Fujisaka 5. Over 100 plants have to date been transferred to soil and appear normal under greenhouse conditions.

Somatic hybridization and genetic transformation using protoplasts will now be possible in rice with the prospect of the efficient regeneration of any hybrid and transformed plants.

Financial support from the Rockefeller Foundation and the Overseas Development Administration is gratefully acknowledged.

Abdullah, R., E.C. Cocking and J.A. Thompson, 1986. Efficient plant regeneration from rice protoplasts through somatic embryogenesis (submitted for publication).

Chu, C.C., C.C. Want, C.S. Sun, C. Hsu, K.C. Yin, C.Y. Chu and F.Y. Bi, 1975. Establishment of an efficient medium for anther culture of rice throuth comparative experiments on the nitrogen sources. Sci. Sinica 18: 659-668.

Coulibaly, M.Y. and Y. Demarly, 1986. Regeneration of plants from protoplasts of rice Oryza sativa L. Z. Pflanzenzuchtg. 96: 79-81.

Fujimura, T., M. Sakurai, H. Akagi, T. Negishi and A. Hirose, 1985. Regeneration of rice plants from protoplasts. Plant Tissue Culture Letters 2: 74-75.

Kao, K.N., 1977. Chromosomal behaviour in somatic hybrids of Soybean-Nicotiana glauca. Molec. Gen. Genet. 150: 225-230.

Linsmaier, E.M. and F. Skoog, 1965. Organic growth factor requirement of tobacco tissue cultures. Physiol. Plant. 8: 100-127.

Muller, A.J. and R. Grafe, 1978. Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase. Mol. Gen. Genet. 161: 67-76.

Thompson, J.A., R. Abdullah and E.C. Cocking, 1986. Protoplast culture of rice using media solidified with agarose. Plant Science (in press).

Yamada, Y., Z.Q. Yang and D.T. Tang, 1986. Plant regeneration from protoplast- derived callus of rice (Oryza sativa L.). Plant Cell Reports 5: 85-88.