35. Plant regeneration from protoplasts of Indica rices

F.J. ZAPATA1, S. ZHANG1, L.B. TORRIZO1, G.C. GHOSH BISWAS2, C.Y. Wu1 and M.V. KUMAR1

1) Tissue Culture Laboratory, Plant Breeding, Genetics and Biochemistry, International Rice Research Institute, P.O. Box 933, Manila, Philippines

2) Swiss Federal Institute of Technology, ETH-Zurich, Institute of Plant Sciences, CH-8092 Zurich, Switzerland.

Among the several methods for delivering foreign genes into rice such as direct introduction into plant protoplasts, targetting competent or embryogenic cells through biolistics, and Agrobacterium-mediated gene transfer, so far, the first one offers the least limitation and highest efficiency. Aside from its use in transformation, protoplasts are also indispensable in somatic hybridization and protoclonal variation studies. Obtaining protoplasts of japonica varieties is now a routine procedure. However, indica rices are still recalcitrant to tissue culture procedures. Therefore we have been trying to identify the optimum conditions for the establishment of embryogenic cell suspensions and subsequent plant re- 

Table 1. Indica varieties with number of plants regenerated from protoplast
culture
===============================================================================
                  Variety                 Plants regenerated (no.)
===============================================================================
                   IR24                                         71
                   IR43                                     >3,000
                   IR52                                         37
                   IR56                                         10
                   IR58                                       >900
                   IR64                                         34
                   IR57311-95-2-3                              251
                   Tetep                                        11
                   Wagwag (Philippines)1                       245
                   VN19 (Vietnam)                               87
===============================================================================
Country of origin
generation from protoplasts of indica cultivars.

Explants used in calli induction included mature seeds, immature embryos and anthers. Embryogenic calli were selected and cell suspensions were established and maintained in the basal media R2 (Ohira et al., 1973), N6 (Chu et al., 1975), or AA2 (Muller and Grafe, 1978), with or without modifications. The cell suspensions were subcultured weekly until friable, densely-cytoplasmic round cells were formed. Suspension cells were digested with mixtures of cellulases and pectinases optimum for each variety. Isolated protoplasts were cultured in Kao's (Kao and Michayluk, 1975), Fujimura's (Fujimura et al., 1985), R2 or N6 medium with nurse cells of Oryza ridleyi, or the Oc cell line obtained from calli derived from roots of Oryza sativa L. C5924 kindly provided by Dr. K. Syono of the University of Tokyo. Plants were regenerated from protoplast-derived calli in modified MS (Murashige and Skooo,, 1962) or N6 medium.

For successful establishment of cell suspension and protoplast culture, we have found the following to be very critical; (1) selection of embryogenic calli for the initiation of cell suspension, (2) use of nurse cells, and (3) the use of maltose instead of sucrose as carbon source especially in plant regeneration (Torrizo and Zapata, 1992; Ghosh Biswas and Zapata, 1992). Furthermore, we have shortened the time for the establishment of cell suspensions from the normal six months to three months which is very important in increasing the efficiency of plant regeneration.

We have regenerated a large number of plants from several indica cultivars (Table 1) which has facilitated the studies on transformation. Establishment of cell suspensions and protoplast culture of other indica varieties are underway.

References

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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.

Ghosh Biswas, G.C. and F.J. Zapata. 1992. High-frequency plant regeneration from protoplasts of indica rice (Oryza sativa L.) using maltose. J. Plt. Physiol. (accepted for publication)

Kao, K.N. and M.R. Michayluk. 1975. Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low density in liquid media. Planta. 126: 105-110.

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.

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Ohira, K., K. Ojima and A. Fujiwara. 1973. Studies on the nutrition of rice cell culture. I. A simple, defined medium for rapid growth in suspension culture. Plant Cell Physiol. 14: 1113-1121.

Torrizo, L.B. and F.J. Zapata. 1992. High efficiency plant regeneration from protoplasts of the indica cultivar IR58. J. Expt. Bot (accepted for publication).