36. Efficient plant regeneration from protoplasts of Japonica rice

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

Protoplasts from five japonica varieties, Zhonghua Nos 6 and 9 (varieties developed through anther culture), Line 02428, Giza 3030-21-2-3 and Taipei 309, were isolated using different sources e.g. suspension cultures, primary calli and suspension cultures derived from protoplasts isolated from primary calli. The protoplasts were cultured in modified Kao's medium (Wu and Zapata, 1992) using the agarose-bead method with nurse cells (Kyozuka et al., 1987).

For the establishment of cell suspensions, calli were induced from immature embryos in MS (Murashige and Skoog, 1962) medium with 2 mg/l 2,4-D. Granular structures on the surface of calli formed in 3-4 weeks were transferred to R2 liquid medium (Ohira et al., 1973) with 2 mg/l 2, 4-D and subcultured weekly.

Protoplasts from suspension culture. Protoplasts isolated from suspension cultures of Zhonghua Nos. 6 and 9, and Line 02428 underwent first division 3-4 days after culture. The plating efficiency was more than 5% in all experiments.

Protoplasts from primary calli. Sufficient protoplasts could be obtained from 3-4-week-old primary calli in all varieties. First division usually occurred 5-6 days after culture. Plating efficiency differed from 0.2-1.3% among varieties with the best response observed in Zhonghua Nos. 6 and 9.

Protoplasts from suspension culture derived from protoplasts of primary calli. Suspension cultures established using 20-day-old colonies derived from protoplasts were ready for protoplast isolation at 10-14 days after subculture. The quality and plating efficiency of the isolated protoplasts were comparable to those isolated from routinely-maintained suspension cells.

For plant regeneration, 2-mm colonies were transferred to N6 (Chu et al., 1975) medium containing 2.5 mg/l kinetin and 0.05 mg/l NAA. Shoot formation was observed at 10-14 days after transfer to regeneration medium regardless of the protoplast source. Protoplasts from primary calli gave the highest regeneration frequencies (53-97%) while those isolated from protoplast-derived suspension cells and several-month-old suspensions produced plants at average frequencies of 45% and 28%, respectively. More than 2,500 plants have been regenerated from these experiments.

Phenotypic variation was observed among plants derived from different protoplast sources. Plants from protoplasts of primary calli grew normally and had high seed set while those regenerated from protoplasts-derived cell suspensions, although looked normal, set seeds at lower rate. More variability was observed among plants obtained from protoplasts isolated from aged suspension cultures and only 40% of those plants were fertile.

Since regeneration ability and protoclonal variation is highly related to the time of in vitro culture, primary callus or young suspension cultures are suggested to be used as source for protoplasts for transformation studies.

Chu C.C., C.C. Wang, C.S. Sun, C. Hsu, C.C., Yin, C.Y. Chu, F.O., Bi, 1975. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci. Sin 16: 659-688.

Kyozuka, J., Y. Hayashi and K. Shimamoto, 1987. High frequency plant regeneration from rice protoplasts by novel nurse culture methods. Mol. Gen. Genet. 206: 408-413.

Murashige, T., F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.

Ohira, K., K. Ojima, A. Fujiwara, 1973. Studies on the nutrition of rice cell culture 1. A simple, defined medium for rapid growth in suspension culture. Plant Cell Physiol. 14: 1113-1121.

Wu, C.Y., F.J. Zapata, 1992. Plant regeneration from protoplasts isolated from primary callus of four japonica rice (Oryza sativa L.) varieties. Plant Science 86: 83-87.