60. Enhancement of division frequency of protoplasts and plant regeneration in rice

Institute of Genetics, Academia Sinica, Beijing 100012, China

Calli were initiated from young inflorescences of Oryza sativa L. on MS medium supplemented with 2mg/l 2,4-D, 3% (w/v) sucrose and 1% (w/v) agar adjusted to pH 5.6-5.8. The calli of 1-1.5 g were introduced into a flask containing 30 ml of AA liquid medium. The suspension cell line was established after being cultured for 7-8 weeks on a gyratory shaker at 100 rpm.

Protoplasts were isolated from established cell line at 3-4 days after subculture. Protoplasts were cultured at densities between 2-5X10⁵/ml in KM8p media solidified with 0.8% (w/v) agarose. KM8p liquid medium was added when the agarose drops were solidifed. The percentage of the protoplasts having divided at least once was determined after 8-10 days. The osmotic pressure of liquid medium surrounding agarose-embedded protoplasts was reduced after two weeks. Ammonium ion was an important factor in obtaining a high rate of protoplast division. We found that at zero concentration of NH\4\⁺ the best colony formation was obtained. Ferric ion was also an important factor for rice protoplast culture. Our experiment indicated that the half concentration of ferric ion used in the standard MS medium was best for the colony formation. Calf serum was als important for rice protoplast culture. We found that an addition of 0.8% calf serum could greatly increase the division frequency. Our experiments indicated that 0.35 mg glucose was the best for rice protoplast culture. When rice protoplasts were cultured in liquid KM8p media, the division frequency was only 1-3% and many protoplasts aggregated and died later. When they were cultured on KM8p media solidified with 0.8% (w/v) agarose, the aggregation of protoplasts was avoided and the divisio frequency was increased to 38%.

The vigor of calli was enhanced when cultured on NMB media and greenspots could be induced easily. Our experiments indicated that a high concentration of 2, 4- D was good for the formation of embryo morphology in calli subculture. Kinetin and 6BA were effective for the induction of green spots.