Studies on plant regeneration from rice (Oryza sativa L.) protoplasts
1989
Gadab Chandra Ghosh Biswas
The cells at four to six days after subculture were used for isolation. Optimum conditions for isolation in Taipei 309, Taipei 177 and Kulu was achieved using an enzyme mixture of 2% cellulase "onozuka" RS and 0.2% pectolyase Y-23 in CPW13M with incubation for 4 to 5 hours. For Tetep, three percent cellulase "onozuka" RS and 0.1% pectolyase Y-23 in CPW13M incubated 6 to 7 hours was optimum. Protoplasts can be isolated from Tetep until 12 days after subculture. Various phytohormone combinations were suitable for protoplast culture depending on the variety. For Taipei 177, 1.0 mg/l 2,4-D was found to be suitable while for Tetep, Taipei 309 and Kulu the combination of 1.0 mg/l 2,4-D, 0.5 mg/l IAA and 0.5 mg/l BAP was optimum. Protoplast grew well with mannitol but 0.55M glucose as the osmotic stabilizer improved plating efficiency. Protoplast had significantly improved the plating efficiency in 0.5-1% Sea Plaque Agarose than in liquid medium. "Bead" culture method plating efficiency of Taipei 177 but not that of Tetep. Protoplasts cultured in NO3-containing medium did not support sustained division, signifying the importance of NH4+ ion for successful rice protoplast culture. The optimum ammonium nitrate concentration was 600 mg/1 and 900 mg/1 for Tetep and Taipei 177, respectively. Plant regeneration was obtained from protoplasts - derived calli in all the four genotypes tested. The quantity of green plants was in the order of Taipei 177 Taipei 309 Tetep. No green plant was obtained from protoplast - derived calli of Kulu.
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