Development of the proper medium for the high plating efficiency in Indica rice protoplast and cell suspension culture

We successfully developed the proper medium/condition for embryogenic callus induction and proliferation, cell suspension establishment, protoplast isolation and culture of rice cv. Khao Dok Mali 105. MS medium supplemented with 2 mg/l 2,4-D was found superior to the N6 medium for the embryogenic callus induction from mature seeds. The addition of 0.3 mg/l kinetin, 30 mg/l tryptophan and 9 mM proline did not affect the frequency of callus formation however, they could enhance the embryogenic callus proliferation. On the other hands, only the non-embryogenic calli could be observed when casein hydrolysate was added to the medium. MS medium supplemented with 0.3 mg/l kinetin, 30 mg/l tryptophan, 9 mM proline, and 2 mg/l 2,4-D was concluded to be the best medium for the callus induction as well as N6 medium supplemented with 10 mM proline, 10 mg/l AgNO3, 35 mg/l cystein and 1 mM arginine was noted to be the proper medium for the suspension culture. Cell lines growing on the modified N6 medium showed a rapid growth rate with a small cluster of densely cytoplasmic cells. A number of viable protoplasts around 6.1*10*[7) P/g fresh weight was obtained by the following isolation method, 4 grams of the cultured fresh tissue in liquid N6 medium (supplemented with 10 mg/l AgNO3 and 9 mM arginine) was plasmolysed in 0.6 M mannitol solution for 1 hour, and two grams of preplasmolysed cells were then incubated in 10 ml enzyme solution (1.5 percent cellulase RS, 0.2 percent pectolyase Y23 and 0.6 percent macerozyme R10 dissolved in CPW solution supplemented with 0.4 M mannitol and 35 mg/l cystein). Protoplasts at the density of 1*10*[5) protoplast/ml were cultured in 1.2 percent agarose blocks (MS medium supplemented with 0.4 M sucrose, 1 mg/l 2,4-D and 0.5 mg/l NAA) immersed in liquid medium and supported with nurse cells was most effective way of culture, and small cluster of cells could be developed after 28 days of setting in the medium.