Plant Regeneration from Protoplast-derived Callus of Sugarcane.

In 5 sugarcane clones, cell suspension cultures were initiated and maintained in modified N6 Iiquid medium containing 2 mg/l 2, 4-D, 500 mg/l casein hydrolysate and 3% sucrose. These suspension cultures were able to regenerate plants for more than three months. Protoplasts isolated from these suspensions were embedded in 1.2% agarose (modified KM8P medium) and cultured in modified KM8P medium with the addition of nurse culture cells from the suspension culture. After several weeks of culttl[re, calluses were obtained in all the 5 clones. Calluses derived from protoplasts were cultured for 2 weeks on PR4 medium (pre-culture medium for regeneration), then transferred to R9 regeneration medium. In the subsequent 30-day period of culture, protoplast-derived calluses of US 76-9 and NiF 8 regenerated green shoots and albino shoots, respectively, while the other 3 clones did not form any organs. In the 6 experiments using suspension cultures of US 76-9 differing in age (8 to 31 weeks after initiation), regeneration of fertile green shoots from protoplast-derived calluses was observed. After the transfer to R1 medium for rooting, a few shoots developed to plants, while most of the shoots died without rooting.