Plant regeneration from protoplast-derived callus of sugarcane

Abstract

In 5 sugarcane clones, cell suspension cultures were initiated and maintained in modified N6 liquid 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 culture, 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.