Factors Affecting Protoplast Isolation and the Regeneration of Shoot-Like Structures From Protoplast-Derived Callus of Sugarcane (Saccharum spp Hybrids)

High yields of protoplasts were isolated from non-regenerable, homogeneous cell suspension cultures of sugarcane, compared with regenerable, heterogeneous cell suspension cultures after incubation in an enzyme composition containing Cellulase RS, Pectinase, Macerozyme and Driselase. Higher yields of protoplasts were released from heterogeneous cell suspension cultures after the addition of 10 mg L-1 silver nitrate to the culture medium; however, ethylene production was not involved in protoplast isolation. Use of 0-05-2% Pectolyase Y23 pectinase rather than other pectinases resulted in higher yields of protoplasts from heterogeneous cell suspension cultures. These results suggest that there are differences in the cell walls between cells from heterogeneous and homogeneous cell suspension cultures which affect the isolation of protoplasts. Protoplasts isolated from heterogeneous cell suspension cultures failed to develop beyond the cell division stage. Protoplasts isolated from homogeneous cell suspension cultures and cultured in agarose droplets bathed in modified 8p medium, reformed cell walls, divided and developed into microcallus. Microcallus transferred to solid modified MS medium containing 1 mg -1 .2,4-D developed into callus. Protoplast-derived callus from one cultivar formed compact nodular callus when subcultured onto the same medium containing 1% activated charcoal. Incubation of this callus on MS medium containing BAP at 0.5 mg L-1, then a combination of BAP and fluridone each at 0.5 mg L-1, resulted in the regeneration of small, chlorophyll-containing shoot-like structures. As yet no intact plants have developed from these shoot-like structures.