Sugarcane protoplasts: factors affecting division and plant regeneration

Sugarcane cell suspensions were initiated from leaf callus and sub-cultured every 7 to 10 days by alternate transfer to MS based medium with 3.0 and 1.0 mg 1(-1)2,4-D. Suspensions older than 3 months gave the most reproducible yields of protoplasts. Isolated protoplasts required 50 mM Ca(2+) in the washing solution and 100 mM Ca(2+) in the culture medium to prevent lysis. At plating densities of 2.0-3.0×10(5) ml(-1), 18% or more of the isolated protoplasts produced cell colonies when cultured in droplets or sectors of Kao and Michayluk (1975) based medium with 1.2% w/v Sea Plaque agarose. Cell colonies were of two morphological types. Those consisting of small, tightly packed cells developed into morphogenic callus. The latter produced an abundance of green meristems from which shoots and whole plants were regenerated.