ISOLATION AND CULTURE OF PROTOPLASTS FROM CELL SUSPENSIONS OF CACTUS PEAR (OPUNTIA FICUS-INDICA MILL.)

Tissue culture techniques are essential to facilitate the application of modern biotechnological methods to cactus pear genetic improvement. Viable protoplasts from cell suspensions of Opuntia ficus-indica, were obtained by incubating cells in medium containing 1% cellulase, 0.5% pectinase, 0.1 mM CaCl₂ and 0.7 M glycine, for 10 h in the dark at 28°C. After transferring protoplasts to a medium consisting of full-strength MS salts and vitamins, 0.7 M glucose, 0.5 mM MES, 0.9 µM kinetin, 1.0 µM picloram, 2.3 µM 2,4-D and 0.2 g/dm^-3 casein hydrolysate, high levels of cell division as well as cell wall regeneration were observed. Under the optimized conditions described here, we obtained a yield of 5 x 10⁵ viable protoplasts for each cm3 of cell suspension and these isolated protoplasts were viable as estimated by Evans blue staining.