Protoplast Isolation from Euphorbia tirucalli L. Cell Suspension Cultures and Sustained Cell Division

Callus of Euphorbia tirucalli L. was initiated with stem segments cultured on MS medium containing 2,4-D (1 ppm) and NAA (2 ppm) (1-MS-N medium), and was maintained on the same medium plus kinetin (0.5 ppm) (1-MS-NK medium). A fine suspension culture was obtained by subculturing the fast growing callus in liquid medium made up of three volumes of 1-MS-NK medium and one volume of modified B5 medium (medium 8p) as described by Kao and Michayluk (1975). Cells then were subcultured in 1-B5 liquid medium. Protoplasts were isolated by digesting the walls of cells cultured as suspension by Driselase (1%) and Pectolyase (0.1%). When transferred to medium 8p the protoplasts divided and formed large cell clusters.