SINGLE-CELL CLONING OF A CROWN GALL FROM PROTOPLASTS REGENERATED IN HORMONE-FREE MEDIUM - ESTABLISHMENT OF PURE TRANSFORMED CELL-LINES OF CATHARANTHUS-ROSEUS

Protoplasts enzymatically isolated from cell line of Catharanthus rose us G. Don crown gall, were cultured at high density (10(5) P ml(-1)) i n modified B5 liquid medium (Gamborg et al. 1976). In the absence of g rowth regulators C. roseus protoplasts were able to regenerate a cell- wall, divide and, subsequently, yield very numerous clones in the abse nce of growth regulators. After two weeks, the cultures were greatly d iluted in order to obtain clones of single-cell origin. Most of the cl ones individually transferred onto solid medium can proliferate indefi nitely, without growth regulators. Among analyzed clones, 90% were nop aline positive. Their ajmalicine and serpentine content was compared w ith that of the parental crown gall line, and was found to be low. The CR10 protoplasts were very easy to grow, they were an interesting mod el for the development of pure tumorous lines. Moreover, we found that the tumorous protoplasts were useful for cell fusion experiments or f or the delicate culture of tree protoplasts.