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A Catharanthus roseus cell line was selected that synthesised catharanthine exclusively under elicitation.From the first day of culture, treatment with very low concentrations of a Pythium extract did not alter the growth of the suspension but, within 24 hours, induced the synthesis of catharanthine and stimulated the production of ajmalicine. Kinetic analysis showed that serpentine then began to accumulate and that all of these effects lasted more than 7 days. Elicitation also induced changes in the cell/medium distribution of the alkaloids. Higher, although non-lethal, concentrations of the fungal elicitor were shown to impair alkaloid production. This cell line will serve as a model to study the conditions for the expression of catharanthine synthesis at the molecular level.

Inter and intraspecific variation was analyzed in two Catharanthus species with regard to isozyme polymorphism and indole alkaloid content in roots and leaves. No significant differences in alkaloid production were observed in three groups of C. roseus plants individualized for their flower color. Conversely, comparisons between C. trichophyllus and C. roseus, showed large differences of alkaloid profiles in both roots and leaves. Specific isozyme markers on four presumed loci were found allowing us to establish that natural hybridizations could occur between the two species when grown together. Experimental hybridizations confirmed that introgressions were feasible but suggested that a reproductive barrier was acting and involved interspecific incompatibility. The identification and assay of the main alkaloid compounds in natural interspecific hybrids displayed such a high hybrid vigor that interspecific hybridization may present a new and successful way of improving alkaloid production in Catharanthus species.

The treatment of a CATHARANTHUS ROSEUS cell suspension culture with a low concentration of PYTHIUM elicitor stimulated the alkaloid production. When these pretreated cells were resuspended in a medium that did not contain the fungal extract, the positive effects of the treatment on alkaloid synthesis and excretion were lost and, moreover, the standard level of production was not recovered. A second treatment of these cells with PYTHIUM elicitor at day 5 of the second culture cycle greatly impaired growth kinetics, but did not stimulate the alkaloid production observed with standard cultures. Repeated treatments with a low concentration of fungal elicitor seemed to have a negative long-term effect on both growth and alkaloid synthesis and did not appear to be a useful process for production purposes.

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