PRODUCTION AND SECRETION OF INDOLE ALKALOIDS IN HAIRY ROOT CULTURES OF CATHARANTHUS-ROSEUS - EFFECTS OF IN-SITU ADSORPTION, FUNGAL ELICITATION AND PERMEABILIZATION

Various hairy root clones were derived by transforming two kinds of Ca tharanthus roseus plants (Catharanthus roseus cvs. Little Linda and Li ttle Delicata) with Agrobacterium rhizogenes ATCC 15834. Hairy root gr owth and indole alkaloid production were then investigated to select a high-yielding hairy root clone. Among three Amberlite resins (XAD-2, XAD-4, and XAD-7), XAD-7 greatly enhanced the release of catharanthine and ajmalicine from hairy root cultures, with an increase in total pr oduction. To enhance alkaloid production and secretion, a permeabilizi ng agent (dimethyl sulfoxide) and a fungal elicitor to provide physica l and biochemical stress, respectively, together with in situ adsorpti on. Dimethyl sulfoxide (0.5% v/v) treatment with in situ adsorption us ing XAD-7 was found appropriate for releasing indole alkaloids from ha iry roots without affecting cell viability. In addition, fungal elicit ation by Penicillium sp. enhanced both alkaloid production and secreti on. By combining in situ adsorption sequentially with these techniques , the release ratio of catharanthine and ajmalicine was enhanced up to 20 and 70%, respectively, which was 3.4 and 2 times higher than that obtained with in situ adsorption by XAD-7 alone. Over a 27-d culture p eriod, the total amounts of catharanthine and ajmalicine produced were 67 and 30.15 mg/l, respectively. These results indicated that in situ adsorption sequentially applied with permeabilization and fungal elic itation have a synergistic effect on the production and secretion of i ndole alkaloids.