INTERACTIVE EFFECTS OF NITRATE AND PHOSPHATE SALTS, SUCROSE, AND INOCULUM CULTURE AGE ON GROWTH AND SESQUITERPENE PRODUCTION IN ARTEMISIA-ANNUA HAIRY ROOT CULTURES

We analyzed four factors (phosphate and nitrate salts, sucrose, and cu lture inoculum age), simultaneously at three levels using a fractional factorial design method to determine the most suitable conditions for maximizing both root biomass and terpenoid production in transformed Artemisia annua root cultures. Optimal growth conditions were determin ed to be: nitrate (15 mM), phosphate (1.0 mM), sucrose content (5% wt/ vol), and inoculum age (8 d-old). Determination of optimal conditions for sesquiterpene production was more complicated than for biomass pro duction. For most experiments artemisinic acid was undetectable especi ally in experiments where phosphate was greater than 0.5 mM and for ne arly all culture inoculum ages of 14 d. Artemisinic acid was also neve r detected whenever arteannuin B was present. Arteannuin B was the maj or artemisinic compound detected in these experiments, sometimes at le vels exceeding 300 mu g/g fresh weight. When the sum of artemisinin an d its three precursors is analyzed, three factors (sucrose, nitrate, a nd inoculum age) are heavily dependent on one another, and in conjunct ion with possible degradation of artemisinin by peroxidase, the curren t analysis does not provide a clear picture regarding the most effecti ve conditions for maximizing the production of artemisinin.