Interactive effects of nitrate and phosphate salts, sucrose, and inoculum culture age on growth and sesquiterpene production in Artemisia annua hairy root cultures

Abstract

We analyzed four factors (phosphate and nitrate salts, sucrose, and culture 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 Artemis& annua root cultures. Optimal growth conditions were determined to be: nitrate (15 mM), phosphate (1.0 nrM), sucrose content (5% wt/vol), and inoculum age (8 d-old). Determination of optimal conditions for sesquiterpene production was more complicated than for biomass production. For most experiments artemisinic acid was undetectable especially in experiments where phosphate was greater than 0.5 mM and for nearly all culture inoculum ages of 14 d. Artemisinic acid was also never detected whenever arteannuin B was present. Arteannuin B was the major artemisinic compound detected in these experiments, sometimes at levels exceeding 300 ~tg/g fresh weight. When the sum of artemisinin and its three precursors is analyzed, three factors (sucrose, nitrate, and inoculum age) are heavily dependent on one another, and in conjunction with possible degradation of artemisinin by peroxidase, the current analysis does not provide a clear picture regarding the most effective conditions for maximizing the production of artemisinin.

Yield enhancement strategies for the production of picroliv from hairy root culture ofPicrorhiza kurroaRoyle ex Benth.

“…The growth promoting effect of 4% sucrose level was also noted earlier in case of hairy root cultures of Artemisia annua 28 and Plumbago zeylanica.…”

Tetraploid Artemisia annua hairy roots produce more artemisinin than diploids

“…Initially we used inoculum from late-log cultures (14 day) to compare biomass yields in flasks. However, previous studies with hairy root cultures showed that the age of the inoculum culture might affect root growth as well as secondary metabolite production (Takahashi et al 2001;Weathers et al 1996Weathers et al , 1997. A common result of polyploidy in plants is a reduced number of cell divisions during growth and development (Adaniya and Shira 2001;Otto and Whitton 2000), so overall reduced growth was not surprising.…”

Artemisia species in vitro cultures for production of biologically active secondary metabolites

“…Four factors: phosphate and nitrate salts, sucrose, and culture inoculum age were analyzed simultaneously to determine the most suitable conditions for maximizing both root biomass and sesquiterpene lactones production in transformed A. annua root cultures by Weathers et al (1997). The authors concluded that optimal growth conditions were determined to be: nitrate (15 mM), phosphate (1.0 mM), sucrose content (5% w/v), and inoculum age (8 day-old) whereas the determination of optimal conditions for sesquiterpene production was more complicated and did not provide the most effective solutions for maximizing the production of artemisinin.…”