Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium. consisting of a half concentration of MS-salts with vitamins, 0.2 mg l-1 BAP, 0.05 mg l-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydrolysate; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid, by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l-1 gibberellic acid, 0.5 g l-1 casein hydrolysate, 10 mg l-1 or 20 mg l-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.