The effects of phytohormones on growth and artemisinin production in hairy root cultures of artemisia annua l.

by McCoy, Mark Christopher.

The in vitro addition of plant growth regulators (i.e. phytohormones) to Agrobacterium transformed hairy root cultures affects morphological and biochemical changes, resulting in altered growth and secondary metabolite accumulation rates in root tissues. Significant increases in both growth and secondary product accumulation have been observed, upon incubation with phytohormones, in some species. Consequently, the use of phytohormones in vitro has received increasing attention as a potential means for increasing those plant secondary products notoriously produced in small quantities. However, currently little is known about the specific effects of phytohormones on growth and secondary metabolism. The Chinese herb Artemisia annua L. produces artemisinin, an effective antimalarial therapeutic. Efforts to increase the amount of artemisinin via chemical synthesis or fieldgrown crops have met with huge costs and disappointingly low yields, respectively. Agrobacterium transformed hairy root cultures of A. annua (Clone YUT16) produce artemisinin and undergo rapid growth compared to non-transformed, making them a good model system to study secondary metabolite production. Demonstrated herein is the first definitive evidence, by any hairy root species, of a favorable response to exogenous combinatorial hormone application as well as the development of a two-stage culture system alluding to optimal growth and artemisinin production conditions in A. annua hairy roots. Furthermore, analysis of artemisinin and biomass accumulation in A. annua hairy roots in the presence of phytohormones has revealed effective individual as well as combinatorial phytohormone concentrations suitable for increasing single and bulk root growth, and artemisinin production. The effectiveness of an i optimal phytohormone combination, with respect to time of addition, its relationship to inoculum size, and its combination with the provision of fresh nutrients and or mechanical stress to the roots is also described resulting in artemisinin yields of up to 0.8 µg/g F.W. Although the findings contained herein are not yet optimized they do, however, argue for the potential usefulness of a two-stage production scheme using phytohormones to increase plant secondary metabolite production in vitro. ii