Production of triterpenoids in liquid-cultivated hairy roots of Galphimia glauca.

The production of three triterpenoids from Galphimia glauca hairy root cultures, the sedative principle galphimine E (2), the recently described glaucacetalin A (3), and maslinic acid (6), was quantified by HPLC in the biomass and the culture medium. Batch cultures of the hairy root line VYT, obtained through infecting cotyledons with Agrobacterium rhizogenes ATCC 15 834, were grown for 41 days in shake flasks containing B5 medium without phytohormones. A maximum biomass of 11 g/L DW was obtained on day 33, while the doubling time was 6 days. Throughout the growth cycle fresh and dry weights as well as triterpene production were registered. Glaucacetalin A (3), excreted into the culture media, reached a maximum amount of 2.14 mg/L after 21 days while galphimine E (2) and maslinic acid (6) were recovered from the root biomasses reaching maximum concentrations of 0.11 and 0.43 mg/g, respectively, on day 39.

Genetic transformation of Glaphimia glauca by Agrobacterium rhizogenes and the production of norfriedelanes.

Transformed root cultures of Galphimia glauca (Malpighiaceae) were established by infecting cotyledons and hypocotyls with Agrobacterium rhizogenes ATCC 15 834. Cotyledon-derived cell lines were grown in liquid B5 nutrient medium without phytohormones and have shown the typical hairy roots phenotype over two years of continuous subculturing. PCR analysis was used to confirm the integration of rol A and rol C genes into the plant genome. The transformed cultures synthesized three major norfriedelanes, the new glaucacetalins A-C (1-3), which were secreted into the nutrient medium. The structural elucidation of these in vitro produced metabolites was performed by the application of high resolution NMR techniques that proved them to be triterpenoids related to the known galphimines, the sedative principles of this plant species. These results suggest the possibility of further biotechnological exploration of sedative friedelane biosynthesis by in vitro plant organ cultures.