Enhancement of cell growth and saponin production in panax ginseng cell culture by nutrient feeding and elicitation

Abstract

Plants are the most important sources of natural products, such as food flavors, dyes, fragrances and pharmaceuticals, which are mostly secondary metabolites of plants. Plant tissue and cell cultures are feasible and promising alternatives for the production of secondary metabolites of plants, particularly those naturally rare and difficult-to-cultivate plant species. Panax ginseng is a famous and valuable oriental herb, and its root is widely used in traditional medicines and health foods. Although ginseng can be cultivated on farms, the cultivation process takes 5-7 years from seeding to final harvest of the mature ginseng roots. Therefore, plant tissue and cell cultures may be more efficient processes for the production of ginseng root and its components. The main objectives of this research project are to study the kinetics of P. ginseng cell suspension culture and the strategies for enhancement of ginseng cell biomass and secondary metabolite (ginseng saponins or ginsenosides) production in cell suspension cultures. The main factors and strategies being investigated included medium composition, nutrient feeding, and stimulation with osmotic stress and chemical elicitors. Statistic methods were applied in some parts of the project for the design and analysis of experiments and for the identification of optimal culture conditions. The results showed that the basic kinetics of growth and nutrient metabolism of the P. ginseng cells in suspension culture was basically similar to that of most other plant cell cultures. The feeding of sucrose and some other nutrients to the culture near the stationary growth phase extended the growth period and increased the biomass growth index by about 100%. The introduction of osmotic stress (by increasing medium osmalality) to the culture at the time of inoculation (day 0) with osmotica such as sorbitol and sodium chloride stimulated the secondary metabolite biosynthesis, but depressed the cell growth. As the osmotica were fed together with the limiting growth nutrients, sucrose and casein hydrolysate, to the culture around the stationary growth phase, secondary metabolite biosynthesis could be improved without significant cell growth depression. Statistical design and analysis of the nutrient feeding and osmotic stress experiments indicted that nutrient (sucrose) feeding time is a major factor affecting the biomass productivity and sorbitol concentration is a major factor affecting both biomass productivity and saponin production. A 4-fold increase in the volumetric saponin yield was achieved with the feeding of 45 g/l sucrose (+ 1.5 g/l casein hydrolysate) and 0.2 M sorbitol to the culture at day 10 post inoculation. The feeding of chemical elicitors to the culture, such as methyl jasmonate (MJ) and yeast extract (YE) was even more effective than the application of osmotic stress to stimulate the saponin production. With 120 uM MJ, for example, the saponin content of cell was increased by more than 4-fold. When MJ was fed together with sucrose to the culture, the volumetric saponin yield was increased by more than 10-fold. The chemical elicitors, which were fed to the culture around the stationary growth phase, caused only minor inhibiting effect on the cell growth. Further investigation showed that the osmotic stress and elicitor treatment induced the general plant defense responses to biotic and abiotic stresses in the ginseng cell cultures, i.e., the increase in the activity of phenylalanine ammonium-lyase (PAL), a key enzyme in plant secondary metabolic pathways, and the transient production of active oxygen species, H2O2, known as the oxidative burst which is an early and signal event in plant defense responses. Therefore, the enhanced saponin biosynthesis of ginseng cells may be a result of the defense responses of plant cells induced by the osmotic stress and chemical elicitors. In conclusion, the application of nutrient feeding and elicitation is a simple and effective strategy for enhancement of ginseng biomass and saponin production in plant cell cultures.