Enhancement of Tanshinone Production in Salvia miltiorrhiza Hairy Root Culture by Ag⁺ Elicitation and Nutrient Feeding

Abstract

Ag⁺ was employed as an abiotic elicitor to stimulate the secondary metabolite production in hairy root culture of Salvia miltiorrhiza, a valuable herbal plant. The addition of Ag₂S₂O₃ at 15 - 40 μM to the culture between 12 and 22 days post inoculation resulted in more than 2-fold increase in the yields of the three diterpenoid tanshinones, tanshinone I, tanshinone IIA and cryptotanshinone. This stimulating effect was dependent on the Ag⁺ dose, the day of Ag⁺ addition to the culture and the tanshinone species, while the total yield of the three tanshinones (TT) was mainly dependent on the Ag⁺ dose, with the highest being attained at 30 μM. The Ag⁺ treatment also caused a dose-dependent inhibition of hairy root growth. Sucrose feeding or medium renewal before the addition of Ag⁺ to the culture effectively prevented the growth inhibition, and significantly increased the biomass concentration and volumetric tanshinone yield. With combined medium renewal and Ag⁺ treatment, in particular, the TT yield was increased 6.6-fold relative to that of the control (55.7 mg/L versus 7.3 mg/L). The elicitor function of Ag⁺ in the hairy root culture was validated by its ability to induce the characteristic elicitor responses of plants, the increase in cross-cell membrane ion fluxes and the production of reactive oxygen species.

Key words

Hairy root culture - Salvia miltiorrhiza - Lamiaceae - tanshinones - Ag⁺ elicitation - nutrient feeding

References

• 1 Giri A, Narasu L. Transformed hairy roots: recent trends and applications.  Biotechnol Adv. 2000;  18 1-22
• 2 Tang W. Chinese drugs of plant origin: chemistry, pharmacology, and use in traditional and modern medicine. Berlin; Springer-Verlag 1992: 902
• 3 Hu Z B, Alfermann A W. Diterpenoid production in hairy root cultures of Salvia miltiorrhiza .  Phytochemistry. 1993;  32 699-703
• 4 DiCosmo F, Misawa M. Eliciting secondary metabolism in plant cell cultures.  Trends Biotechnol. 1985;  3 318-22
• 5 Threlfall D R, Whitehead I M. The use of metal ions to induce the formation of secondary products in plant tissue cultures. In: Robins RJ, Rhodes MJC, editors Cambridge; Cambridge University Press 1988: pp 51-6
• 6 Choi H K, Yun J H, Kim S I, Son J S, Kim H R, Kim J H, Choi H J, Hong S S. Enhanced production of paclitaxel by semi-continuous batch process (SCBP) in suspension culture of Taxus chinensis .  Enzyme Microb Technol. 2001;  29 583-6
• 7 Pitta-Alvarez S I, Spollansky T C, Giulietti A M. The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida .  Enzyme Microbial Technol. 2000;  26 252-8
• 8 Furze J M, Rhodes M JC, Parr A J, Robins R J, Withehead I M, Threlfall D R. Abiotic factors elicit sesquiterpenoid phytoalexin production but not alkaloid production in transformed root cultures of Datura stramonium .  Plant Cell Rep. 1991;  10 111-4
• 9 Wang C, Wu J, Mei X. Enhancement of taxol production and excretion in Taxus chinensis cell culture by fungal elicitation and medium renewal.  Appl Microbiol Biotechnol. 2001;  55 404-10
• 10 Zhong J J. Plant cell culture for production of paclitaxel and other taxanes.  J Biosci Bioeng. 2002;  94 591-9
• 11 Murashige T, Skoog F A. A revised medium for rapid growth and biosynthesis with tobacco tissue cultures.  Physiol Plant. 1962;  15 473-97
• 12 Chen H, Chen F, Zhang Y L, Song J Y. Production of lithospermic acid B and rosmarinic acid in hairy root cultures of Salvia miltiorrhiza .  J Ind Microbiol Biotechnol. 1999;  22 133-8
• 13 Zhang C H, Wu J Y. Enhancement of elicitor-induced paclitaxel biosynthesis in plant (Taxus spp.) cell cultures by ethylene inhibitors.  Enzyme Microb Technol. 2003;  32 71-7
• 14 Bond P A. Production systems. In: Pollard JW, Walker JM, editors Clifton; Humana Press 1990: pp 503-11
• 15 Wang M Q, Chen Y H. HPLC analysis of common Chinese herbs (in Chinese). Beijing; Science Press 1999: pp 70-4
• 16 Trigiano R N, Gray D J. Plant tissue culture concepts and laboratory exercises. 2nd ed Boca Raton; CRC Press 2000: p 377
• 17 Yahraus T, Chandra S, Legendre L, Low P S. Evidence for a mechanically induced oxidative burst.  Plant Physiol. 1995;  109 1259-66
• 18 Chen H, Chen F, Chiu F CK, Lo C MY. The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza .  Enzyme Microbial Technol. 2001;  28 100-5
• 19 Ebel J, Mithöfer A. Early events in the elicitation of plant defense.  Planta. 1998;  206 335-48