Ginsenoside Production by Hairy Root Cultures of Panax ginseng C.A. Meyer in Bioreactors

Hairy roots infected with Agrobacterium rhizogenes were induced from roots, stems, and leaves of Korean ginseng (Panax ginseng C.A. Meyer) on half strength MS medium supplemented with 300 mg·L Cefotaxim sodium. DNA extraction was carried out and PCR results confirmed that the hairy roots induced by A. rhizogenes KCTC 2703 have the rol C gene in their T-DNA. Selected root lines were propagated in 5-litre cone type bubble bioreactors containing MS media supplemented with 2.0 mg·L NAA and 30 mg·L sucrose. To increase ginsenoside content, jasmonic acid in various concentrations was added to the culture medium after 30 days of culture and the roots were then cultured for 7 more days until harvest. Total ginsenoside content increased with increasing jasmonic acid concentration, but high concentrations inhibited the root growth. Ginsenosid productivity was greatest at 2.0 mg·L jasmonic acid. On the other hand, ginsenosides in the Rb group mainly increased, while those in the Rg group did not. Particularly, high concentrations (5 and 10 mg·L) of jasmonic acid decreased Rg1 content to almost half of that in the control, but significantly increased the Rb1 content of the Rb group. Among the ginsenosides in the Rb group, the Rb1 content increased more than Rb2, Rc, and Rd. There were gradual increases in ginsenosides in the Rb group, but those in the Rg group fluctuated then slightly decreased at the end of the culture period. Further studies are required to raise the contents of ginsenosides in the Rg group and to determine the optimal type of elicitors as well as optimal time for the elicitor treatment for accelerating ginsenoside production. INTRODUCTION Panax ginseng C.A. Meyer belongs to the Araliaceae family and is traditionally considered one of the most potent medicinal plants in the Orient, where it has been used for centuries as a health tonic. The most important active component of ginseng roots is ginsenoside and more than 20 different ginsenosides have been identified (Lee et al., 1995). The demand for ginseng roots and its extracts has been increasing but cultivated ginseng roots are expensive since seeding is still a principal propagation method, which takes about 4 to 6 years to harvest. Disease control remains the central problem in commercial cultivation of ginseng. Consequently, the commercial cultivation of ginseng has required the application of pesticides, which resulted in the serious problem of pesticide residues (Yu and Ohh, 1995). Another major problem in ginseng cultivation is replant diseases. Due to the problems, traditional cultivation cannot meet the increasing demand of the ginseng market, especially that for ginsenosides. There have been alternative approaches for ginsenoside production through cell culture and tissue culture technology and there are many reports on ginsenoside production in cell cultures by controlling growth regulators (Zhong et al., 1996), the nitrogen source (Zhong and Wang, 1998), sucrose concentration and inoculum cell size (Akalezi et al., 1999). From the view of biomass production, on the other hand, root culture is more efficient compared to cell culture due to fast growth and stable metabolite productivity (Carvalho and Curtis, 1998). For commercial production of ginsenoside, adventitious roots were cultured in large-scale bioreactors and the ginsenoside profiles of the adventitious roots were similar to those of field-grown ginseng roots (Seon et al., 1999; Son et al, 1999). Hairy roots infected by Agrobacterium rhizogenes also produce Proc. Int. Conf. on MAP Eds. J. Bernáth et al. Acta Hort. 597, ISHS 2003 238 the same secondary metabolite as those synthesized in intact parent plant roots with similar or higher yields (Zehra et al., 1999). However, the ginsenoside content of the roots remains low and should be increased (Yoshimatsu et al., 1996; Pinol et al., 1999). A wide variety of elicitors has been employed to increase the content of secondary metabolites in plant cell cultures (Dornenburg and Knorr, 1995; Chang et al., 1998) and we found jasmonic acid to be efficient for ginsenoside production (Yu et al., 2000a). Apart from elicitors, there are many factors that affect the content of secondary metabolites, e.g., medium components, growth regulators, carbon source, medium pH, and air temperature. In this experiment, concentrations of sucrose, benzyl adenine (BA), and jasmonic acid were varied at different levels in ginseng hairy root cultures to determine their effects on biomass increase and ginsenoside production. MATERIALS AND METHODS Establishment of Hairy Root Culture Roots, stems, and leaves of ginseng were sterilized, cut into 5 mm lengths and then placed on NS solid medium (Nutrient broth, DIFCO) infected with Agrobacterium rhizogenes (KCTC 2703, 2704, and 2743), where they were kept overnight. Explants were transferred to half strength MS (Murashige and Skoog, 1962) media supplemented with 300 mg·L Cefotaxim sodium to induce roots. After root induction, DNA extraction was carried out by the method reported by White and Sinkar (1987). The PCR results were checked by agarose gel electrophoresis with marker Hind-ш-digested-λDNA, which confirmed that ginseng hairy roots induced by A. rhizogenes KCTC 2703 (ATCC 15383) had the rol C gene in its T-DNA. Root lines were selected according to root growth and ginsenoside content and suspension cultured in 400 mL conical flasks containing 100 mL half strength MS medium with 3% sucrose at 23±2 oC in darkness. Experiment on Sucrose Concentration Two grams of hairy roots were inoculated in a 400 mL conical flask containing 100 mL of half strength MS medium, in which sucrose concentrations were varied at 1, 2, 3, 5, 7, and 9%. Cultures were maintained at 23±2 oC in darkness. Biomass increase, ginsenoside content, and ginsenoside productivity were investigated after 5 weeks of culture. Experiment on BA Concentration Forty grams of hairy roots were inoculated in a 10 L drum type airlift bioreactor containing 8 L of half strength MS medium supplemented with 30 mg·L sucrose. BA concentrations were varied at 0, 1, and 3 mg·L and the volume of the input air was adjusted to 0.1 vvm. Culture conditions and the procedure used were the same as in the experiment on sucrose. Experiment on Jasmonic Acid Concentration Twenty grams of hairy roots were inoculated in a 5 L drum type airlift bioreactor containing 4 L of half strength MS medium supplemented with 30 mg·L sucrose. The roots were treated with different jasmonic acid concentrations: 0, 1, 2, and 5 mg·L 7 days before harvest. Culture conditions and the procedure used were the same as in the experiment on sucrose. RESULTS AND DISCUSSION Sucrose concentrations affected root growth and ginsenoside content in different ways. Dry weight and percentage dry weight increased with increasing sucrose concentration but ginsenoside production was promoted by low sucrose concentrations (2-5%), while high sucrose concentrations (7-9%) inhibited ginsenoside production (Table 1). One % sucrose, on the other hand, inhibited both root growth and ginsenoside production. The maximum ginsenoside content (8.01 mg·g·dw) as well as the maximum