INCREASE OF SAPONIN CONTENTS VIA ELICITOR TREATMENTS IN BIOREACTOR CULTURE OF GINSENG (PANAX GINSENG C.A. MEYER) ADVENTITIOUS ROOTS

Ginseng (Panax ginseng C.A. Meyer) is one of the most potent medicinal plants and has been used for centuries as a health tonic. Saponin is considered one of the most important active components in ginseng root, which shows anabolic, adaptogenic, antibiotic, minor hyperglycemic, and anti-cancer activities. More than 20 different types of saponin have been identified. Current advances in plant biotechnology allowed production of saponin through adventitious root culture of ginseng in large-scale bioreactors. However, one problem still to be resolved is the fact that although ginseng adventitious root is an efficient means of saponin production due to fast root growth and stable metabolite productivity, saponin content in cultured ginseng root is still lower than that found in field-cultivated ginseng root. To increase saponin content, ginseng adventitious roots were cultured in a 20-liter balloon type airlift bioreactor for 40 days followed by different types and concentrations of elicitor treatments: methyl dihydro jasmonate, methyl epijasmonate, methyl epi-dihydro jasmonate, jasmonic acid and methyl jasmonate. Methyl epi-jasmonate (100 μmol) resulted in the highest content of saponin accumulation followed by methyl jasmonate, while methyl dihydro jasmonate was not effective for saponin accumulation. Based on the results and the price of elicitors, further research is under way to maximize saponin content by applying methyl jasmonate. INTRODUCTION Ginseng (Panax ginseng C.A. Meyer) is one of the most potent medicinal plants used for centuries as a health tonic. Saponin is considered one of the most important active components in ginseng root with anabolic, adaptogenic, antibiotic, minor hyperglycemic, and anti-cancer activities. More than 20 different types of saponin have been identified. Current advances in plant biotechnology allow for the production of saponin through adventitious root culture of ginseng in large-scale bioreactors. Ginseng adventitious root is an efficient source for saponin production due to its fast root growth and stable metabolite productivity. On the other hand, saponin content in cultured ginseng root is still lower than that in field-cultivated ginseng root, a main problem still to be resolved. Saponin content can be increased by increasing biomass growth through optimization of culture conditions such as medium component, type and concentration of auxin, nitrogen source in the culture medium, medium supply method, air volume, etc. On the other hand, to maximize saponin accumulation elicitation is necessary. Since it was first characterized in the late 1980’ (Eilert, 1987), there have been many reports on the activation of useful compounds through biotic and abiotic elicitations: Monoterpenic indole alkaloid biosynthesis by jasmonates in C. roseus and C. ledgeriana seedlings (Aerts et al., 1994; Gantet et al., 1998), increased ajmalicine, catharanthine, and tryptamine levels. The addition of vanadyl sulfate to cell suspension cultures of C. roseus increased ajmalicine, catharanthine, and tryptamine levels (Smith et al., 1987; Tallevi and DiCosmo, 1988). In case of saponin accumulation, yeast had a synergistic effect on saponin synthesis in hairy roots of C. roseus (Hwang et al., 1996). This experiment was Proc. WOCMAP III, Vol. 5: Quality, Efficacy, Safety, Processing & Trade in MAPs Eds. E. Brovelli, S. Chansakaow, D. Farias, T. Hongratanaworakit, M. Botero Omary, S. Vejabhikul, L.E. Craker and Z.E. Gardner Acta Hort. 679, ISHS 2005 146 conducted to increase saponin accumulation in ginseng adventitious root cultures by applying different types and concentrations of elicitors. MATERIALS AND METHODS Calli were induced from ginseng roots on MS (Murashige and Skoog, 1962) medium containing 1.0 mg L 2,4-D and 0.1 mg L kinetin. The calli were cultured on modified MS solid media (without NH4NO3) containing 2.0 mg L NAA and 50 g L sucrose in the dark to induce adventitious roots (Yu et al., 2002). Thirty grams of adventitious roots were cultured for 40 days in a 20 L balloon type airlift bioreactor (Fig. 1) containing 5 L modified MS medium supplemented with 2.0 mg L IBA and 50 g L sucrose. The adventitious roots were treated with different types and concentrations of elicitors: Methyl dihydro jasmonate, methyl epi-jasmonate, methyl epi-dihydro jasmonate, jasmonic acid and methyl jasmonate were respectively added to the media with the concentrations of 50, 100 and 150 μM. Cultures were maintained for 7 days at 22±1°C in the dark. Extraction and determination of ginsenosides were carried out by the modification of ginsenoside analysis method (Kim, 2002). The ginsenoside fraction was analyzed using an HPLC (Waters 2690 separation module; Waters 996 photodiode array detector; Waters millennium 2010 chromatography manager) on an Altec Platinum C18 column with water and acetonitrile as mobile phase. RESULTS AND DISCUSSION The highest saponin content was obtained utilizing methyl epi-jasmonate (MEJ) at a concentration of 100 μM (Table 1) but this chemical is too expensive for commercial use. Methyl jasmonate (MeJA) is an effective chemical for large-scale cultures since it is less expensive and also has a demonstrated good effect on saponin accumulation. Methyl dihydro jasmonate (MDJ) and jasmonic acid (JA) were determined to have little effect on saponin accumulation. The PD/PT ratio was also higher in the treatment of MEJ and MeJA. With elicitations by 50 and 100 μM MeJA, saponin content increased more than 6 fold compared with control. Utilizing histological observation, we found that treatment with MeJA induced a high vacuolation pattern in root cells (data not shown). Further research is still needed to reveal the exact cellular and biochemical changes occurred after treatment with MeJA. Jasmonic acid and its derivatives are considered to be involved in the signal transduction pathway that induces particular enzymes catalyzing biochemical reactions to form defense compounds of low molecular weight in plants, such as polyphenols, alkaloids, quinones, terpenoids, and polypeptides (Mizukami et al., 1993; William et al., 1996). In Lithospermum cell cultures, jasmonates caused a rapid increase in the activities of enzymes involved in the biosynthesis of shikonin such as p-hydroxybenzoate geranyltransferase (Urbanek et al., 1996). Ginsenosides belong to triterpenoide saponins. They are originated from acetyl-Co A through more than 15 metabolic steps, some of which are not clearly known (Yazaki et al., 1997). Until now, enzymes related to the synthesis of protopanaxadiol and protopanaxatriol ginsenosides have not been determined. In our experiment, jasmonic acid derivatives specifically promoted the accumulation of protopanaxadiol ginsenosides rather than the accumulation of protopanaxatriol ginsenosides. This result suggests that jasmonic acid may trigger enzyme activities for the synthesis of protopanaxadiol ginsenosides. Further work is required to investigate the enzymes involved in the enzymatic biosynthesis of ginsenosides. ACKNOWLEDGEMENTS This work was supported in part by the Korea Science and Engineering Foundation (KOSEF) through the Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju, 361-763, Korea.