The improvement of ginsenoside accumulation in Panax ginseng as a result of γ-irradiation

Abstract

In this study, gamma rays were used to irradiate embryogenic calli induced from cotyledon explants of Panax ginseng Meyer. After the embryogenic calli were irradiated, they were transferred to adventitious roots using an induction medium; next, mutated adventitious root (MAR) lines with a high frequency of adventitious root formations were selected. Two MAR lines (MAR 5-2 and MAR 5-9) from the calli treated with 50 Gy of gamma rays were cultured on an NH4NO3-free Murashige and Skoog medium with indole-3-butyric acid 3 mg/L. The expression of genes related to ginsenoside biosynthesis was analyzed using reverse transcription polymerase chain reaction with RNA prepared from native ginseng (NG), non-irradiated adventitious root (NAR) and 2 MAR lines. The expression of the squalene epoxidase and dammarenediol synthase genes was increased in the MAR 5-2 line, whereas the phytosterol synthase was increased in the MAR 5-9 line. The content and pattern of major ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1) were analyzed in the NG, NAR, and 2 MAR lines (MAR 5-2 and MAR 5-9) using TLC and HPLC. In the TLC analysis, the ginsenoside patterns in the NG, NAR, and 2 MAR lines were similar; in contrast, the MAR 5-9 line showed strong bands of primary ginsenosides. In the HPLC analysis, compared with the NG, one new type of ginsenoside was observed in the NAR and 2 MAR lines, and another new type of ginsenoside was observed in the 2 MAR lines irradiated with gamma rays. The ginsenoside content of the MAR 5-9 line was significantly greater in comparison to the NG.

Keywords: Gamma irradiation; Ginsenoside; HPLC; Panax ginseng; TLC.

Fig. 1.. Adventitious root production from gamma irradiated mutant lines: (A) ginseng seed was germinated by in vitro culture, (B) amorphous embryogenic calli were induced from ginseng cotyledons, (C) embryogenic callus formation on the callus induction medium, (D) selected high frequency of adventitious root formation, (E) proliferation of the adventitious root in the liquid adventitious root induction medium. NAR, non-irradiated adventitious root; MAR, mutated adventitious root (5-line).

Fig. 2.. The fresh mass of adventitious roots of the non-irradiated adventitious root (NAR) and 2 mutated adventitious root (MAR) lines after 4 wk.

Fig. 3.. The biosynthetic pathway of ginsenoside in Panax ginseng. FPP, farnesyl diphosphate, SS, squalene synthase; SE, squalene epoxidase; PNX, cycloartenol synthase; PNY1, oxidosqualene cyclase 1; PNY2, oxidosqualene cyclase 2; DDS, dammarenediol synthase; NAR, non-irradiated adventitious root; MAR, mutated adventitious root.

Fig. 4.. The expression of genes related to ginsenoside biosynthesis in the mutated adventitious root (MAR). NG, native ginseng; NAR, non-irradiated adventitious root; SS, squalene synthase; SE, squalene epoxidase; PNX, cycloartenol synthase; PNY1, oxidosqualene cyclase 1; PNY2, oxidosqualene cyclase 2; DDS, dammarenediol synthase.

Fig. 5.. A ginsenoside analysis using TLC and HPLC. (A) TLC analysis of ginsenosides. The star indicates a new type of ginsenoside. (B) Ginsenoside contents in the native ginseng (NG), non-irradiated adventitious root (NAR) and 2 mutated adventitious root (MAR) lines. Black arrow was a new band.

Fig. 6.. The HPLC chromatograms of ginseng extracts in the non-irradiated adventitious root (NAR) and the 2 mutated adventitious root (MAR) lines. Seven ginsenosides including the Rb1, Rb2, Rc, and Rd of the Rb groups and the Re, Rf and Rg1 of the Rg group, were marked at each peak area. The star indicates a new type ginsenoside. NG, native ginseng.