Tissue Culture and Micropropagation of Mongolian Licorice (Glycyrrhiza uralensis Fisch.)
Yu. Oyunbileg1, Kh. Altanzul1,Ts. Oyunsuren1, D. Bayarlkhagva2
1Laboratory of Molecular Biology, Institute of Biology, Mongolian Academy of Sciences, Ulaanbaatar 210351, Mongolia, e-mail: firstname.lastname@example.org
2Department of Genetics and Molecular Biology, Faculty of Biology, National University of Mongolia, Ulaanbaatar 210646, Mongolia
Mongolian licorice (Glycyrrhiza uralensis Fisch.) is a pharmacologically important plant rich in flavonoids and saponins. For tissue culture, root and cotyledon explants from seedlings were used. Sterilized explants with one node were used for micropropagation. Half-strength Murasige-Skuge medium and Gamborg‘s B5 medium with different supplements were used for the induction of calluses and multiple shoots. Conditions for tissue culture and micropropagation of G. uralensis were determined.
Keyword: callus,Glycyrrhiza uralensis,in vitro,micropropagation,Mongolia,nodal culture
Currently, there is much international interest in increasing plant resources, plant productivity and the ability to synthesize specific compounds, especially various secondary metabolites useful for medicinal practices. Licorice (Glycyrrhiza uralensis Fisch.), belonging to the family Fabaceae, is recognised as one of the most valuable and widely used oriental herbs. During the last few years, use of licorice has increased rapidly. However, due to human influence and natural desertification processes in Mongolia licorice resources may become exhausted in the near future. In vitro culture and propagation are useful tools in the conservation of this important plant. Use of in vitro cultures of G. uralensis and G. glabra L. have been reported by a number of authors (Thengane et al., 1998; Kovalenko & Kurchii, 1998; Kohjyouma et al., 1995). The roots and isolated active components of these plants are widely used for treatment of viral infections, inflammatory diseases and prevention of different cancers (Arase et al., 1997; Numazaki, 2003; Shiraki et al., 2003). The aim of our research is to determine methods of in vitro culture and micropropagation of Mongolian licorice, in order to increase the bioresources of G. uralensis in Mongolia.
Material and Methods
Plant materials. Seed samples of G. uralensis were provided by researchers from the Institute of Botany at the Mongolian Academy of Sciences. Two and four year old G. uralensis leaves, roots and whole plants were also collected from Dashinchilen district in Bulgan province, and Bogd and Baatsagaan districts in Bayankhongor province during a field study.
Culture conditions. The seeds were soaked in sterilized water for 24 hours, followed by a 70% ethanol bath for 30 seconds. Stratifications were performed at 4oC for 72 hours, and the seed surface sterilized with 2% sodium hypochloride solution with a drop of Tween-20 for 5 minutes, before being rinsed with sterile water 3 times. The prepared seeds were then used for germination and microprogation experiments. For germination of G. uralensis, half-strength basal Murashige-Skoog medium (MS medium; pH 5.8) supplemented with 0.8 % agar and 1.5 % sucrose was used. Autoclaving was performed at +121 0C for 15 minutes (Oyunbileg & Mijidsuren, 2004). Seedlings grown on Gamborg‘s B5 medium and supplemented with 4 different combinations of growth regulators (BAP-1.0 mg/L and 2.0 mg/L, IAA-0.1 mg/L and 1.0 mg/L) (Liao et al., 2004) were used for micropropagation.
Incubation. All cultures were incubated at 25 0C under 8/16 hours photoperiod in a daylight cabinet. Light intensity was 40 μMole·m-2 · s-1.
Naturally grown licorice has poor seed germination, about 1 % to 8 % (Gankhuyag & Ligaa, 1993; Dashjamts, 1983). Gankhuyag and Ligaa (1993) stated that it is possible to achieve 92 % seed germination through scarification, and previous researchers also determined that seed germination could be increased after mechanical damage to the seed coat (Gankhuyag & Ligaa, 1993; Dashjamts, 1983). In this study, seed coats were damaged and the stratification method was used, as a result 55.8 % - 100 % of seeds germinated. There was no significant difference between mediums (1/2 MS, 1/2 B5, 1 % agar) used in the experiment as shown in Figure 1.
One week after germination, root and cotyledon explants from seedlings were transferred to Gamborg’s B5 medium and supplemented with 1 mg/l 2.4-dichlorphenoxyacetic acid (2.4-D), 0.1 mg/l kinetin and 2% sucrose to initiate callus growth. Two to three weeks later, callus initiation occurred (Table 1).
Calluses that grew from root explants were more friable, yellowish in colour and watery compared with those initiated from cotyledon explants (Fig. 2). Propagation of G. uralensis was performed using nodal and shoot tip cultures, on Gamborg’s B5 medium, which was supplemented with 5 different combinations of BAP and IAA growth regulators. There was no significant difference between the lengths of shoots and the addition of BAP and IAA combinations did not increase shoot formation (Table 2).
In addition, the seedlings were grown on halfstrength MS medium supplemented with 4 different combinations of BAP and NAA growth regulators. The results showed that half-strength MS medium with 0.1 mg/l NAA was effective in inducing the root of licorice (Table 3). After 14 days of culture, the medium with no growth regulators was effective in the formation of multiple shoots and roots. Some shoots rooted spontaneously on half-strength MS medium without growth regulators. This medium was therefore selected for rooting and regeneration of whole plantlets. As a result, after 42 days of culture the plantlets developed (Fig. 3), but only 50 plantlets were transplanted into pots.
Currently we are continuing our research, and regenerated plantlets are being acclimatized. The nodal cultivated on MS medium formed new shoots after 10 days. In order to identify the optimal medium for regeneration, cultures from seedlings and multiple shoots from nodal cultures were sub-cultured on half-strength MS medium. The results of our experiments showed that regenerated plantlets could be obtained after about 3 weeks (Fig. 4). These results were similar to those conducted by Thengane et al. (1998) and Kohjyouma et al. (1995) who developed in vitro plantlets of Glycyrrhiza glabra. One problem raised during the in vitro micropropogation was the secretion of phenol-like substances, which inhibited the growth of tissue culture. The same phenomenon was observed by Kovalenko et al. (1998).
In this case, the secretion of phenolics was regulated by abscisic acid, which acts as an antioxidant. In our experiment, it was found that frequently transferring the new shoots to new medium prevented the phenol-like substances from influencing culture growth. The results obtained from this study would be useful for increasing G. uralensis resources and for secondary metabolism research.
We would like to thank the Asia Research Center at the National University of Mongolia and the Korea Foundation for Advanced Studies for supporting this research. Thanks also to Drs. D. Magsar and G. Ochirbat from the Institute of Botany, Mongolian Academy of Sciences for supplying the seed samples.
- Arase, Y., Ikeda, K. & Murashima, N. 1997. The long-term efficiency of glycyrrhizin in chronic hepatitis C patients. Cancer 79: 1494-1500.
- Dashjamts, Y. 1983. Ecological and biological characteristics of Mongolian Glycyrrhiza uralensis. Ph.D. Dissertation. Ulaanbaatar. (in Russian)
- Gankhuyag, G. & Ligaa, U. 1993. Mongolian Licorice. Bayankhongor. (in Mongolian)
- Kohjyouma, M., Kohda, H., Tani, N., Ashida, K., Sugino, M., Yamamoto, A. & Horikoshi, T. 1995. In-vitro propagation from axillary buds of Glycyrrhiza glabra L. Plant Tissue Culture Letters 12(2): 145-149.
- Kovalenko, P.G. & Kurchii, B.A. 1998. Use of abscisic acid in the plant tissue culture of licorice Glycyrrhiza glabra L. electroporated protoplasts. In: Proceedings of II International Symposium on Plant Biotechnology, Ukraine. p. 65.
- Liao, Zh., Chen, M., Tan, F., Sun, X. & Tang, K. 2004. Micropropagation of endangered Chinese aloe. Plant Cell Tissue and Organ Culture 76: 83-86.
- Numazaki, K. 2003. Glycyrrhizin therapy for viral infections. African Journal of Biotechnology 2(10): 392-393.
- Oyunbileg, Yu. & Mijidsuren, A. 2004. Studies on tissue culture of Glycyrrhiza uralensis Fisch. In: Proceedings of the Second Workshop on Korea-Mongolia Science and Technology Cooperation, Biotechnology, Seoul, Korea, p.106-112.
- Shiraki, K., Sato, H., Kurokawa, M. & Yoshida, Y. 2003. Glycyrrhizin: the molecule and present knowledge of action in various liver diseases. In: Prevention of progression in chronic liver disease. (Ed. by N. Hayashi & M.P. Manns). Falk workshop, Freiburg, Germany. p.76-89.
- Thengane, S.R., Kulkarni, D.K. & Krishnamurthy, K.V. 1998. Micropropagation of licorice (Glycyrrhiza glabra L.) through shoot tip and nodal cultures. In Vitro Cellular and Developmental Biology of Plants 34(4): 331- 334.