Somatic embryogenesis of Panax ginseng in liquid cultures: A role for polyamines and their metabolic pathways

Plant Growth Regulation 31: 209–214, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 209 Somatic embryogenesis of Panax ginseng in liquid cultures: a role for polyamines and their metabolic pathways Claire Kevers*, Nathalie Le Gal, Marta Monteiro, Jacques Dommes & Thomas Gaspar Plant Molecular Biology and Hormonology, Institute of Botany B 22, University of Liège, Sart Tilman, B – 4000 Liège, Belgium (*author for correspondence, e-mail: c.kevers@ulg.ac.be) Received 8 December 1999; accepted in revised form 16 December 1999 Key words: Panax ginseng, polyamines, somatic embryogenesis Abstract A callus with embryogenic capacity was generated from root sections of Panax ginseng and used as an inoculum source for embryogenic liquid cultures in a three-step process: – a suspension culture of cell aggregates in the presence of an auxin/cytokinin mixture, – an induction medium containing auxin only (for 5 to 30 days), – a regeneration medium containing cytokinin only (for one month). Up to 25 embryos were recovered per 2.5 g of aggregates in these conditions. Incorporation of polyamines or their precursors arginine and ornithine into either the induction or regeneration media increased the number of embryos produced by up to 4 times. Inhibitors of both biosynthesis and biodegradation of polyamines reduced the number of embryos. These results support earlier findings of the role of polyamines in the process of somatic embryogenesis. The success of these liquid cultures opens up the possibility of producing somatic embryos of Panax ginseng in bioreactors. Abbreviations: AG – aminoguanidine; BSAA – benzoselenienyl-3 acetic acid; CHA – cyclohexylamine; 2,4-D – 2,4-dichlorophenoxyacetic acid; DFMA – difluoromethylarginine; DFMO – difluoromethylornithine; IAA – indoleacetic acid; Kin – kinetin; ZR – zeatin riboside 1. Introduction Panax root has been used in Oriental medicine since ancient times. The crude root extract is known to have tonic, stimulatory and adaptogenic properties [21] due to the presence of a wide range of saponins and sapogenins [23]. Recently, ginseng has also become a popular tonic and health food complement in Western countries. Therefore, the demand for the plant has increased dramatically worldwide. Ginseng is very expensive because of its long-term conventional (5–7 years) and troublesome production cycle. Propagation methods of ginseng by plant tissue culture and particularly by somatic embryogenesis have been investigated. Somatic embryogenesis has been successfully induced on solid media, directly from root [2, 3, 9, 30], leaf [30] or flower bud [28] derived calli or directly from zygotic embryos or cotyledons [10, 11]. Culture of ginseng tissues in bioreactors was developed in order to produce fresh material containing ginsenoside saponins [4]. However somatic embryogenesis induction in liquid culture has not been described. Polyamines are small polycations found in most organisms and are essential for cellular proliferation and normal cellular function. The diamine putrescine is the precursor of the polyamines spermidine and spermine. In higher plants, putrescine can be derived through two different pathways from ornithine via ornithine decarboxylase (EC 4.1.1.17) or from arginine via arginine decarboxylase (EC 4.1.1.19), both of which are specific, rate-limiting enzymes [16]. Many of the studies demonstrating enzyme and polyamine functions have been made possible due to the use of inhibitors, such as difluoromethylarginine (DFMA, a specific and irreversible inhibitor of arginine decarboxylase), difluoromethylornithine (DFMO, an inhibitor of ornithine decarboxylase), cyclohexylamine (CHA, an inhibitor of spermidine 210 synthase) and aminoguanidine (AG, an inhibitor of the conversion of putrescine into 11 -pyrroline and γ -aminobutyric acid). It has been demonstrated in various plant tissues that the ornithine decarboxylase pathway is particularly active in cell proliferation and that the arginine decarboxylase pathway is involved in embryo and organ differentiation and stress responses [13, 14, 29]. Treatments that modify polyamine levels, such as the exogenous application of polyamines [1, 18], specific polyamine anabolism [24] and catabolism inhibitors [17, 19], are interesting ways for improving morphogenesis [22]. The present study was undertaken with two purposes: first, in order to develop an efficient process for the initiation of somatic embryos of P. ginseng from cell aggregates in suspension cultures, and second, to check the possible use of polyamines to improve the production. The physiological role of the polyamines was indirectly evaluated by exogenous applications of these compounds, of their metabolic precursors and of inhibitors of their metabolism. 2. Materials and methods 2.1 Plant material One-year-old roots of Panax ginseng C.A. Meyer were used throughout the study. Roots were surfaced sterilized in ethanol (70%) for 3 min then in sodium hypochlorite (3%) for 20 min, and rinsed three times with sterile distilled water. Disinfected roots were cut into 3 mm thick sections. 2.2 Induction of embryogenic callus The callus induction medium was MS basal (mineral and organic compounds) medium [27] supplemented with 1 mg l−1 2,4-D. The pH was adjusted to 5.8 prior to autoclaving. The cultures were incubated in darkness at 25 ± 2 ◦ C in 9 cm plastic Petri dishes sealed with polyethylene film for 6 weeks. The embryogenic callus formed in Panax ginseng is a compact, opaque white callus. It was transferred to half strength MS medium (MS/2) and supplemented with benzoselenienyl-3 acetic acid (BSAA, 1 mg l−1 ) and kinetin (Kin, 0.3 mg l−1 ) in darkness, at 25 ◦ C for multiplication. Embryogenic potential of the callus was maintained by 4-week subculturing on the same medium. 2.3 Establishment of suspension cultures Liquid cultures were initiated by transferring approximately 2.5 g of finely minced 6-month-old embryogenic calli (without embryos) in 100 ml Erlenmeyer flasks in 50 ml liquid MS/2 medium supplemented with BSAA 1 mg l−1 and Kin 0.3 mg l−1 for one month. The flasks were shaken at 80 rpm at 25 ± 2 ◦ C in darkness. The suspension cultures obtained consisted of cell aggregates (± 3 mm in diameter). 2.4 Somatic embryogenesis The suspension aggregates were transferred to embryogenesis induction medium. This induction medium is a MS/2 liquid medium supplemented with auxins (BSAA or IAA, 3 mg l−1 ). After 5 days (or more as specified in the results) in the induction medium, the aggregates were transferred to regeneration medium. Organised structures were regenerated in MS/2 liquid medium supplemented with cytokinins (Kin or ZR, 0.2 mg l−1 ). After one month on this medium, the number of embryogenic regenerated structures from the aggregates was estimated under a microscope as the number of embryogenic structures obtained from 2.5 g of aggregates transferred to the induction medium. All cultures were maintained in darkness, at 25 ◦ C. In order to check the role or effect of polyamines, the induction medium or the regeneration medium was modified by the addition of putrescine, spermidine, spermine, arginine, ornithine, DFMO, DFMA, CHA or AG at various concentrations (10−5 to 10−3 M) after autoclaving. These additives did not appear affect the general health of the cultures, except that there was some delayed browning at the highest concentration (10−3 M). Each experiment (three erlenmeyer flasks) was repeated at least three times. 3. Results 3.1 Induction of embryogenesis Calli (2.5 g) were cultured under various conditions to induce somatic embryogenesis. Addition of either of two auxins, BSAA and IAA, at 3 mg l−1 , and different induction times (5, 15 or 30 days) were tested. After treatment, the calli were transferred for the regeneration of somatic embryos in the presence or absence of the cytokinins Kin or ZR at 211 Table 1. Number of embryos of ginseng formed from 2.5 g of callus, after various treatments: pretreatment for one month on MS/2 without regulators, induction in auxin medium (BSAA or IAA 3 mg l−1 ) for 5, 15 days or one month, and regeneration in the presence or absence of a cytokinin (Kin or ZR 0.2 mg l−1 ) for one month. Values are the means of at least three samples ± standard deviation Pretreatment MS/2 0 (months) Induction Regeneration Time Auxin Cytokinin (days) (3 mg l−1 ) (0.2 mg l−1 ) Number of embryos 1 1 1 – – – 1 1 1 – – – – – – – – – – – – – – – 5 5 5 5 5 5 5 5 5 5 5 5 15 15 15 15 15 15 30 30 30 30 30 30 14 ± 3 10 ± 1 12 ± 1 24 ± 1 18 ± 1 8±2 6±2 11 ± 2 1±1 10 ± 2 22 ± 2 14 ± 3 16 ± 2 25 ± 1 20 ± 3 10 ± 1 8±1 6±2 16 ± 1 13 ± 1 10 ± 2 14 ± 2 9±1 4±2 BSAA BSAA BSAA BSAA BSAA BSAA IAA IAA IAA IAA IAA IAA BSAA BSAA BSAA IAA IAA IAA BSAA BSAA BSAA IAA IAA IAA Kin ZR – Kin ZR – Kin ZR – Kin ZR – Kin ZR – Kin ZR – Kin ZR – Kin ZR – 0.2 mg l−1 . In some cases, a pretreatment of one month in liquid medium without hormone was also tested before induction. The number of embryogenic structures formed was counted after one month of culture (Table 1). These embryogenic structures were embryos at various developmental stages: globular, heart-shaped, torpedo or cotyledonary. The embryogenic structures regenerated from aggregates were generally more numerous when the auxin used for the induction was BSAA rather than IAA. Duration of induction affected the regeneration process. The results were very similar when induction lasted 5 or 15 d but the formation of embryogenic structures decreased if the induction time was longer (one month). In the regeneration medium, cytokinin Figure 1. Number of somatic embryos of ginseng formed from 2.5 g of callus, when polyamines (putrescine, spermidine and spermine) were added at various concentrations (µM) to the induction (A, 5 days) or the regeneration (B, one month) medium. was needed to regenerate embryogenic structures. Pretreatment on MS/2 0 (without regulators) for one month did not improve the formation of somatic embryos. The best results were obtained with a 15 d – induction in the presence of 3 mg l−1 BSAA and with a regeneration medium containing 0.2 mg l−1 ZR. Induction in the presence of BSAA for 5 days, followed by regeneration in the presence of Kin, produced similar results. These last conditions were used in all subsequent experiments. 3.2 Influence of exogenous polyamines Putrescine, spermidine and spermine were added to the induction (5 days) or the regeneration (one month) medium at various concentrations (10−5 to 10−3 M). The resulting data are presented in Figure 1. Promotion of somatic embryogenesis by polyamines added to induction or regeneration medium were observed at all concentrations tested. A 5- and 4fold increase in the number of embryogenic structures 212 Figure 2. Number of somatic embryos of ginseng formed from 2.5 g of callus, when precursors of polyamines (arginine or ornithine) were added at various concentrations (µM) to the induction (A, 5 days) or the regeneration (B, one month) medium. Figure 3. Number of somatic embryos of ginseng formed from 2.5 g of callus, when inhibitors of polyamines biosynthesis (DFMA, DFMO) were added at various concentrations (µM) to the induction (A, 5 days) or the regeneration (B, one month) medium. has been recorded with spermidine (10−3 M) added to induction or regeneration medium respectively. esis process but the effect was lower than that observed with DFMO and DFMA at the same concentrations. 3.3 Influence of exogenous precursors of polyamines 4. Discussion Arginine and ornithine were added at the same concentrations as polyamines into the induction (5 days) or the regeneration (one month) medium (Figure 2). Arginine and ornithine, at concentration above 10−5 M had a beneficial effect on the formation of embryogenic structures, but this effect was lower than that observed with polyamines. This effect was very similar when the precursors were added to the induction or the regeneration medium. 3.4 Influence of polyamine biosynthesis inhibitors All the tested polyamine biosynthesis inhibitors reduced somatic embryogenesis when added to the induction or the regeneration medium (Figures 3 and 4). DFMA and DFMO drastically reduced the formation of somatic embryos at all the tested concentrations (10−5 to 10−3 M) (Figure 3). CHA or AG (10−3 M) completely blocked somatic embryogenesis when added to the induction medium (Figure 4). In the other cases, the inhibitors inhibited the somatic embryogen- Somatic embryogenesis from ginseng cell aggregates in liquid culture was induced by cultivation for 5 d in the presence of auxin and further regenerated after a transfer into cytokinin supplemented media for one month. The addition of one of the three polyamines to the induction or the regeneration media did not significantly affect callus growth but promoted the formation of somatic embryos. The stimulation of somatic embryogenesis by exogenous polyamines has already been reported in a number of other plant tissue and cell cultures [1, 12, 15, 26, 31]. In the present work, the most active polyamine on ginseng aggregates was spermidine in contrast to other tissues where putrescine was the most active [26, 31]. This promoting action of polyamines on somatic embryogenesis may be due to an efficient conversion of competent cells into embryos as suggested by Bajaj and Rajam [5]. The stimulatory effect of putrescine in somatic embryogenesis of Solanum melongena has 213 The present results demonstrate the possibility of programming somatic embryogenesis of P. ginseng in liquid cultures. The data presented also confirm that production of somatic embryos can be regulated by the manipulation of polyamine levels and metabolism either by using exogenous polyamines or their specific metabolic inhibitors. Under appropriate conditions, the embryogenic structures obtained can further develop into plantlets with green leaves and a root, which opens up the possibility of mass production of somatic embryos of Panax ginseng in bioreactors. Acknowledgements Figure 4. Number of somatic embryos of ginseng formed from 2.5 g of callus, when inhibitors of polyamines metabolism (CHA, AG) were added at various concentrations (µM) to the induction (A, 5 days) or the regeneration (B, one month) medium. This research was financed by ORTIS Laboratories (Elsenborn, Belgium) and the Wallonian Ministry for Technology. We thank the Merrell Dow Research Institute (Cincinnati, Ohio, USA) for providing the DFMO and DFMA inhibitors. References been shown not to be due to the added nitrogen [31]. The present counter-experiments using inhibitors of polyamine biosynthesis indirectly confirm this fact. The endogenous polyamine level of the tissue and the stage at which polyamine was applied influenced the action of exogenous putrescine on differentiation of somatic embryos [25], of roots [20] or of stem meristema [7]. In ginseng cell suspensions, we showed that addition of polyamine biosynthesis inhibitors (DFMA and DFMO) to either induction or regeneration medium significantly reduced the number of regenerated structures, as already shown in Hevea [12]. This observation suggests that polyamines are involved in the induction and regeneration of somatic embryos. In carrot, the expression of a mouse ornithine decarboxylase cDNA promoted somatic embryogenesis [6]. In the same way, the inhibition of somatic embryogenesis by CHA at high concentration could be due to the dramatic reduction of the ratio between spermidine and putrescine levels [8]. The inhibitory effect of AG is interpreted as the further involvement of the putrescine catabolic 11 -pyrroline pathway in the inductive phase as previously demonstrated in another organogenic program, the rooting process [18]. 1. 2. 3. 4. 5. 6. 7. 8. Altman A, Nadel B, Falash Z and Levin N (1990) Somatic embryogenesis in celery: induction, control and changes in polyamines and proteins. In: Nijkamp HJJ, Van Der Plas LHW and Van Aartrijk J (eds) Progress in Plant Cellular and Molecular Biology. Dordrecht: Kluwer Academic Publishers, pp 454–459 Asaka I, Ii I, Yoshikawa T, Hirotani M and Furuya T (1992) Embryoid formation by high temperature treatment from multiple shoots of Panax ginseng. Planta Med 59: 345–346 Asaka I, Ii I, Hirotani M, Asada Y, Yoshikawa T and Furuya T (1993) Mass production of ginseng (Panax ginseng) embryoids on media containing high concentrations of sugar. Planta Med 60: 146–148 Asaka I, Ii I, Hirotani M, Asada Y and Furuya T (1993) Production of ginsenoside saponins by culturing ginseng (Panax ginseng) embryogenic tissues in bioreactors. Biotechnol Lett 15: 1259–1264 Bajaj S and Rajam MV (1995) Efficient plant regeneration from long-term callus cultures of rice by spermidine. Plant Cell Rep 14: 717–720 Bastola D and Minocha SC (1995) Increased putrescine biosynthesis through transfer of mouse ornithine decarboxylase cDNA in carrot promotes somatic embryogenesis. Plant Physiol 109: 63–71 Bernet E, Claparols I, Santos MA and Torné JM (1998) Role of putrescine metabolic pathways in the diffentiation process of maize meristematic callus. Plant Physiol Biochem 36: 759– 766 Bharti P and Rajam MV (1995) Effects of the polyamine biosynthesis inhibitor difluoromethylornithine on growth, polyamine levels, chromosome behaviour and polygenic traits in wheat (Triticum aestivum L.). Ann of Bot 76: 297–301 214 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Chang WC and Hsing YI (1980) Plant regeneration through somatic embryogenesis in root-derived callus of ginseng (Panax ginseng C.A. Meyer). Theor Appl Genet 57: 133–135 Choi YE and Soh WY (1994) Origin of somatic embryo induced from cotyledons of zygotic embryos at various developmental stages of ginseng. J Plant Biol 37: 365–370 Choi YE and Soh WY (1996) Effect of plumule and radicule on somatic embryogenesis in the cultures of ginseng zygotic embryos. Plant Cell Tissue Organ Culture 45: 137–143 El Hadrami I and D’Auzac J (1992) Effects of polyamine biosynthetic inhibitors on somatic embryogenesis and cellular polyamines in Hevea brasiliensis. J Plant Physiol 140: 33–36 Feirer RP, Mignon G and Litway JD (1984) Arginine decarboxylase and polyamines required for embryogenesis in the wild carrot. Science 223: 1433–1435 Flores HE (1990) Polyamines and plant stress. In: Flores HE (ed) Stress Responses in Plants: Adaptation and Acclimation Mechanism. New York: Wiley-Liss Inc., pp 217–239 Galston AW and Flores HE (1991) Polyamines and plant morphogenesis. In: Slocum RD and Flores HE (eds) Biochemistry and Physiology of Polyamines in Plants. Boca Raton: CRC Press, pp 175–186 Galston AW and Kaur-Sawhney R (1990) Polyamines in plant physiology. Plant Physiol 94: 406–410 Hausman JF, Kevers C, Evers D and Gaspar T (1997) Conversion of putrescine into aminobutyric acid, an essential pathway for root formation by poplar shoots in vitro. In: Altman A and Waisel Y (eds) Biology of Root Formation and Development. Plenum Press, pp 133–140 Hausman JF, Kevers C and Gaspar T (1994) Involvement of putrescine in the inductive rooting phase of poplar shoots in vitro. Physiol Plant 92: 201–206 Hausman JF, Kevers C and Gaspar T (1995) Auxin-polyamine interaction in the control of the rooting inductive phase of poplar shoots in vitro. Plant Sci 110: 63–71 Hausman JF, Kevers C and Gaspar T (1995) Putrescine control of peroxidase activity in the inductive phase of rooting in poplar shoots in vitro, and the adversary effect of spermidine. J. Plant Physiol 146: 681–685 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. Hu SY (1976) The genus Panax (ginseng) in Chinese medicine. Econ Bot 30: 11–28 Kevers C, Bringaud C, Hausman JF and Gaspar T (1997) Putrescine involvement in the inductive phase of walnut shoots rooting in vitro. Saussurea 28: 47–57 Li TSC (1995) Asian and American ginseng: a review. Hort Technology 5: 27–34 Mengoli M, Rosell P and Bagni N (1989) Effect of long term treatment on growth, bud formation and free amine and hydroxycinamoyl putrescine levels in leaf explants of Nicotiana tabacum cultivated in vitro. Plant Physiol Biochem 27: 1–8 Minocha R, Kvaalen H, Minocha SC and Long S (1993) Polyamines in embryogenic cultures of Norway spruce (Picea abies) and red spruce (Picea rubens). Tree Physiol 13: 365– 377 Minocha SC and Minocha R (1995) Role of polyamines in somatic embryogenesis. In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry. Vol. 30. Somatic Embryogenesis and Synthetic Seeds 1. Berlin: Springer-Verlag, pp 53–70 Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15: 473–497 Shoyama Y, Zhu XX, Nakai R, Shiraishi S and Kohda H (1997) Micropropagation of Panax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets. Plant Cell Rep 16: 450–453 Tiburcio AF, Kaur-Sawhney R and Galston AW (1988) Polyamine biosynthesis during vegetative and floral bud differentiation in thin-layer tobacco tissue cultures. Plant Cell Physiol 29: 1241–1249 Tirajoh A, Kyung TS and Punja ZK (1998) Somatic embryogenesis and plantlet regeneration in american ginseng (Panax quinquefolium L.). In Vitro Cell Dev Biol 34: 203–211 Yadav JS and Rajam MV (1997) Spatial distribution of free and conjugated polyamines in leaves of Solanum melongena L. associated with differential morphogenetic capacity: efficient somatic embryogenesis with putrescine. J Exp Bot 48: 1537–1545