In Vitro Cellular & Developmental Biology - Plant

, Volume 47, Issue 6, pp 710–718 | Cite as

In vitro propagation of North American ginseng (Panax quinquefolius L.)

  • Esther E. Uchendu
  • Gopinadhan Paliyath
  • Dan C. W. Brown
  • Praveen K. Saxena
Plant Tissue Culture

Abstract

North American ginseng (NAG) (Panax quinquefolius L.) is a medicinally important plant with multiple uses in the natural health product industry. As seed propagation is time-consuming because of the slow growth cycle of the plant, in vitro propagation using a bioreactor system was evaluated as an effective approach to accelerate plant production. An efficient method was developed to multiply nodal explants of NAG using liquid-culture medium and a simple temporary immersion culture vessel. The effects of plant growth regulators, phenolics, and chemical additives (activated charcoal, melatonin, polyvinylpolypyrrolidone, and ascorbic acid) were evaluated on in vitro-grown NAG plants. The highest number (12) of shoots per single node was induced in half-strength Schenk and Hildebrandt basal medium containing 2.5 mg/l kinetin, in which 81% of the cultured nodes responded. In a culture medium with 0.5 mg/l α-naphthalene acetic acid (NAA), roots were induced in 78% of the explants compared to 50% with a medium containing indole-3-acetic acid. All of the resulting plants appeared phenotypically normal, and 93% of the rooted plants were established in the greenhouse. Phenolic production increased significantly (P < 0.05) over a 4-wk culture period with a negative impact on growth and proliferation. Activated charcoal (AC; 50 mg/l) significantly reduced total phenolic content and was the most effective treatment for increasing shoot proliferation. Shoot production increased as the phenolic content of the cultures decreased. The most effective treatment for NAG development from cultured nodal explants in the bioreactor was 2.5 mg/l kinetin, 0.5 mg/l NAA, and 50 mg/l AC in liquid culture medium. This protocol may be useful in providing NAG tissues or plants for a range of ginseng-based natural health products.

Keywords

Antioxidant Bioreactor Liquid medium Micropropagation Node Phenolic 

References

  1. Birmeta G.; Welander M. Efficient micropropagation of Ensete ventricosum applying meristem wounding: a three-step protocol. Plant Cell Rep 23: 277–283; 2004.PubMedCrossRefGoogle Scholar
  2. Castillo-Sanchez J. X.; Garcia-Falcon M. S.; Garrido J.; Martinez-Carballo E.; Martins-Dias L. R.; Mejuto X. C. Phenolic compounds and colour stability of Vinhao wines: Influence of wine making protocol and fining agents. Food Chem 106: 18–26; 2008.CrossRefGoogle Scholar
  3. Chen G.; Huo Y.; Tan D. X.; Liang Z.; Zhang W.; Zhang Y. Melatonin in Chinese medicinal herbs. Life Sci 73: 19–28; 2003.PubMedCrossRefGoogle Scholar
  4. Choi K. T.; Lee M. G.; Kwon W. S.; Lee J. H. Strategy for high quality ginseng breeding. Korean J Breeding 26: 83–91; 1994.Google Scholar
  5. Choi S. M.; Son S. H.; Yun S. R.; Kwon O. W.; Seon J. H.; Paek K. Y. Pilot-scale culture of adventitious roots of ginseng in a bioreactor system. Plant Cell, Tissue Organ Cult 62: 187–193; 2000.CrossRefGoogle Scholar
  6. Choi Y. E.; Yang D. C.; Yoon E. S.; Choi K. T. Plant regeneration via adventitious bud formation from cotyledon explants of Panax ginseng C.A.Meyer. Plant Cell Rep 17: 731–736; 1998.CrossRefGoogle Scholar
  7. Christensen L. P. Ginsenosides: Chemistry, Biosynthesis, Analysis and Potential Health Effects. Adv Food Nutr Res 55: 1–99; 2008.CrossRefGoogle Scholar
  8. Dubbels R.; Reiter R. J.; Klenke E.; Goebel A.; Schnakenberg E.; Ehlers C.; Schiwara H. W.; Schloot W. Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry. J Pineal Res 18: 28–31; 1995.PubMedCrossRefGoogle Scholar
  9. Ebert A.; Taylor H. F. Assessment of the changes of 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal. Plant Cell, Tissue Organ Cult 20: 165–172; 1990.Google Scholar
  10. Feeney M.; Punja Z. K. Production of somatic embryos of American ginseng in suspension culture and regeneration of plantlets. Acta Hort 625: 225–231; 2003.Google Scholar
  11. Fournier A. R.; Charest P.; Gosselin A.; Khanizadeh S.; Dorais M. Growing American ginseng organically in a North American broadleaf forest. Acta Hort 765: 77–86; 2008.Google Scholar
  12. Fridborg G.; Pedersen M.; Landstrom L. E.; Eriksson T. The effect of activated charcoal on tissue cultures: Adsorption of metabolites inhibiting morphogenesis. Physiol Plantarum 43: 104–106; 1978.CrossRefGoogle Scholar
  13. Garcia G.; Fernandez-Galiano E.; Mauri P. V. Liquid medium culture of Quercus suber L. somatic embryos comparation between liquid and solid culture and among different growing media. Acta Hort 447: 149–151; 1997.Google Scholar
  14. Habibi N.; Suthar R. K.; Purohit S. D. Role of PGRs and inhibitors in induction and control of somatic embryogenesis in Themeda quadrivalvis. Indian J Exp Biol 47: 198–203; 2009.PubMedGoogle Scholar
  15. Hahn E. J.; Kim Y. S.; Yu K. W.; Jeong C. S.; Paek K. Y. Adventitious root cultures of Panax ginseng C.A. Meyer and ginsenoside production through large-scale bioreactor system. J. Plant Biotechnol 5: 1–6; 2003.Google Scholar
  16. Harlan J. R.; de Wet J. M. J. Toward a rational classification of cultivated plants. Taxonomy 20: 509–517; 1971.CrossRefGoogle Scholar
  17. He C. N.; Gao W. W.; Yang J. X.; Bi W.; Zhang X. S.; Zhao Y. J. Identification of autotoxic compounds from fibrous roots of Panax quinquefolium L. Plant Soil 318: 63–72; 2009.CrossRefGoogle Scholar
  18. Hovius M. H. Y.; Proctor J. T. A.; Saxena P. K. Effects of date and growth regulators on the culture of immature zygotic embryos of North American ginseng. J Ginseng Res 31: 14–22; 2007.CrossRefGoogle Scholar
  19. Hu W. W.; Yao H.; Zhong J. J. Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors. Biotechnol Progr 17: 838–846; 2001.CrossRefGoogle Scholar
  20. Jhang J. J.; Staba E. J.; Kim J. Y. American and Korean ginseng tissue cultures: growth, chemical analysis and plantlet production. In Vitro 9: 253–259; 1974.CrossRefGoogle Scholar
  21. Kefeli V. I.; Kadyrov C. S. Natural growth inhibitors, their chemical and physiological properties. Annu Rev Plant Physiol 22: 185–196; 1971.CrossRefGoogle Scholar
  22. Kevers C.; Gal N. L.; Monteiro M.; Dommes J.; Gasper T. Somatic embryogenesis of Panax ginseng in liquid cultures: a role for polyamines and their metabolic pathways. Plant Growth Regul 31: 209–214; 2000.CrossRefGoogle Scholar
  23. Kevers C.; Jacques P.; Thonart P.; Gaspar T. In vitro root cultures of Panax ginseng and P. quinquefolium. Plant Growth Regul 27: 173–178; 1999.CrossRefGoogle Scholar
  24. Kishira H.; Takada M.; Shoyama Y. Micropropagation of Panax ginseng C.A. Meyer by somatic embryos. Acta Hort 319: 197–202; 1992.Google Scholar
  25. Kolar J.; Johnson C. H.; Machackova I. Exogenously applied melatonin affects flowering of the short-day plant Chenopodium rubrum. Physiol Plantarum 118: 605–612; 2003.CrossRefGoogle Scholar
  26. Kolar J.; Machackova I.; Eder J.; Prinsen E.; van Dongen W.; van Onckelen H.; Illnerova H. Melatonin: Occurrence and daily rhythm in Chenopodium rubrum. Phytochemistry 44: 1407–1413; 1997.CrossRefGoogle Scholar
  27. Linington I. M. In vitro propagation of Dipterocarpus alatus and Dipterocarpus intricatus. Plant Cell, Tissue Organ Cult 27: 81–88; 1991.CrossRefGoogle Scholar
  28. Luo S. W.; Huang W. H.; Cao G. Y.; Yu R. C. Tissue culture of Panax schinseng. Plant Physiol Comm. 2: 26–38; 1964.Google Scholar
  29. Manjula S.; Thomas A.; Daniel B.; Nair G. M. In vitro plant regeneration of Aristolochia indica through axillary shoot multiplication and organogenesis. Plant Cell, Tissue Organ Cult 51: 145–148; 1997.CrossRefGoogle Scholar
  30. Mathur A.; Shukla Y. N.; Pal M.; Ahuja P. S.; Uniyal G. C. Saponin production in callus and cell suspension cultures of Panax quinquefolium. Phytochemistry 35: 1221–1225; 1994.CrossRefGoogle Scholar
  31. Matsingou T. C.; Petrakis N.; Kapsokefalou M.; Salifoglou A. Antioxidant activity of organic extracts from aqueous infusions of sage. J Agric Food Chem 51: 6696–6701; 2003.PubMedCrossRefGoogle Scholar
  32. Molyneux P. The use of stable free radical diphenyl picrylhydrazyl (DPPH) for estimating antioxidant activity. J Sci Technol 26: 211–219; 2004.Google Scholar
  33. Monteiro M.; Kevers C.; Dommes J.; Gaspar T. A specific role for spermidine in the initiation phase of somatic embryogenesis in Panax ginseng CA Meyer. Plant Cell, Tissue Organ Cult 68: 225–232; 2002.CrossRefGoogle Scholar
  34. Murashige T.; Skoog F. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15: 473–497; 1962.CrossRefGoogle Scholar
  35. Murch S. J.; Liu C.; Romero R. M.; Saxena P. K. In vitro culture and temporary immersion bioreactor production of Crescentia cujete. Plant Cell, Tissue Organ Cult 78: 63–68; 2004.CrossRefGoogle Scholar
  36. Murch S. J.; Saxena P. K. A melatonin-rich germplasm line of St John’s wort (Hypericum perforatum L.). J Pineal Res 41: 284–287; 2006.PubMedCrossRefGoogle Scholar
  37. Murch S. J.; Alan A. R.; Cao J.; Saxena P. K. Melatonin and serotonin in flowers and fruits of Datura metel L. J Pineal Res 47: 277–283; 2009.PubMedCrossRefGoogle Scholar
  38. Nilprapruck P.; Yodmingkhwan P. Effect of exogenous methyl jasmonate on the internal browning of pineapple fruit (Ananas comosus L.) cv. Pattavia. KKU Res J 14: 489–498; 2009.Google Scholar
  39. Pan M. J.; van Staden J. The use of charcoal in in vitro culture-a review. Plant Growth Regul 26: 155–163; 1998.CrossRefGoogle Scholar
  40. Posmyk M. M.; Bałabusta M.; Wieczorek M.; Sliwinska E.; Janas K. M. Melatonin applied to cucumber (Cucumis sativus L.) seeds improves germination during chilling stress. J Pineal Res 46: 214–223; 2009.PubMedCrossRefGoogle Scholar
  41. Proctor J. T. A.; Bailey W. G. Ginseng: industry, botany and culture. Hort Rev 9: 187–236; 1987.Google Scholar
  42. Proctor J. T. A.; Slimmon T.; Saxena P. K. Modulation of root growth and organogenesis in thidiazuron-treated ginseng (Panax quinquefolium L.). Plant Growth Regul 20: 201–208; 1996.CrossRefGoogle Scholar
  43. Schenk R. U.; Hildebrandt A. C. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50: 199–204; 1972.CrossRefGoogle Scholar
  44. Shi W.; Wang Y.; Li J.; Zhang H.; Ding L. Investigation of ginsenosides in different parts and ages of Panax ginseng. Food Chem 102: 664–668; 2007.CrossRefGoogle Scholar
  45. Shoyama Y.; Matsushita H.; Zhu X. X.; Kishira H. Somatic embryogenesis in ginseng (Panax species). In: Bajaj Y. P. S. (ed) Biotechnology in Agriculture and Forestry, Somatic Embryogenesis and Synthetic Seed II, vol. 31. Springer, New York, pp 344–356; 1995.Google Scholar
  46. Shoyama Y.; Zhu X. X.; Nakai R.; Shiraishi S.; Kohda H. Micropropagation of Panax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets. Plant Cell Rep 16: 450–453; 1997.Google Scholar
  47. Singleton V. L.; Orthofer R.; Lamuela-Raventos R. M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu Reagent. Methods Enzymol 299: 152–178; 1999.CrossRefGoogle Scholar
  48. Symons G. M.; Reid J. B. Interactions between light and plant hormones during de-etiolation. J Plant Growth Regul 22: 3–14; 2003.CrossRefGoogle Scholar
  49. Tan D. X.; Manchester L. C.; Helton P.; Reiter R. J. Phytoremediative capacity of plants enriched with melatonin. Plant Signal Behav 2: 514–516; 2007.PubMedCrossRefGoogle Scholar
  50. Thomas T. D. The role of activated charcoal in plant tissue culture. Biotechnol Adv 26: 618–631; 2008.PubMedCrossRefGoogle Scholar
  51. Tirajoh A.; Kyung T. S.; Punja Z. K. Somatic embryogenesis and plantlet regeneration in American ginseng (Panax quinquefolium L.). In Vitro Cell Dev Biol Plant 34: 203–211; 1998.Google Scholar
  52. Tisserat B. Propagation of Date Palm (Phoenix dactylifera L.) in vitro. J Exp Bot 30: 1275–1283; 1979.CrossRefGoogle Scholar
  53. Trautmann I. A.; Visser J. H. The possible role of phenolic substances in the establishment of suspension cultures of guayule (Parthenium argentatum Gray). Bioresource Technol 35: 133–139; 1991.CrossRefGoogle Scholar
  54. Velioglu Y. S.; Mazza G.; Gao L.; Oomah B. D. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J. Agric. Food Chem. 46: 4113–4117; 1998.CrossRefGoogle Scholar
  55. Wang A. S. Callus induction and plant regeneration of American ginseng. HortScience 25: 571–572; 1990.Google Scholar
  56. Wang C. Z.; Aung H. H.; Zhang B.; Sun S.; Li X. L.; He H.; Xie J. T.; He T. C.; Du W.; Yuan C. S. Chemopreventive effects of heat-processed Panax quinquefolius root on human breast cancer cells. Anticancer Res 28: 2545–2551; 2008.PubMedGoogle Scholar
  57. Wang J.; Gao W. Y.; Zhang J.; Huang T.; Cao Y.; Zhao Y. X. Dynamic change of metabolites and nutrients in suspension cells of Panax quinquefolium L. in bioreactor. Acta Physiol Plant 32: 463–467; 2010.CrossRefGoogle Scholar
  58. Wu J.; Zhong J. J. Production of ginseng and its bioactive components in plant cell culture: current technological and applied aspects. J Biotechnol 68: 89–99; 1999.PubMedCrossRefGoogle Scholar
  59. Young P. S.; Murthy H. N.; Yoeup P. K. Mass multiplication of protocorm-like bodies using bioreactor system and subsequent plant regeneration in Phalaenopsis. Plant Cell, Tissue Organ Cult 63: 67–72; 2000.CrossRefGoogle Scholar
  60. Yuan C. S.; Attele A. S.; Wu J. A.; Lowell T. K.; Gu Z.; Lin Y. Panax quinquefolium L. inhibits thrombin-induced endothelin release in vitro. Am J Chin Med 27: 331–338; 1999.PubMedCrossRefGoogle Scholar
  61. Zhong J. J.; Bai Y.; Wang S. J. Effects of plant growth regulators on cell growth and ginsenoside saponin production by suspension cultures of Panax quinquefolium. J Biotechnol 45: 227–234; 1996.CrossRefGoogle Scholar
  62. Zhou S.; Brown D. C. W. High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants. Plant Cell Rep. 25: 166–173; 2006.PubMedCrossRefGoogle Scholar
  63. Zhou S.; Brown D. C. W. et al. A highly efficient protocol for micropropagation of North American ginseng. In: Xu Z. (ed) Biotechnology and Sustainable Agriculture 2006 and Beyond. Proceedings of the 11th IAPTC&B Congress. Springer Netherlands, Beijing, China, pp 425–428; 2007.CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2011

Authors and Affiliations

  • Esther E. Uchendu
    • 1
  • Gopinadhan Paliyath
    • 1
  • Dan C. W. Brown
    • 2
  • Praveen K. Saxena
    • 1
  1. 1.Department of Plant AgricultureUniversity of GuelphGuelphCanada
  2. 2.Agriculture and Agri-Food CanadaLondonCanada

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