Modification of the droplet-vitrification method of cryopreservation to enhance survival rates of adventitious roots of Panax ginseng

  • Kim-Cuong Le
  • Haeng-Hoon Kim
  • So-Young ParkEmail author
Research Report


Panax ginseng Meyer is an important medicinal plant producing bioactive compounds. A droplet-vitrification method was developed for cryopreserving adventitious root cultures of mountain ginseng, and variations of this procedure were tested to determine their effects on regrowth rates and osmotic stress responses. Root regrowth rates were examined after exposing root segments to two pre-culture treatments, three loading solutions, and two vitrification solutions with different exposure periods. Pre-culturing excised segments of adventitious roots with 0.3 M sucrose produced the highest rate of regrowth after cryopreservation. Loading for 20 min with 17.5% (w/v) sucrose and 17.5% (w/v) glycerol at room temperature increased the regrowth rate of cryopreserved adventitious roots fourfold, compared with non-loaded samples. Treatments involving different vitrification solutions and exposure periods were compared, and the highest rate of regrowth (15%) after cryopreservation was achieved by incubating adventitious roots in modified plant vitrification solution 3 containing 40% (w/v) glycerol and 40% (w/v) sucrose for 10 min at room temperature, suggesting that ginseng adventitious root tips were sensitive to osmotic stress. Further study is necessary to develop optimal vitrification solutions that enhance the survival rate of cryopreserved adventitious roots of mountain ginseng.


Adventitious root Cryopreservation Droplet-vitrification Panax ginseng Long-term conservation Vitrification 



This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Advanced Production Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (Grant No. 315013-4).

Author contributions

K-CL contributed to data acquisition and wrote the manuscript. H-HK and K-YP participated in interpreted data and revising for intellectual content. S-YP made substantial contributions to the conception and design of the study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.


  1. Ahmad P, Sarwat M, Sharma S (2008) Reactive oxygen species, antioxidants and signaling in plants. J Plant Biol 51:167–173CrossRefGoogle Scholar
  2. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399CrossRefGoogle Scholar
  3. Benson EE, Lynch PT, Jones J (1992) The detection of lipid peroxidation products in cryoprotected and frozen rice cells: consequences for post-thaw survival. Plant Sci 85:107–114CrossRefGoogle Scholar
  4. Bisht SS, Sharma A, Chaturvedi K (1989) Certain metabolic lesions of chromium toxicity in radish. Indian J Agric Biochem 2:109–115Google Scholar
  5. Chen G, Ren L, Zhang J, Reed BM, Zhang D, Shen XH (2015) Cryopreservation affects ROS-induced oxidative stress and antioxidant response in Arabidopsis seedlings. Cryobiology 70:38–47CrossRefGoogle Scholar
  6. Christensen LP (2008) Ginsenosides: chemistry, biosynthesis, analysis, and potential health effects. Adv Food Nutr Res 55:1–99CrossRefGoogle Scholar
  7. Da Silva Cordeiro L, Simões-Gurgel C, Albarello N (2015) Multiplication and cryopreservation of adventitious roots of Cleome rosea Vahl. Vitro Cell Dev Biol 51:249–257CrossRefGoogle Scholar
  8. Engelman F, Takagi H (2000) Cryopreservation of tropical plant germplasm current research progress and application. Japan International Research Center for Agriculture Sciences, TsukubaGoogle Scholar
  9. Engelmann F, Dussert S (2013) Cryopreservation. In: Normah MN, Chin HF, Reed BM (eds) Conservation of tropical plant species. Springer, Berlin, pp 107–119CrossRefGoogle Scholar
  10. Fábián A, Jäger K, Darkó É, Barnabás B (2008) Cryopreservation of wheat (Triticum aestivum L.) egg cells by vitrification. Acta Physiol Plant 30:737–744CrossRefGoogle Scholar
  11. Fu C, Li L, Wu W, Li M, Yu X, Yu L (2012) Assessment of genetic and epigenetic variation during long-term Taxus cell culture. Plant Cell Rep 31:1321–1331CrossRefGoogle Scholar
  12. Fujikawa S, Steponkus PL (1991) Plasma membrane ultrastructural changes by vitrification procedures. Jpn J Free Dry 37:25–29Google Scholar
  13. Hahn EJ, Yu KW, Paek KY (2003) Adventitious root cultures of Panax ginseng CV Meyer and ginsenoside production through large-scale bioreactor system. J Plant Biotechnol 5:1–6Google Scholar
  14. Halliwell B (2006) Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life. Plant Physiol 141:312–322CrossRefGoogle Scholar
  15. Hirata K, Mukai M, Goda S, Ishio-Kinugasa M, Yoshida K, Sakai A, Miyamoto K (2002) Cryopreservation of hairy root cultures of Vinca minor (L.) by encapsulation-dehydration. Biotechnol Lett 24:371–376CrossRefGoogle Scholar
  16. Hughes ZE, Mancera RL (2014) Molecular mechanism of the synergistic effects of vitrification solutions on the stability of phospholipid bilayers. Biophys J 106:2617–2624CrossRefGoogle Scholar
  17. Ibrahim S, Normah MN (2013) The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification. Plant Growth Regul 70:237–246CrossRefGoogle Scholar
  18. Jia MX, Shi Y, Di W, Jiang XR, Xu J, Liu Y (2017) ROS-induced oxidative stress is closely related to pollen deterioration following cryopreservation. Vitro Cell Dev Biol 53:433–439CrossRefGoogle Scholar
  19. Jung DW, Sung CK, Touno K, Yoshimatsu K, Shimomura K (2001) Cryopreservation of Hyoscyamus niger adventitious roots by vitrification. J Plant Physiol 158:801–805CrossRefGoogle Scholar
  20. Kaczmarczyk A, Funnekotter B, Menon A, Phang PY, Al-Hanbali A, Bunn E, Mancera RL (2012) Current issues in plant cryopreservation. In: Katov II (ed) Current frontiers in cryobiology. Croatia, InTech, pp 417–438Google Scholar
  21. Kim HH, Lee YG, Park SU, Lee SC, Baek HJ, Cho EG, Engelmann F (2009a) Development of alternative loading solutions in droplet-vitrification procedures. CryoLetters 30:291–299Google Scholar
  22. Kim HH, Lee YG, Shin DJ, Ko HC, Gwag JG, Cho EG, Engelmann F (2009b) Development of alternative plant vitrification solutions and loading solutions in droplet-vitrification procedures. Cryobiology 59:320–334CrossRefGoogle Scholar
  23. Kim HH, Popova EV, Yi JY, Cho GT, Park SU, Lee SC, Engelmann F (2010) Cryopreservation of hairy roots of Rubia akane (Nakai) using a droplet-vitrification procedure. CryoLetters 31:473–484Google Scholar
  24. Kim DS, Song M, Kim SH, Jang DS, Kim JB, Ha BK, Kim SH, Lee KJ, Kang SY, Jeong IY (2013) The improvement of ginsenoside accumulation in Panax ginseng as a result of γ-irradiation. J Ginseng Res 37:332–340CrossRefGoogle Scholar
  25. Kiselev KV, Shumakova OA, Tchernoded GK (2011) Mutation of Panax ginseng genes during long-term cultivation of ginseng cell cultures. J Plant Physiol 168:1280–1285CrossRefGoogle Scholar
  26. Kulus D, Zalewska M (2014) Cryopreservation as a tool used in long-term storage of ornamental species–a review. Sci Hortic (Amsterdam) 168:88–107CrossRefGoogle Scholar
  27. Kulus D, Abratowska A, Mikuła A (2018) Morphogenetic response of shoot tips to cryopreservation by encapsulation-dehydration in a solid mutant and periclinal chimeras of Chrysanthemum × grandiflorum/Ramat./Kitam. Acta Physiol Plant 40:18CrossRefGoogle Scholar
  28. Kuranuki Y, Sakai A (1995) Cryopreservation of in vitro-grown shoot tips of tea (Camellia sinensis) by vitrification. CryoLetters 16:345–352Google Scholar
  29. Lambert E, Geelen D (2008) Cryopreservation of hairy root cultures from Maesa lanceolata. In: Laamanen J, Uosukainen M, Häggman H, Rantala ANS (eds) Cryopreservation of Cropspecies in Europe CRYOPLANET COST Action 871, 20th–23rd Feb 2008, Oulu, FinlandGoogle Scholar
  30. Lambert E, Goossens A, Panis B, van Labeke MC, Geelen D (2009) Cryopreservation of hairy root cultures of Maesa lanceolata and Medicago truncatula. Plant Cell, Tissue Organ Cult 96:289–296CrossRefGoogle Scholar
  31. Lee KH (1998) The pharmacology of Chinese herbs. J Nat Prod 61:1575–1576CrossRefGoogle Scholar
  32. Lü JM, Yao Q, Chen C (2009) Ginseng compounds: an update on their molecular mechanisms and medical applications. Curr Vasc Pharmacol 7:293–302CrossRefGoogle Scholar
  33. Makowska Z, Keller J, Engelmann F (1999) Cryopreservation of apices isolated from garlic (Allium sativum L.) bulbils and cloves. CryoLetters 20:175–182Google Scholar
  34. Martinez-Montero ME, Harding K (2015) Cryobionomics: evaluating the concept in plant cryopreservation. In: Barh D, Khan MS, Davies E (eds) Plant omics: the omics of plant science. Springer, Berlin, pp 655–682Google Scholar
  35. Matsumoto T, Sakai A, Yamada K (1994) Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Rep 13:442–446CrossRefGoogle Scholar
  36. Nishizawa S, Sakai A, Amano Y, Matsuzawa T (1993) Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Sci 91:67–73CrossRefGoogle Scholar
  37. Oh SY, Wu CH, Popova E, Hahn EJ, Paek KY (2009) Cryopreservation of Panax ginseng adventitious roots. J Plant Biol 52:348–354CrossRefGoogle Scholar
  38. Pandhair V, Sekhon BS (2006) Reactive oxygen species and antioxidants in plants: an overview. J Plant Biochem Biotechnol 15:71–78CrossRefGoogle Scholar
  39. Panis B, Piette B, Swennen R (2005) Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci 168:45–55CrossRefGoogle Scholar
  40. Park SU, Kong H, Shin DJ, Bae CH, Lee SC, Bae CH, Rha ES, Kim HH (2014) Development of vitrification protocol in Rubia akane (Nakai) hairy roots using a systematic approach. CryoLetters 35:138–144Google Scholar
  41. Poobathy R, Sinniah UR, Xavier R, Subramaniam S (2013) Catalase and superoxide dismutase activities and the total protein content of protocorm-like bodies of Dendrobium Sonia-28 subjected to vitrification. Appl Biochem Biotechnol 170:1066–1079CrossRefGoogle Scholar
  42. Quain MD, Berjak P, Acheampong E, Kioko JI (2009) Sucrose treatment and explant water content: critical factors to consider in development of successful cryopreservation protocols for shoot tip explants of the tropical species Dioscorea rotundata (yam). CryoLetters 30:212–223Google Scholar
  43. Ren L, Zhang D, Jiang XN, Gai Y, Wang WM, Reed BM, Shen XH (2013) Peroxidation due to cryoprotectant treatment is a vital factor for cell survival in Arabidopsis cryopreservation. Plant Sci 212:37–47CrossRefGoogle Scholar
  44. Sakai A, Engelmann F (2007) Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLetters 28:151–172Google Scholar
  45. Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. Brasiliensis Tanaka) by vitrification. Plant Cell Rep 9:30–33CrossRefGoogle Scholar
  46. San José MC, Valladares S, Janeiro LV, Corredoira E (2014) Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. Acta Physiol Plant 36:109–116CrossRefGoogle Scholar
  47. Steponkus PL, Langis R, Fujikawa S (1992) Cryopreservation of plant tissues by vitrification. In: Steponkus PL (ed) Advances in low-temperature biology. JAI Press, London, pp 1–61Google Scholar
  48. Suzuki M, Ishikawa M, Okuda H, Noda K, Kishimoto T, Nakamura T, Ogiwara I, Shimura I, Akihama T (2006) Physiological changes in gentian axillary buds during two-step preculturing with sucrose that conferred high levels of tolerance to desiccation and cryopreservation. Ann Bot 97:1073–1081CrossRefGoogle Scholar
  49. Takagi H, Thinh NT, Islam OM, Senboku T, Sakai A (1997) Cryopreservation of invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Rep 16:594–599CrossRefGoogle Scholar
  50. Thinh NT (1997) Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Dr Pap Fac Agric Kobe Univ JapanGoogle Scholar
  51. Touno K, Yoshimatsu K, Shimomura K (2006) Characteristics of Atropa belladonna hairy roots cryopreserved by vitrification method. CryoLetters 27:65–72Google Scholar
  52. Turgut-Kara N, Kahraman BÜ (2015) Effects of long-term culture of Astragalus chrysochlorus callus onmorphology, genetic structure, gene expression and metabolism. Plant Biosyst 149:329–336CrossRefGoogle Scholar
  53. Uchendu EE, Muminova M, Gupta S, Reed BM (2010) Antioxidant and anti-stress compounds improve regrowth of cryopreserved Rubus shoot tips. Vitro Cell Dev Biol 46:386–393CrossRefGoogle Scholar
  54. Varghese B, Naithani SC (2008) Oxidative metabolism-related changes in cryogenically stored neem (Azadirachta indica A. Juss.) seeds. J Plant Physiol 165:755–765CrossRefGoogle Scholar
  55. Wang RR, Gao XX, Chen L, Huo LQ, Li MF, Wang QC (2014) Shoot recovery and genetic integrity of Chrysanthemum morifolium shoot tips following cryopreservation by droplet-vitrification. Sci Hortic (Amsterdam) 176:330–339CrossRefGoogle Scholar
  56. Wen B, Wang R, Cheng H, Song S (2010) Cytological and physiological changes in orthodox maize embryos during cryopreservation. Protoplasma 239:57–67CrossRefGoogle Scholar
  57. Wesley-Smith J, Berjak P, Pammenter NW, Walters C (2013) Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures. Ann Bot 113:695–709CrossRefGoogle Scholar
  58. Wu YL, Shen XH (2011) Cryopreservation of Dendrobium wardianum Warner. protocorms by vitrification. Chin J Cell Bio 33:279–287Google Scholar
  59. Xie JT, Attele AS, Yuan CS (2006) Ginseng: beneficial and potential adverse effect. In: Yuan CS, Beiber E, Bauer BA (eds) A textbook of complementary and alternative therapies. CRC Press Company, Boca Raton, London, New York, Washington, DC, pp 71–89Google Scholar
  60. Xue SH, Luo XJ, Wu ZH, Zhang HL, Wang XY (2008) Cold storage and cryopreservation of hairy root cultures of medicinal plant Eruca sativa Mill., Astragalus membranaceus and Gentiana macrophylla Pall. Plant Cell, Tissue Organ Cult 92:251–260CrossRefGoogle Scholar
  61. Yi JY, Sylvestre I, Colin M, Salma M, Lee SY, Kim HH, Park HJ, Engelmann F (2012) Improved cryopreservation using droplet-vitrification and histological changes associated with cryopreservation of Madder (Rubia akane Nakai). Korean J Hortic Sci Technol 30:79–84CrossRefGoogle Scholar
  62. Yoon JW, Kim HH, Ko HC, Hwang HS, Hong ES, Cho EG, Engelmann F (2006) Cryopreservation of cultivated and wild potato varieties by droplet vitrification: effect of subculture of mother-plants and of preculture of shoot tips. CryoLetters 27:211–222Google Scholar
  63. Yoshimatsu K, Touno K, Shimomura K (2000) Cryopreservation of medicinal plant resources: retention of biosynthetic capabilities in transformed cultures. In: Cryopreservation of tropical plant germplasm: current research progress and application. Proceedings of an international workshop, Tsukuba, Japan, October 1998. International Plant Genetic Resources Institute (IPGRI), pp 77–88Google Scholar
  64. Zhang D, Ren L, Gq Chen ZJ, Reed BM (2015) ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox. Plant Cell Rep 34:1499–1513CrossRefGoogle Scholar

Copyright information

© Korean Society for Horticultural Science 2019

Authors and Affiliations

  1. 1.Department of Horticultural Science, Division of Animal, Horticulture and Food SciencesChungbuk National UniversityCheongjuRepublic of Korea
  2. 2.Department of Well-being ResourcesSunchon National UniversitySuncheonRepublic of Korea

Personalised recommendations