Skip to main content
SpringerLink
Log in

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

  • Research Report
  • Published:
Horticulture, Environment, and Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Ahmad P, Sarwat M, Sharma S (2008) Reactive oxygen species, antioxidants and signaling in plants. J Plant Biol 51:167–173

    Article  CAS  Google Scholar 

  • Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399

    Article  CAS  PubMed  Google Scholar 

  • 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 85:107–114

    Article  CAS  Google Scholar 

  • Bisht SS, Sharma A, Chaturvedi K (1989) Certain metabolic lesions of chromium toxicity in radish. Indian J Agric Biochem 2:109–115

    CAS  Google Scholar 

  • 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 70:38–47

    Article  CAS  PubMed  Google Scholar 

  • Christensen LP (2008) Ginsenosides: chemistry, biosynthesis, analysis, and potential health effects. Adv Food Nutr Res 55:1–99

    Article  CAS  Google Scholar 

  • Da Silva Cordeiro L, Simões-Gurgel C, Albarello N (2015) Multiplication and cryopreservation of adventitious roots of Cleome rosea Vahl. Vitro Cell Dev Biol 51:249–257

    Article  CAS  Google Scholar 

  • Engelman F, Takagi H (2000) Cryopreservation of tropical plant germplasm current research progress and application. Japan International Research Center for Agriculture Sciences, Tsukuba

    Google Scholar 

  • Engelmann F, Dussert S (2013) Cryopreservation. In: Normah MN, Chin HF, Reed BM (eds) Conservation of tropical plant species. Springer, Berlin, pp 107–119

    Chapter  Google Scholar 

  • Fábián A, Jäger K, Darkó É, Barnabás B (2008) Cryopreservation of wheat (Triticum aestivum L.) egg cells by vitrification. Acta Physiol Plant 30:737–744

    Article  CAS  Google Scholar 

  • 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 31:1321–1331

    Article  CAS  PubMed  Google Scholar 

  • Fujikawa S, Steponkus PL (1991) Plasma membrane ultrastructural changes by vitrification procedures. Jpn J Free Dry 37:25–29

    Google Scholar 

  • 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 5:1–6

    Google Scholar 

  • Halliwell B (2006) Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life. Plant Physiol 141:312–322

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 24:371–376

    Article  CAS  Google Scholar 

  • Hughes ZE, Mancera RL (2014) Molecular mechanism of the synergistic effects of vitrification solutions on the stability of phospholipid bilayers. Biophys J 106:2617–2624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ibrahim S, Normah MN (2013) The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification. Plant Growth Regul 70:237–246

    Article  CAS  Google Scholar 

  • 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 53:433–439

    Article  CAS  Google Scholar 

  • Jung DW, Sung CK, Touno K, Yoshimatsu K, Shimomura K (2001) Cryopreservation of Hyoscyamus niger adventitious roots by vitrification. J Plant Physiol 158:801–805

    Article  CAS  Google Scholar 

  • 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 417–438

    Google Scholar 

  • 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 30:291–299

    CAS  PubMed  Google Scholar 

  • 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 59:320–334

    Article  Google Scholar 

  • 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 31:473–484

    PubMed  Google Scholar 

  • 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 37:332–340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kiselev KV, Shumakova OA, Tchernoded GK (2011) Mutation of Panax ginseng genes during long-term cultivation of ginseng cell cultures. J Plant Physiol 168:1280–1285

    Article  CAS  PubMed  Google Scholar 

  • Kulus D, Zalewska M (2014) Cryopreservation as a tool used in long-term storage of ornamental species–a review. Sci Hortic (Amsterdam) 168:88–107

    Article  Google Scholar 

  • 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 40:18

    Article  CAS  Google Scholar 

  • Kuranuki Y, Sakai A (1995) Cryopreservation of in vitro-grown shoot tips of tea (Camellia sinensis) by vitrification. CryoLetters 16:345–352

    Google Scholar 

  • 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, Finland

  • 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 96:289–296

    Article  Google Scholar 

  • Lee KH (1998) The pharmacology of Chinese herbs. J Nat Prod 61:1575–1576

    Article  CAS  Google Scholar 

  • Lü JM, Yao Q, Chen C (2009) Ginseng compounds: an update on their molecular mechanisms and medical applications. Curr Vasc Pharmacol 7:293–302

    Article  PubMed  PubMed Central  Google Scholar 

  • Makowska Z, Keller J, Engelmann F (1999) Cryopreservation of apices isolated from garlic (Allium sativum L.) bulbils and cloves. CryoLetters 20:175–182

    Google Scholar 

  • 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 655–682

    Google Scholar 

  • 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 13:442–446

    Article  CAS  PubMed  Google Scholar 

  • 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 91:67–73

    Article  CAS  Google Scholar 

  • Oh SY, Wu CH, Popova E, Hahn EJ, Paek KY (2009) Cryopreservation of Panax ginseng adventitious roots. J Plant Biol 52:348–354

    Article  CAS  Google Scholar 

  • Pandhair V, Sekhon BS (2006) Reactive oxygen species and antioxidants in plants: an overview. J Plant Biochem Biotechnol 15:71–78

    Article  CAS  Google Scholar 

  • Panis B, Piette B, Swennen R (2005) Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci 168:45–55

    Article  CAS  Google Scholar 

  • 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 35:138–144

    CAS  PubMed  Google Scholar 

  • 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 170:1066–1079

    Article  CAS  PubMed  Google Scholar 

  • 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 30:212–223

    CAS  PubMed  Google Scholar 

  • 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 212:37–47

    Article  CAS  PubMed  Google Scholar 

  • Sakai A, Engelmann F (2007) Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLetters 28:151–172

    CAS  PubMed  Google Scholar 

  • 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 9:30–33

    Article  CAS  PubMed  Google Scholar 

  • 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 36:109–116

    Article  CAS  Google Scholar 

  • 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 1–61

    Google Scholar 

  • 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 97:1073–1081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 16:594–599

    Article  CAS  PubMed  Google Scholar 

  • Thinh NT (1997) Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Dr Pap Fac Agric Kobe Univ Japan

  • Touno K, Yoshimatsu K, Shimomura K (2006) Characteristics of Atropa belladonna hairy roots cryopreserved by vitrification method. CryoLetters 27:65–72

    CAS  PubMed  Google Scholar 

  • Turgut-Kara N, Kahraman BÜ (2015) Effects of long-term culture of Astragalus chrysochlorus callus onmorphology, genetic structure, gene expression and metabolism. Plant Biosyst 149:329–336

    Article  Google Scholar 

  • 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 46:386–393

    Article  CAS  Google Scholar 

  • Varghese B, Naithani SC (2008) Oxidative metabolism-related changes in cryogenically stored neem (Azadirachta indica A. Juss.) seeds. J Plant Physiol 165:755–765

    Article  CAS  PubMed  Google Scholar 

  • 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) 176:330–339

    Article  CAS  Google Scholar 

  • Wen B, Wang R, Cheng H, Song S (2010) Cytological and physiological changes in orthodox maize embryos during cryopreservation. Protoplasma 239:57–67

    Article  PubMed  Google Scholar 

  • 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 113:695–709

    Article  PubMed  PubMed Central  Google Scholar 

  • Wu YL, Shen XH (2011) Cryopreservation of Dendrobium wardianum Warner. protocorms by vitrification. Chin J Cell Bio 33:279–287

    Google Scholar 

  • 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 71–89

    Google Scholar 

  • 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 92:251–260

    Article  Google Scholar 

  • 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 30:79–84

    Article  Google Scholar 

  • 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 27:211–222

    PubMed  Google Scholar 

  • 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 77–88

  • 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 34:1499–1513

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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 information

Authors and Affiliations

  1. Department of Horticultural Science, Division of Animal, Horticulture and Food Sciences, Chungbuk National University, Cheongju, 28466, Republic of Korea

    Kim-Cuong Le & So-Young Park

  2. Department of Well-being Resources, Sunchon National University, Suncheon, Jeonnam, 57922, Republic of Korea

    Haeng-Hoon Kim

Authors
  1. Kim-Cuong Le
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Haeng-Hoon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. So-Young Park
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to So-Young Park.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interests.

Additional information

Communicated by Inhwa Yeam.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Le, KC., Kim, HH. & Park, SY. Modification of the droplet-vitrification method of cryopreservation to enhance survival rates of adventitious roots of Panax ginseng. Hortic. Environ. Biotechnol. 60, 501–510 (2019). https://doi.org/10.1007/s13580-019-00150-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13580-019-00150-8

Keywords

Search

Navigation