Modification of the droplet-vitrification method of cryopreservation to enhance survival rates of adventitious roots of Panax ginseng
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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.
KeywordsAdventitious 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).
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.
- Bisht SS, Sharma A, Chaturvedi K (1989) Certain metabolic lesions of chromium toxicity in radish. Indian J Agric Biochem 2:109–115Google Scholar
- Engelman F, Takagi H (2000) Cryopreservation of tropical plant germplasm current research progress and application. Japan International Research Center for Agriculture Sciences, TsukubaGoogle Scholar
- Fujikawa S, Steponkus PL (1991) Plasma membrane ultrastructural changes by vitrification procedures. Jpn J Free Dry 37:25–29Google 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 5:1–6Google 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 417–438Google 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 30:291–299Google 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 31:473–484Google Scholar
- Kuranuki Y, Sakai A (1995) Cryopreservation of in vitro-grown shoot tips of tea (Camellia sinensis) by vitrification. CryoLetters 16:345–352Google 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, FinlandGoogle Scholar
- Makowska Z, Keller J, Engelmann F (1999) Cryopreservation of apices isolated from garlic (Allium sativum L.) bulbils and cloves. CryoLetters 20:175–182Google 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 655–682Google 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 35:138–144Google 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 30:212–223Google Scholar
- Sakai A, Engelmann F (2007) Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLetters 28:151–172Google 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 1–61Google 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 97:1073–1081CrossRefGoogle Scholar
- Thinh NT (1997) Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Dr Pap Fac Agric Kobe Univ JapanGoogle Scholar
- Touno K, Yoshimatsu K, Shimomura K (2006) Characteristics of Atropa belladonna hairy roots cryopreserved by vitrification method. CryoLetters 27:65–72Google Scholar
- Wu YL, Shen XH (2011) Cryopreservation of Dendrobium wardianum Warner. protocorms by vitrification. Chin J Cell Bio 33:279–287Google 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 71–89Google 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 27:211–222Google 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 77–88Google Scholar