Cryopreservation of Grapevine Shoot Tips from In Vitro Plants Using Droplet Vitrification and V Cryo-plate Techniques

  • Jean Carlos Bettoni
  • Ranjith PathiranaEmail author
  • Remi Bonnart
  • Ashley Shepherd
  • Gayle Volk


The availability of and easy access to Vitis genetic resources are essential for future breeding program advances. Cryopreservation is currently considered an ideal means for the long-term preservation of clonally propagated plant genetic resources. When robust methods for cryopreservation of Vitis spp. are available, there is an opportunity to preserve collections for extended lengths of time with minimal cost and labor requirements and a low risk of loss. This chapter describes the droplet vitrification and V cryo-plate protocols that have been shown to be effective for the cryopreservation of multiple V. vinifera genotypes and other Vitis species.


Vitis Tissue culture Conservation Genetic resources Germplasm 



This research was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the US Department of Energy (DOE) and the US Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the author’s and do not necessarily reflect the policies and views of USDA, DOE, or ORAU/ORISE. The authors are grateful to the ORISE/USDA for the Postdoctoral Researcher stipend granted to Jean Carlos Bettoni to perform research at the USDA-ARS National Laboratory for Genetic Resources Preservation (NLGRP). NLGRP Vitis cryopreservation research is funded in part by the Science and Technology Development Fund (Egypt). Ranjith Pathirana acknowledges the funding by Royal Society Te Apārangi for collaboration with USDA under the Catalyst Seeding project CSG-PAF1702.


  1. Alleweldt G, Dettweiler E (1994) The genetic resources of Vitis: world list of grapevine collections, 2nd edn. Institut für Rebenzüchtung Geilweilerhof, Siebeldingen, GeilweilwehofGoogle Scholar
  2. Benelli C, De Carlo A, Engelmann F (2013) Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis. Biotechnol Adv 31(2):175–185. CrossRefPubMedGoogle Scholar
  3. Benson EE (2008) Cryopreservation of phytodiversity: a critical appraisal of theory and practice. Crit Rev Plant Sci 27:141–219CrossRefGoogle Scholar
  4. Benson EE, Harding K (2012) Cryopreservation of shoot tips and meristems: an overview of contemporary methodologies. Methods Mol Biol 877:191–226. CrossRefPubMedGoogle Scholar
  5. Bettoni JC, Dalla Costa M, Gardin JPP, Kretzschmar AA, Pathirana R (2016) Cryotherapy: a new technique to obtain grapevine plants free of viruses. Rev Bras Frutic 38(2).
  6. Bettoni JC, Bonnart R, Shepherd AN, Kretzschmar AA, Volk GM (2019a) Cryopreservation of grapevine (Vitis spp.) shoot tips from growth chamber-sourced plants and histological observations. Vitis 58(2):71–78. CrossRefGoogle Scholar
  7. Bettoni JC, Bonnart R, Shepherd AN, Kretzschmar AA, Volk GM (2019b) Successful cryopreservation of Vitis vinifera ‘chardonnay’ from both in vitro and growth chamber source plants. Acta Hortic 1234:211–218. CrossRefGoogle Scholar
  8. Bettoni JC, Bonnart R, Shepherd AN, Kretzschmar AA, Volk GM (2019c) Modifications to a Vitis shoot tip cryopreservation procedure: effect of shoot tip size and use of cryoplates. CryoLetters 40(2):103–112PubMedGoogle Scholar
  9. Bi W-L, Pan C, Hao X-Y, Cui Z-H, Kher MM, Marković Z, Wang Q-C, Teixeira da Silva JA (2017) Cryopreservation of grapevine (Vitis spp.)—a review. In Vitro Cell Dev Biol Plant 53(5):449–460. CrossRefGoogle Scholar
  10. Bi W-L, Hao X-Y, Cui Z-H, Volk GM, Wang Q-C (2018a) Droplet-vitrification cryopreservation of in vitro-grown shoot tips of grapevine (Vitis spp.). In Vitro Cell Dev Biol Plant 54:590–599. CrossRefGoogle Scholar
  11. Bi W-L, Hao X-Y, Cui Z-H, Pathirana R, Volk GM, Wang Q-C (2018b) Shoot tip cryotherapy for efficient eradication of Grapevine leafroll-associated virus-3 from diseased grapevine in vitro plants. Ann Appl Biol 173(3):261–270. CrossRefGoogle Scholar
  12. Carimi F, Pathirana R, Carra A (2011) Biotechnologies for germplasm management and improvement. In: Szabo PV, Shojania J (eds) Grapevines – varieties, cultivation and management. Nova Science, New York, pp 199–249Google Scholar
  13. Carimi F, Carra A, Panis B, Pathirana R (2016) Strategies for conservation of endangered wild grapevine (Vitis vinifera L. subsp. sylvestris (C.C. Gmel.) Hegi). Acta Hortic 1115:81–86. CrossRefGoogle Scholar
  14. Dussert S, Engelmann F, Noirot M (2003) Development of probabilistic tools to assist in the establishment and management of cryopreserved plant germplasm collections. CryoLetters 24(3):149–160PubMedGoogle Scholar
  15. Eibach R, Zyprian E, Töpfer R (2009) The use of molecular markers for pyramidizing resistance genes in grapevine breeding. Acta Hortic 827:551–558. CrossRefGoogle Scholar
  16. Markovic Z, Chatelet P, Sylvestre I, Kontic JK, Engelmann F (2013) Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips. Cent Eur J Biol 8(10):993–1000. CrossRefGoogle Scholar
  17. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  18. Pathirana R, McLachlan A, Hedderley D, Panis B, Carimi F (2016) Pre-treatment with salicylic acid improves plant regeneration after cryopreservation of grapevine (Vitis spp.) by droplet vitrification. Acta Physiol Plant 38(1):1–11. CrossRefGoogle Scholar
  19. Reed BM (2014) Antioxidants and cryopreservation, the new normal? Acta Hortic 1039:41–48CrossRefGoogle Scholar
  20. Reed BM, Kovalchuk I, Kushnarenko S, Meier-Dinkel A, Schoenweiss K, Pluta S, Straczynska K, Benson EE (2004) Evaluation of critical points in technology transfer of cryopreservation protocols to international plant conservation laboratories. CryoLetters 25(5):341–352PubMedGoogle Scholar
  21. 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(1):30–33. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Volk GM, Henk AD, Jenderek MM, Richards CM (2016) Probabilistic viability calculations for cryopreserving vegetatively propagated collections in genebanks. Genet Resour Crop Evol 64(7):1–10. CrossRefGoogle Scholar
  23. Volk GM, Shepherd AN, Bonnart R (2018) Successful cryopreservation of Vitis shoot tips: novel pre-treatment combinations applied to nine species. CryoLetters 39(5):322–330PubMedGoogle Scholar
  24. Wang B, Wang R-R, Cui Z-H, Bi W-L, Li J-W, Li B-Q, Ozudogru EA, Volk GM, Wang Q-C (2014) Potential applications of cryogenic technologies to plant genetic improvement and pathogen eradication. Biotechnol Adv 32(3):583–595. CrossRefPubMedGoogle Scholar
  25. Wang M-R, Chen L, Teixeira da Silva JA, Volk GM, Wang Q-C (2018) Cryobiotechnology of apple (Malus spp.): development, progress and future prospects. Plant Cell Rep 37(5):689–709. CrossRefPubMedGoogle Scholar
  26. Yamamoto S, Rafique T, Priyantha WS, Fukui K, Matsumoto T, Niino T (2011) Development of a cryopreservation procedure using aluminium cryo-plates. CryoLetters 32(3):256–265Google Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Jean Carlos Bettoni
    • 1
    • 2
  • Ranjith Pathirana
    • 3
    Email author
  • Remi Bonnart
    • 2
  • Ashley Shepherd
    • 2
  • Gayle Volk
    • 2
  1. 1.Oak Ridge Institute for Science and Education (ORISE)Oak RidgeUSA
  2. 2.USDA-ARS National Laboratory for Genetic Resources PreservationFort CollinsUSA
  3. 3.The New Zealand Institute for Plant & Food Research LimitedPalmerston NorthNew Zealand

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