An Introduction to the Cryopreservation of Clonally Propagated Plants
Jean Carlos Bettoni, Independent Researcher, 35 Brasil Correia Street, Videira, SC 89560510, Brazil. jcbettoni@gmail.com
Katheryn Chen, Department of Soil and Crop Sciences, Colorado State University, 307 University Ave., Fort Collins, Colorado 80523.
Gayle M. Volk, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521. Gayle.Volk@usda.gov
The purpose of this chapter is to introduce the concept of cryopreserving plant genetic resources for crops that are clonally/vegetatively propagated.
Outline
1. Introduction
Cryopreservation is the secure storage of biological materials at ultra-low temperatures. Cryopreservation in liquid nitrogen (LN) or in its vapor phase (LNV) is a safe and cost-effective strategy for the long-term storage of plant genetic resources that cannot be stored as orthodox seeds. Once cryopreserved, materials remain safe and available at a low annual cost. In LN or LNV, plant tissues or organs are preserved in a state where cellular division and metabolic processes cease, preserving the structure and function of the biological system and minimizing the risk of contamination.
Cryopreservation is routinely implemented to preserve the diverse genetic resources of many plant species. Effective procedures have been identified for cryopreserving seeds, pollen, cell cultures, dormant buds, and shoot tips. Because each of these plant forms fulfills different roles in ex situ plant genetic resources collections, the most appropriate propagule to cryopreserve depends on the plant species and program targets, as well as the available infrastructure and technical skills. In plant species that produce few to no seeds, intermediate or recalcitrant seeds, or in plants that are vegetatively propagated to maintain a specific gene combination, vegetative propagules such as shoot tips and dormant buds are the most suitable source for preservation (Video 1).
Video 1. A video introducing clonal cryopreservation narrated by Jennifer Kendall.
2. Dormant bud cryopreservation
For clonal cryopreservation of winter-hardy woody species, dormant buds might be the best choice. Dormant bud cryopreservation capitalizes on the natural state of dormancy that enables propagules to survive low temperatures. Unlike traditional shoot tip cryopreservation methods, this approach does not rely on tissue culture introduction and multiplication to provide propagules. Although it is generally a much faster approach, its applicability is much more limited; it completely depends on seasonal availability of dormant buds and it is only successful for some cold-hardy temperate crops such as apple (Tanner et al., 2021).
Video 2. A video summarizing dormant bud cryopreservation narrated by Katheryn Chen.
3. Shoot tip cryopreservation
Unlike dormant buds, shoot tip cryopreservation can effectively preserve a wide range of vegetatively-propagated plants from both temperate and tropical origins. Shoot tips, the small growing points in plants, can be preserved using relatively little space, and can regenerate entire plants. Although they are labor-intensive and require specific tissue culture skills, the versatility provided by shoot tip cryopreservation enables cryobanks across the world to securely back up a wide range of economically important plant species including fruit crops, root and tuber crops, as well as rare and endangered species (Panis et al., 2020).
Video 3. A video summarizing shoot tip cryopreservation narrated by Remi Bonnart.
4. References
Panis B, Nagel M, Van den houwe I. 2020. Challenges and prospects for the conservation of crop genetic resources in field genebanks, in in vitro collections and/or in liquid nitrogen. Plants 9:1634. DOI: 10.3390/plants9121634
Tanner JD, Chen KY, Bonnart RM, Minas IS, Volk GM. 2021. Considerations for large-scale implementation of dormant budwood cryopreservation. Plant Cell, Tissue and Organ Culture 144:35-48. DOI: 10.1007/s11240-020-01884-5
5. Acknowledgments
Chapter citation: Bettoni JC, Chen K, Volk GM. 2024. An Introduction to the Cryopreservation of Clonally Propagated Plants. In: Volk GM (Eds.) Training in Plant Genetic Resources: Cryopreservation of Clonal Propagules. Fort Collins, Colorado: Colorado State University. Date accessed. https://colostate.pressbooks.pub/clonalcryopreservation/chapter/an-introduction-to-the-cryopreservation-of-clonally-propagated-plants/
This project was funded by the USDA-ARS and by the USDA-NIFA Higher Education Challenge Program grant 2020-70003-30930, with support for associated videos from IMLS Grant # MG-70-18-0063-18 to the Center of Plant Conservation.
USDA is an equal opportunity provider, employer, and lender. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
Nitrogen in the liquid phase maintains stable temperatures of -196 °C, cold enough for cryopreservation of many plant tissues
When liquid nitrogen in cryogenic tanks evaporates, it forms a cold vapor that is typically around -180 °C (+/- 20°C); this vapor layer can be used to cryopreserve plant tissues
Seeds that are desiccation- and cold-tolerant, and therefore amenable to traditional storage methods