Plant cryopreservation explained

Plant cryopreservation is a genetic resource conservation strategy that allows plant material, such as seeds, pollen, shoot tips or dormant buds to be stored indefinitely in liquid nitrogen.^[1] After thawing, these genetic resources can be regenerated into plants and used on the field. While this cryopreservation conservation strategy can be used on all plants, it is often only used under certain circumstances: 1) crops with recalcitrant seeds e.g. avocado,^[2] coconut^[3] 2) seedless crops such as cultivated banana and plantains^[4] or 3) crops that are clonally propagated such as cassava, potato, garlic and sweet potato.^{[5] [6]}

History

The history of plant cryopreservation started in 1965 when Hirai was studying the biology activities that happened when biological samples were frozen. Three years later, there was the first successful attempt cryopreserving callus cells. The following years, new methods to cryopreserve were developed, such as direct immersion, slow freezing and vitrification, as well as applied to more and more plants species and plant tissues.^[7]

Methods

• Direct immersion. This is the immersion of plant material directly in liquid nitrogen, or after desiccation. This is often done with (orthodox) seeds that already have a low moisture content or pollen.^[8] This method cannot be used with tissues that contain a lot of water or are sensitive to dehydration.
• Slow freezing. This method relies on the mechanism of freeze dehydration to pull water out of the cells and thus prevent ice formation in the cell.^[9]
• Vitrification. By freezing at an ultra-fast rate and using osmotic dehydration, the water that is still present in the cell is unable to form crystals and will be part of a glass-like or vitrified solution. This method can be further split in different variants e.g. droplet vitrification, encapsulation dehydration and plate vitrification. These techniques were successfully used with several economically-important crops, such as chrysanthemum^[10] or bleeding heart.^[11]

Hurdles and limitations

Aside from the challenges involved with cryopreservation in general, an important hurdle, when developing cryopreservation protocols for storage of plant germplasm, is that plants within a species can have a different tolerance to cryopreservation.^[12] This difference seems to be linked with the drought resistance of the different cultivars within the species.^[13] Even within the plant itself there can be noticeable differences depending on the tissue that is used, as both structure and composition play an important role during cryopreservation.

Organizations relying on plant cryopreservation

• Alliance of Bioversity International and Ciat
• International Potato Center
• Huntington Garden
• Rural development administration of Korea
• Leibniz Institute of Plant Genetics and Crop Plant Research

Notes and References

1. Reed BM . 2017-08-01. Plant cryopreservation: a continuing requirement for food and ecosystem security . In Vitro Cellular & Developmental Biology - Plant. en. 53. 4. 285–288. 10.1007/s11627-017-9851-4. 32177737. 1475-2689. free.
2. O'Brien C, Hiti-Bandaralage JC, Folgado R, Lahmeyer S, Hayward A, Mitter N . 2020. Developing a cryopreservation protocol for avocado (Persea americana Mill.) apical shoot tips using different antioxidants. Acta Horticulturae. 1285. 15–22. 10.17660/ActaHortic.2020.1285.3. 226751510 . 0567-7572.
3. Sopade PA, Samosir YM, Rival A, Adkins SW . Dehydration improves cryopreservation of coconut (Cocos nucifera L.) . Cryobiology . 61 . 3 . 289–96 . December 2010 . 20959171 . 10.1016/j.cryobiol.2010.09.007 .
4. Book: Panis B, Swennen R . Cryopreservation of Germplasm of Banana and Plantain (Musa Species). 1995. Cryopreservation of Plant Germplasm I. Biotechnology in Agriculture and Forestry. 32. 381–397. Berlin, Heidelberg. Springer Berlin Heidelberg. 978-3-642-08184-2 . 10.1007/978-3-662-03096-7_27.
5. 6. Wilms H, Fanega Sleziak N, Van der Auweraer M, Brands M, Verleije M, Hardeman D, Andre E, Panis B. 7 September 2020. Development of a fast and user-friendly cryopreservation protocol for sweet potato genetic resources. Scientific Reports. 10. 1. 14674. 10.1038/s41598-020-70869-3. 7477159. 32895398. 2020NatSR..1014674W.
6. Zimnoch-Guzowska E., Chmielarz P., Wawrzyniak M.K., Plitta-Michalak B.P., Michalak M., Pałucka M., Wasileńczyk U., Kosek P., Kulus D., Rucińska A., Mikuła A., 2022. Polish cryobanks: research and conservation for plant genetic resources. Acta Societatis Botanicorum Poloniae 91: 9121. https://doi.org/10.5586/asbp.9121
7. Kulus D., Zalewska M., 2014. Cryopreservation as a tool used in long-term storage of ornamental species – a review. Scientia Horticulturae 168: 88-107. https://doi.org/10.1016/j.scienta.2014.01.014
8. da Silva RL, de Souza EH, de Jesus Vieira L, Pelacani CR, Souza FV . 2017-05-17. Cryopreservation of pollen of wild pineapple accessions . Scientia Horticulturae. en. 219. 326–334. 10.1016/j.scienta.2017.03.022. 0304-4238.
9. Book: Kartha KK . Facultad de Ciencias Agrarias, Instituto de Botanica del Nordeste . Casilla de Correos 209, Corrientes (3400) Argentina.. In vitro growth responses and plant regeneration from cryopreserved meristems of cassava (Manihot esculenta Crantz)*) . 1982. 709654438.
10. Kulus, D., Rewers, M., Serocka, M. et al. Cryopreservation by encapsulation-dehydration affects the vegetative growth of chrysanthemum but does not disturb its chimeric structure. Plant Cell Tiss Organ Cult 138, 153–166 (2019). https://doi.org/10.1007/s11240-019-01614-6
11. Kulus, D. Shoot Tip Cryopreservation of Lamprocapnos spectabilis (L.) Fukuhara Using Different Approaches and Evaluation of Stability on the Molecular, Biochemical, and Plant Architecture Levels. Int. J. Mol. Sci. 2020, 21, 3901. https://doi.org/10.3390/ijms21113901
12. Panis B, Piette B, Swennen R . 2005-01-01. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Science. en. 168. 1. 45–55. 10.1016/j.plantsci.2004.07.022. 0168-9452.
13. Escobar RH, Mafla G, Roca WM . A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen . Plant Cell Reports . 16 . 7 . 474–478 . April 1997 . 30727635 . 10.1007/s002990050263 .