Seed cryopreservation of Corymbia citriodora: effects of cryoprotective treatments on seed vigor and germination
DOI:
https://doi.org/10.33158/ASB.r270.v12.2026Keywords:
Forest germplasm, orthodox seeds, vitrification, plant genetic resources, biodiversity, forest production, forest species, germplasm preservationAbstract
Efficient ex situ conservation of forest genetic resources depends on protocols that preserve seed viability during ultra-low-temperature storage. This study evaluated the effects of cryoprotective solutions on the germination and vigor of Corymbia citriodora (Hook.) K.D. Hill & L.A.S. Johnson seeds after storage in liquid nitrogen (-196 °C). A completely randomized design was used with six cryoprotective treatments and four replicates of 25 seeds each. Seeds were packed in cryotubes and subjected to the following treatments: control, 0.4 mol L−1 sucrose, 2.0 mol L−1 glycerol, PVS1, PVS2, and PVS3 before storage in liquid nitrogen (-196 °C). After 70 days of storage, the seeds were thawed at 40 °C for 1 min and tested for germination on Germitest paper under controlled conditions. Initial vigor, final germination, abnormal seedlings, and hard seeds were assessed. Data were analyzed by analysis of variance, Tukey's test, generalized linear models with binomial distribution, principal component analysis, and hierarchical clustering. Initial vigor did not differ among treatments; however, final germination declined in the sucrose, glycerol, and PVS3 treatments. PVS1 performed similarly to the control and showed the best overall post-thaw response. These results indicate that less aggressive vitrification solutions are more suitable for maintaining the viability of C. citriodora seeds under cryogenic storage.
Downloads
References
Benson, E. E. (2008). Cryopreservation theory. In: Reed, B. M. (Ed.). Plant cryopreservation: A practical guide. p. 15–32. Novo Mexico, USA: Springer.
Boland, D. J., Brooker, M. I. H., Chippendale, G. M., Hall, N., Hyland, B. P. M., Johnston, R. D., Kleinig, D. A., McDonald, M. W., & Turner, J. D. (2006). Forest trees of Australia. (5th ed.). Clayton, Vitória, Austrália: CSIRO Publishing. https://doi.org/10.1071/9780643069701
Cai, H., Shao, J., & Shen, Y. (2025). Seed storability in forest trees: Research progress and future perspectives. MDPI: Forests, 16(3), 467. https://doi.org/10.3390/f16030467
Figueiredo, M. A., Rosa, S. D. V. F., Ricaldoni, M. A., Pereira, C. C., Coelho, S. V. B., & Silva, L. C. (2021). Physiological, biochemical, and ultrastructural aspects of Coffea arabica L. seeds under different cryopreservation protocols. Ciência e Agrotecnologia, 45, e027020. https://doi.org/10.1590/1413-7054202145027020
MAPA - Ministério da Agricultura, Pecuária e Abastecimento. (2009). Regras para análise de sementes. Brasília, DF: MAPA/ACS.
Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nadarajan, J., & Pritchard, H. W. (2014). Biophysical characteristics of successful oilseed embryo cryoprotection and cryopreservation using vacuum infiltration vitrification: An innovation in plant cell preservation. PLoS ONE, 9(5), e96169. https://doi.org/10.1371/journal.pone.0096169
Nagel, M., Pence, V., Ballesteros, D., Lambardi, M., Popova, E., & Panis, B. (2024). Plant cryopreservation: Principles, applications, and challenges of banking plant diversity at ultralow temperatures. Annual Review of Plant Biology, 75, 797–824. https://doi.org/10.1146/annurev-arplant-070623-103551
Pence, V. C. (2014). Tissue cryopreservation for plant conservation: Potential and challenges. International Journal of Plant Sciences, 175(1), 40–45. https://doi.org/10.1086/673301
Pritchard, H. W. (2007). Cryopreservation of desiccation-tolerant seeds. In: Day, J. G., & Stacey, G. N. (Eds.). Cryopreservation and freeze-drying protocols. p. 185–201. Totowa, Nova Jersey, USA: Humana Press. https://doi.org/10.1007/978-1-59745-362-2_13
Reed, B. M. (2008). Plant cryopreservation: A practical guide. Novo Mexico, USA: Springer. https://doi.org/10.1007/978-0-387-72276-4
Roque-Borda, C. A., Kulus, D., Souza, A. V., Kaviani, B., & Vicente, E. F. (2021). Cryopreservation of agronomic plant germplasm using vitrification-based methods: An overview of selected case studies. MDPI: International Journal of Molecular Sciences, 22(11), 6157. https://doi.org/10.3390 ijms22116157
Rosa, T. A. O., Pedro Junior, S., Tejo, D. P., Paula, J. C. B., Shimizu, G. D., & Faria, R. T. de. (2024). Cryopreservation of Adenium obesum seeds in liquid nitrogen. Pesquisa Agropecuária Brasileira, 59, e03442. https://doi.org/10.1590/S1678-3921.pab2024.v59.03442
Sakai, A., & Engelmann, F. (2007). Vitrification, encapsulation-vitrification and droplet-vitrification: A review. Cryo Letters, 28(3), 151–172.
Sakai, A., Kobayashi, S., & Oiyama, I. (1990). Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. Var. brasiliensis Tanaka) by vitrification. Plant Cell Reports, 9, 30–33. https://doi.org/10.1007/BF00232130
Solberg, S. Ø., Yndgaard, F., Andreasen, C., von Bothmer, R., Loskutov, I. G., & Asdal, Å. (2020). Long-term storage and longevity of orthodox seeds: A systematic review. Frontiers in Plant Science, 11, article 1007. https://doi.org/10.3389/fpls.2020.01007
Souza, A. C., Costa, M. C., Figueiredo, M. A., Pereira, C. C., Coelho, S. V. B., Vilela, A. L. O., Pereira, D. S., & Rosa, S. D. V. F. (2024). Steps of cryopreservation of coffee seeds: Physiological responses and antioxidant systems. Ciência Rural, 54(2), e20220480. https://doi.org/10.1590/0103-8478cr20220480
Walters, C. (2015). Orthodoxy, recalcitrance and in-between: Describing variation in seed storage characteristics using threshold responses to water loss. Planta, 242(2), 397–406. https://doi.org/10.1007/s00425-015-2312-6
Walters, C., Ballesteros, D., & Vertucci, V. A. (2010). Structural mechanics of seed deterioration: Standing the test of time. Plant Science, 179(6), 565–573. https://doi .org/10.1016/j.plantsci.2010.06.016
Walters, C., Wheeler, L. M., & Grotenhuis, J. M. (2005). Longevity of seeds stored in a genebank: Species characteristics. Seed Science Research, 15(1), 1–20. https://doi.org/10.1079/SSR2004195
Zámečník, J., Faltus, M., & Bilavčík, A. (2021). Vitrification solutions for plant cryopreservation: Modification and properties. Plants, 10(12), article number 2623. https://doi.org/10.3390/plants10122623
Zeng, L., Sun, Z., Fu, L., Gu, Y., Li, R., He, M., & Wei, J. (2024). Cryopreservation of medicinal plant seeds: Strategies for genetic diversity conservation and sustainability. MDPI: Plants, 13(18), 2577. https://doi.org/10.3390/plants13182577
Downloads
Published
Issue
Section
License
Copyright (c) 2026 Missio, F. F., Faria, R. T., Soares, R. E., Almeida, B. M. S., & Silva, T. R.
This work is licensed under a Creative Commons Attribution 4.0 International License.
How to Cite
