Zygotic Embryos

Cryopreserving isolated zygotic embryos and embryo axes can be a useful strategy with some species, particularly when the embryo shows more tolerance to desiccation than the intact seed. This method requires isolating the embryos from the seeds before cryopreservation, as well as having an in vitro germination method for recovery. Banking zygotic embryos has the advantage of conserving genetic diversity equivalent to that of seeds.

Video Resources

Challenges associated with zygotic embryo cryopreservation: A case study on Syzygium maire by Jayanthi Nadarajan

Expand for More Info

Jayanthi Nadarajan is the Team Leader for the Germplasm Conservation Team at Plant and Food Research, New Zealand. Jayanthi has over 16 years of experience in plant conservation research with particular focus in seed conservation science. She is particularly interested in researching problematic seeds that are short-lived or not storable under seed bank condition. Jayanthi also has expertise in seed’s water and lipid biophysics research associated with seed longevity at sub-zero temperatures. She promotes research in developing new technologies and possible application of unique cryopreservation techniques. Her recent success was in developing a novel technique of ‘vacuum infiltration vitrification’ for cryopreservation of oily seed embryos. Jayanthi advocates integrated conservation strategies covering pollen and seed storage complemented with in vitro and cryopreservation techniques for conservation of exceptional plant species.

Ex situ conservation of oak genetic resources: Possible techniques and biological limitations of seeds by Paweł Chmielarz

Expand for More Info

Paweł Chmielarz is a Professor (natural sciences) in the Department of Seed Biology of the Institute of Dendrology Polish Academy of Sciences, where he has been since 1989 (PhD–1997 Agricultural University in Poznań). His research interests span both seeds and in vitro culture. Much of his work has been on better understanding of plant ex situ conservation. He is also interested in exploring the underlying molecular mechanisms that decide plant cell fate. Paweł Chmielarz investigated the implications of laboratory results to the forest practice and as a benefit for the society (cloned in vitro monumental oaks, 800 year-old). He has given numerous invited talks and tutorials of preservation of forest genetic resources in gene banks.

Cryobiotechnology of embryonic axes for the preservation of Oak (Quercus spp.) genetic resources by Daniel Ballesteros

Expand for More Info

Daniel Ballesteros is a researcher of the Comparative Seed Biology team at Royal Botanic Gardens, Kew. He has a PhD in plant biology and over 15 years of international experience in plant physiology and genetic resources preservation. He also carries out teaching and training activities for staff and supervise undergraduate, MSc and PhD students. His main research line aims to reveal the fundamental basis of desiccation and low temperature stress tolerance in plants, particularly in relation to successful ex situ conservation (including cryopreservation). In addition to comparative studies on desiccation and low temperature tolerance he is interested in the variation in and mechanisms of longevity in plant propagules, from fern spores as a unicellular model to more complex systems (e.g. seeds). His research is multidisciplinary but often involves making structural and biophysical determinations. In recent years he has developed research to understand the basis of the variation of response to the cryopreservation process of embryonic axes of European recalcitrant seeds, including oaks and buckeyes.

Expand for Selected References

Ballesteros D, Pence VC (2019) Survival and growth of embryo axes of temperate trees after two decades of cryo-storage. Cryobiology 88:110–113. doi: 10.1016/j.cryobiol.2019.04.005

Ballesteros D, Pritchard HW (2020) The cryobiotechnology of oaks : An integration of approaches for the long-term ex situ conservation of Quercus species. Forests 11:1–25. doi: 10.3390/f11121281

Ballesteros D, Sershen, Varghese B, et al (2014) Uneven drying of zygotic embryos and embryonic axes of recalcitrant seeds: Challenges and considerations for cryopreservation. Cryobiology 69:100–109. doi: 10.1016/j.cryobiol.2014.05.010

Chmielarz P, Michalak M, Pałucka M, Wasileńczyk U (2011) Successful cryopreservation of Quercus robur plumules. Plant Cell Rep 30:1405–1414. doi: 10.1007/s00299-011-1049-3

Corredoira E, San-Jose MC, Ballester A, Vieitez AM (2004) Cryopreseravtion of zygotic embryo axes and somatic embryos of European chestnut. CryoLetters 25:33–42.

Da-Silva-Ledo A, Santana FV, Araujo-de-Oliveira AC, et al (2020) Cryopreservation of Brazilian green dwarf coconut plumules by droplet-vitrification. Cienc Rural St Maria 50:1–8.

Engelmann F (2011) Cryopreservation of embryos: An overview. In: Thorpe TA, Yeung EB (eds) Plant Embryo Culture: Methods and Protocols, Methods in Molecular Biology, vol. 710. Springer Science+Business Media, LLC, pp 155–184

Gaidamashvili M, Khurtsidze E, Benelli C, Lambardi M (2019) Development of an efficient “One-Step Freezing” cryopreservation protocol for a georgian provenance of chestnut (Castanea sativa Mill.) zygotic embryos. Not Bot Horti Agrobot Cluj-Napoca 47:1047–1054. doi: 10.15835/nbha47411653

Gaidamashvili M, Khurtsidze E, Kutchava T, et al (2021) Efficient protocol for improving the development of cryopreserved embryonic axes of chestnut (Castanea sativa Mill.) by encapsulation–vitrification. Plants 10:1–9. doi: 10.3390/plants10020231

González-Benito ME, Prieto RM, Herradón E, Martín C (2002) Cryopreservation of Quercus suber and Quercus ilex embryonic axes: In vitro culture, desiccation and cooling factors. CryoLetters 23:283–290.

Malik SK, Choudhary R, Kaur S, et al (2020) Storage behavior and cryopreservation of Citrus cavaleriei , an endangered, cold-resistant species of northeast India with exceptionally large seeds. CryoLetters 41:281–290.

Nadarajan J, Pritchard HW (2014) Biophysical characteristics of successful oilseed embryo cryoprotection and cryopreservation using vacuum infiltration vitrification: An innovation in plant cell preservation. PLoS One 9:e96169. doi: 10.1371/journal.pone.0096169

Pukacki PM, Juszczyk K (2015) Desiccation sensitivity and cryopreservation of the embryonic axes of the seeds of two Acer species. Trees 29:385–396. doi: 10.1007/s00468-014-1118-7

Reed BM (ed) (2008) Plant Cryopreservation: A Practical Guide. Springer, New York

Wen B, Wang X, Tan Y, Song S (2013) Differential responses of Mimusops elengi and Manilkara zapota seeds and embryos to cryopreservation. In Vitro Cell Dev Biol – Plant 49:717–723. doi: 10.1007/s11627-013-9562-4

Wesley-Smith J, Walters C, Pammenter NW, Berjak P (2001) Interactions among water content, rapid (nonequilibrium) cooling to -196 degrees C, and survival of embryonic axes of Aesculus hippocastanum L. seeds. Cryobiology 42:196–206. doi: 10.1006/cryo.2001.2323

Xia K, Hill LM, Li DZ, Walters C (2014) Factors affecting stress tolerance in recalcitrant embryonic axes from seeds of four Quercus (Fagaceae) species native to the USA or China. Ann Bot 114:1747–1759. doi: 10.1093/aob/mcu193