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Title: Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures

Author
item WESLEY-SMITH, JAMES - University Of Kwazulu-Natal
item BERJAK, PATRICIA - University Of Kwazulu-Natal
item PAMMENTER, NORMAN - University Of Kwazulu-Natal
item Walters, Christina

Submitted to: Annals Of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/1/2013
Publication Date: 3/1/2014
Citation: Wesley-Smith, J., Berjak, P., Pammenter, N., Walters, C.T. 2014. Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures. Annals Of Botany. 113(4):695-709. DOI: 10.1093/aob/mct284.

Interpretive Summary: Recalcitrant seeds do not survive drying and so cannot be preserved using conventional storage conditions at -20oC. Thus, cryogenic technologies are required to preserve genetic diversity in species producing recalcitrant seeds such as oaks, some maples and many tropical fruits. The key to cryopreservation is finding the right balance between partial drying and extremely rapid cooling to avoid ice formation and create aqueous glasses that stabilize cellular structures. This study demonstrates that tiny ice crystals form when hydrated embryonic axes are cooled at about 100C/ sec, but cell structure is maintained if only a few crystals form. When any ice crystals form and come in close proximity, recrystallization occurs during warming and cells are damaged by the large crystals. The study shows stringencies and flexibilities required to effect cryopreservation protocols for recalcitrant seeds.

Technical Abstract: Cryogenic technologies are required to preserve embryonic axes of recalcitrant seeds. Formation of potentially lethal intracellular ice limits successful cryopreservation; thus, it is important to understand the relationships among cryo-exposure techniques, water content and survival. In this paper, undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro. Intracellular ice was detected in cells but was not necessarily lethal. Embryo cells survived when crystal diameter was between 0.2-0.4 µm and fewer than 20 crystals were distributed per µm2 in the cytoplasm. Ice crystals were not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Also, intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. We noticed that shoot apices were more sensitive than radicles to cryo-exposure. Our findings challenge the accepted paradigm that intracellular ice formation is always lethal. It is probable that in multicellular tissues freezing damage involves mechanical stresses that deform intracellular constituents and/or disrupt intracellular organization. Cryopreservation treatments that deliver the appropriate combination of water content and cooling and warming rates prevent lethal freezing damage by limiting the size and intracellular location of ice crystals. This work indicates that the combination of water content and cooling and warming rates needs to be ascertained, probably at the tissue level, to maximize survival of cryopreservation. However, the ultimate aim is to understand the over-arching principles needed to optimize treatments without undue reliance on empirical approaches.