Submitted to: Cryobiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/4/2001
Publication Date: 9/1/2001
Citation: Wesley-Smith, J., C. Walters, N.W. Pammenter and P. Berjak. 2001. Interactions of water content, rapid (non-equilibrium) cooling to -196°C and survival of embryonic axes of Aesculus hippocastanum seeds. Cryobiology 42:196-206. Interpretive Summary: To successfully cryopreserve germplasm, cells must reach liquid nitrogen temperatures without intracellular water freezing. This is easily accomplished in dry cells, since there is limited water available to freeze. Successful cryopreservation is also relatively easy for materials that contain very few cells (e.g.spermatoza) because the small size allows sufficiently rapid cooling so that ice crystals do not have time to form. Large hydrated tissues are relatively hard to cryopreserve, because the thermal mass precludes rapid cooling. This study reports successful cryopreservation of hydrated plant embryos of buckeye which contain more than 500,000 cells. The technique optimizes water contents to lower thermal mass and increase the intracellular viscosity while limiting desiccation damage and then uses a plunging device (similar to a guillotine) to achieve cooling rates in excess of 300C per second. Survival rates of 100% demonstrate that this technique can be applied in seedbanks.
Technical Abstract: This study investigated the interactions between water content, rapid (non- equilibrium) cooling to -196 C using isopentane or sub-cooled nitrogen and survival of embryonic axes of A. hippocastanum. Average cooling rates in either cryogen did not exceed 60 C/s for axes containing more than 1.0 g H2O/g dw (g/g). Partial dehydration below 0.5 g/g facilitated faster cooling, averaging about 200 and 580 C/s in sub-cooled nitrogen and isopentane, respectively. The combination of partial drying and rapid cooling led to increased survival and reduced cellular damage in axes. Electrolyte leakage was ten-fold higher from fully hydrated axes cooled in either cryogen compared with control axes that were not cooled. Drying axes to 0.5 g/g reduced electrolyte leakage of cryopreserved axes to levels similar to that of control material. Axis survival was assayed by germination in vitro. Axes with water contents greater than 1.0 g/g did not survive cryogenic cooling. Between 1.0 and 0.75 g/g axes survived cryogenic exposure but developed abnormally. The proportion of axes developing normally increased with increasing dehydration below 0.75 g/g, reaching a maximum between 0.5 and 0.25 g/g following cooling at about 70 C/s in either isopentane or sub-cooled nitrogen. These results support the hypothesis that rapid cooling enhances the feasibility of cryopreserving desiccation-sensitive embryonic axes by increasing the upper limit of allowable water contents and overall survival.