Interactions among Water Content, Rapid (Nonequilibrium) Cooling to −196°C, and Survival of Embryonic Axes of Aesculus hippocastanum L. Seeds

Abstract

This study investigated the interactions among water content, rapid (nonequilibrium) cooling to −196°C using isopentane or subcooled nitrogen, and survival of embryonic axes of Aesculus hippocastanum. Average cooling rates in either cryogen did not exceed 60°C s−1 for axes containing more than 1.0 g H2O g−1dw (g g−1). Partial dehydration below 0.5 g gg−1 facilitated faster cooling, averaging about 200 and 580°C s−1 in subcooled 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 10-fold higher from fully hydrated axes cooled in either cryogen than from control axes that were not cooled. Drying of axes to 0.5 g g−1, reduced electrolyte leakage of cryopreserved axes to levels similar to those of control material. Axis survival was assayed by germination in vitro. Axes with water contents greater than 1.0 g g−1, did not survive cryogenic cooling. Between 1.0 and 0.75 g g−1, axes survived cryogenic exposure but developed abnormally. The proportion of axes developing normally after being cooled in isopentane increased with increasing dehydration below 0.75 g g−1, reaching a maximum between 0.5 and 0.25 g g−1 after being cooled at ≥300°C s−1. Cooling rates attained in subcooled nitrogen did not exceed 250°C s−1, and normal development of axes was observed only at ≤0.4 g g−1. These results support the hypothesis that rapid cooling enhances the feasibility of cryopreservation of desiccation-sensitive embryonic axes by increasing the upper limit of allowable water contents and overall survival.