Interactions among water content, rapid (nonequilibrium) cooling to-196°C, and survival of embryonic axes of Aesculus hippocastanum L. seeds

This study investigated the interactions among water content, rapid (nonequilibrium) cooling to -196 degreesC using isopentane or subcooled nitrogen, and survival of embryonic axes of Aesculus hippocustanum. Average cooling rates in either cryogen did not exceed 60 degreesC s(-1) for axes containing more than 1.0 g H2O g(-1) dw (g g(-1)). Partial dehydration below 0.5 g g(-1) facilitated faster cooling. averaging about 200 and 580 degreesC 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 greater than or equal to 300 degreesC s(-1). Cooling rates attained in subcooled nitrogen did not exceed 250 degreesC s(-1). and normal development of axes was observed only at less than or equal to0.4 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. (C) 2001 Academic Press.