title: Interactions among water content, rapid (nonequilibrium) cooling to -196 degrees 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 degrees C using isopentane or subcooled nitrogen, and survival of embryonic axes of Aesculus hippocastanum. Average cooling rates in either cryogen did not exceed 60 degrees C s(-1) for axes containing more than 1.0 g H(2)O g(-1)dw (g g(-1)). Partial dehydration below 0.5 g gg(-1) facilitated faster cooling, averaging about 200 and 580 degrees 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 > or =300 degrees C s(-1). Cooling rates attained in subcooled nitrogen did not exceed 250 degrees C s(-1), and normal development of axes was observed only at < or =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.
authors.affiliation: Electron Microscope Unit, School of Life and Environmental Sciences, University of Natal, Durban, South Africa.
authors.forename: J
authors.initials: J
authors.lastname: Wesley-Smith
authors.forename: C
authors.initials: C
authors.lastname: Walters
authors.forename: N W
authors.initials: NW
authors.lastname: Pammenter
authors.forename: P
authors.initials: P
authors.lastname: Berjak
mesh_terms: D015925:Cryopreservation / Q000379:methods*; D012639:Seeds / Q000737:chemistry* / Q000196:embryology; D014197:Trees / Q000737:chemistry / Q000196:embryology; D014867:Water / Q000032:analysis
chemical_list: D014867:Water