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A Novel Method of Natural Cryoprotection ¹

Intracellular Glass Formation in Deeply Frozen Populus

American Red Cross, Biomedical Research Laboratories, Bethesda, Maryland 20814

Correlating measurements from differential scanning calorimetry, freeze-fracture freeze-etch electron microscopy, and survival of twigs after two-step cooling experiments, we provide strong evidence that winter-hardened Populus balsamifera v. virginiana (Sarg.) resists the stresses of freezing below –28°C by amorphous solidification (glass formation) of most of its intracellular contents during slow cooling (5°C per hour). It is shown that other components of the intracellular medium go through glass transitions during slow cooling at about –45°C and below –70°C. This `three glass' model was then used to predict the results of differential scanning calorimetry, freeze-fracture freeze-etch electron microscopy, and biological experiments. This model is the first definitive explanation for the resistance of a woody plant to liquid N₂ temperatures even if quench cooling (1200°C per minute) begins at temperatures as high as –20°C and warming is very slow (5°C per hour). It is also the first time high temperature natural intracellular glass formation has been demonstrated.