Protoplasts Surviving Freezing to −196 C and Osmotic Dehydration in 5 Molar Salt Solutions Prepared from the Bark of Winter Black Locust Trees

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Abstract
Free protoplasts were prepared from the living bark tissue of the trunk of summer and winter black locust trees by enzymic digestion of thin slices of the tissue for 3 h in a medium containing 2% Onozuka cellulase, 2% Rhozyme pectinase and 2% Driselase in mannitol solutions using 0.4 M mannitol for summer tissue and 1.0 M mannitol for winter tissues. Cleaned suspensions of protoplasts and also thin slices of tissue with cells intact were frozen to temperatures of -10.degree. C, -20.degree. C, -30.degree. C, -40.degree. C and liquid N in sucrose and balanced salt solutions. Similar suspensions of protoplasts were also subjected to strong osmotic dehydration (plasmorrhysis) in a series of balanced salt solutions of increasing molarity. Tests for survival showed that protoplasts retain the same properties of either extreme susceptibility or extreme resistance to injury by freezing or osmotic dehydration as the cells from which they are prepared. Winter protoplasts showed capability for tolerating freezing to -196.degree. C and plasmorrhysis in 5 M salt solutions. Protoplasts are a valid and useful system for investigating the properties of the protoplasm and surface membranes associated with the seasonal development of extreme hardiness in the cells of woody plants.