Isolation and Freezing Tolerances of Mesophyll Cells from Cold-hardened and Nonhardened Winter Rye

A method is described for the release of large quantities of mesophyll cells from leaves of both cold-hardened and nonhardened winter rye seedlings by a combination of enzymic and physical maceration. Such preparations usually contain a certain percentage of nonviable cells and are thus not suitable for quantitative biochemical studies. A method is also described whereby pure preparations of viable cells could be obtained by centrifugation on Percoll, using the observation that upon replacement of the isolation medium by water the viable and dead cells exhibited very different buoyant densities. The buoyant density of cells isolated from cold-hardened seedlings differed significantly from their nonhardened counter-parts. Survival of the isolated cold-hardened and nonhardened cells following extracellular freezing in water and following plasmolysis in balanced salt solutions was found to be in very close agreement with that of the plants and tissues from which they were isolated.