Isolation of protoplasts from unhardened and hardened tissues of winter rye and wheat

An enzymatic method is described for preparing stable protoplasts in high yield from non-hardened seedlings and hardened seedlings of winter wheat and rye capable in the hardened condition of tolerating −15 and −26 °C, respectively. The protoplasts were prepared from the epicotyl tissues which were found to be highly amenable to enzymic digestion. Metabolic competence and vitality of protoplasts were established by neutral red staining, cyclosis, regeneration of cell wall, and absence of alteration in lipid unsaturation.