Viability of Plant Protoplasts

Abstract

In the context of work with plant protoplasts, viability may be defined as the capacity of an isolated protoplast to continue to grow in culture, to replace its lost cell wall and to form either suspension cells, callus or plantlets. However, for many practical situations, protoplasts need to be checked for viability at an early stage prior to use in a biochemical, genetical or cytological study. In spite of this, most workers often report neither on the viability levels of the protoplasts used in their experiments nor how long the protoplasts are left between preparation and use in an experiment (vide several articles in Pilet 1985 a). The process of removing the cell wall by enzyme digestion followed by washes and centrifugation of the isolated protoplasts must render them considerably changed from their stage in the cell. In addition, it is likely that the plasmalemma will be damaged or very modified from that of the intact cell. It is difficult to say precisely how long after isolation it takes for the plasmalemma to recover, since we are unable to know all of its features when a cell wall is present. However, it is clear that a proportion of the protoplasts in the preparation immediately after isolation will not survive in culture, this proportion depending upon the type of material employed as the protoplast source. Since the production of protoplasts became routine, a number of tests have been employed to check protoplast viability at an early stage after preparation, some having been used in viability tests for almost a century, others being developed for current needs. Unfortunately, the use of the classical test of reversible plasmolysis is not possible with protoplasts, although cyclosis does offer a quick and simple test for viability.