Protoplast Isolation and Staining

The successful isolation of mesophyll protoplasts from plant species has become a versatile tool for in vivo imaging of subcellular structures. Taking advantage of the various cytochemical probes available, the subcellular localization of specific organelles can be visualized in live protoplasts. In an isolated system, monitoring of the dynamics of organelle movement in response to external stimuli, stresses or an exogenous substance can be substantially facilitated. The isolation of a pure population of non-stressed, healthy protoplasts critically affects the reliability and reproducibility of these studies. In this chapter, we detail a standard protocol for the isolation of live mesophyll protoplasts from leaves of the model plant, Arabidopsis thaliana. We also consider the critical factors for empirical optimization of protoplast isolation procedures for succulent species such as Kalanchoe daigremontiana, Bienertia sinuspersici and Lampranthus spectabilis.