Isolation of protoplasts for patch-clamp experiments: an improved method requiring minimal amounts of adult leaf or root tissue from monocotyledonous or dicotyledonous plants

Summary

For the purpose of patch-clamp studies, a protoplast isolation procedure is presented in which an osmotic shock (changing the osmolarity of the medium) and centrifugation step are omitted to limit mechanical stress. Apart from the reduction of mechanical stress factors, protoplast washing is also limited. Protoplasts have been isolated from different monocotyledonous and dicotyledonous plant species and from different tissues (leaves and roots). The seal success rate in patch-clamp experiments was high (about 85% successful seals showing a seal resistance > 10GΩ). To evaluate the electrogenic viability of the protoplasts, fusicoccin and light responses of the plasma membrane and channel activity were tested. The addition of fusicoccin to the bathing medium caused typical high activation of the proton pump. Switching the light on and off caused transient depolarizations and hyperpolarizations, respectively, matching data reported for mesophyll cells in micro-electrode studies. Whole-cell and single-channel recordings of protoplast plasma membranes isolated from intact tobacco andArabidopsis leaves were comparable with data published for protoplasts from corresponding tissue cultures. It is concluded that our isolation procedure yields protoplasts with electrogenic responses and is therefore suitable for patch-clamp studies in physiological research.