Simple method to isolate vacuoles and protoplasts for patch-clamp experiments

It was not possible to obtain protoplasts or vacuoles from the thallus of t he livenwort Conocephalum conicum by applying cell-wall-degrading enzymes. Therefore, a surgical method was developed to isolate protoplasts and vacuo les. A thallus was plasmolyzed and cut. The few protoplasts along the cutti ng edge that were not destroyed emerged from the edge under deplasmolysis a nd became thus accessible for a patch pipette. Whereas under slightly hypoo smolar conditions the emerging protoplast remained largely intact, more hyp oosmolar conditions gave rise to isolated vacuoles. This method to isolate protoplasts and vacuoles could also be applied to other plant tissues like leaves of Arabidopsis thaliana. Patch-clamp measurements were performed wit h isolated vacuoles and excised tonoplast patches. A slowly activating vacu olar channel in C. conicum displayed the characteristic features of higher- plant slowly activating vacuolar channels.