ISOLATION OF PROTOPLASTS FOR PATCH-CLAMP EXPERIMENTS - AN IMPROVED METHOD REQUIRING MINIMAL AMOUNTS OF ADULT LEAF OR ROOT-TISSUE FROM MONOCOTYLEDONOUS OR DICOTYLEDONOUS PLANTS

For the purpose of patch-clamp studies, a protoplast isolation procedu re is presented in which an osmotic shock (changing the osmolarity of the medium) and centrifugation step are omitted to limit mechanical st ress. Apart from the reduction of mechanical stress factors, protoplas t washing is also limited. Protoplasts have been isolated from differe nt monocotyledonous and dicotyledonous plant species and from differen t tissues (leaves and roots). The seal success rate in patch-clamp exp eriments was high (about 85% successful seals showing a seal resistanc e > 10 G Omega). To evaluate the electrogenic viability of the protopl asts, fusicoccin and light responses of the plasma membrane and channe l activity were tested. The addition of fusicoccin to the bathing medi um caused typical high activation of the proton pump. Switching the li ght on and off caused transient depolarizations and hyperpolarizations , respectively, matching data reported for mesophyll cells in micro-el ectrode studies. Whole-cell and single-channel recordings of protoplas t plasma membranes isolated from intact tobacco and Arabidopsis leaves were comparable with data published for protoplasts from correspondin g 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.