Protoplast patch-clamping using an upright microscope with a movable stage

Abstract

Investigating membrane properties of plants is a challenging task, considering that success of experiments is highly dependent on the possibility to isolate metabolically active protoplasts that can withstand membrane current recordings. The aim of the present work is to obtain viable protoplasts derived from root cells of Pisum sativum that can be used for the whole-cell patchclamp. We designed the procedure of the pea protoplasts isolation that delivers stable protoplasts with preserved membrane integrity suitable for electrophysiological experiments. We applied a custom approach for patch-clamping protoplasts using a microscope with a movable microscope stage. We recorded prominent inward and prominent outward types of membrane current profiles of protoplasts. Obtained data indicate that optimized isolation protocol and custom system for patchclamping, can be applied to study membrane properties of root protoplasts.