Patch clamping protoplasts from vascular plants : method for the quick isolation of protoplasts having a high success rate of gigaseal formation

Abstract

A method is described for the isolation of protoplasts (Pisum sativum, Phaseolus vulgaris, Avena sativa, Arabidopsis thaliana) in preparation for ion flux studies using patch clamp electrophysiology. Protoplasts that have been exposed to hydrolytic, cell wall degrading, enzymes for as little as 5 minutes form gigaseals (seal resistance higher than 10 giga Ohm) with the patch pipette with success rates greater than 40%. Sealing of these protoplasts is fast, averaging less than 2 minutes. This method yields high rates of gigaseal formation in a variety of tissues from both monocots and dicots and will enhance data collection in ion flux studies of plasma membranes of vascular plants.