AbstractProtoplasts are plant cells lacking cell walls. They can be generated from stationary callus cultures derived from Arabidopsis thaliana seedlings. After treatment of the callus with cellulase and pectinase, protoplasts are inoculated with viral RNAs using polyethylene glycol. After various times postinoculation, total RNA is extracted and subjected to electrophoresis on nondenaturing agarose gels for further analysis. Stationary callus cultures are simpler to maintain than more traditional suspension cultures and yield high‐quality, uniform protoplasts. Protoplasts prepared in this fashion can also be used for uptake of DNA.