| Summary: | Abstract Background Guard cell protoplasts (GCPs) isolated from various plants have proven to be especially useful for studies of signal transduction pathways and plant development. But it is not easy to isolate highly purified preparations of large numbers of GCPs from plants. In this research, our focus is on a method to isolate large numbers of guard cells from tomato leaves. The protocols described yield millions of highly purified, viable GCPs, which are also suitable for studies on guard cell physiology. Results We developed an efficient method for isolating GCPs from epidermal fragments of tomato leaves. The protocol requires a two-step digestion to isolate high-quality tomato GCPs. In this procedure, cellulysin (in method L) was replaced by cellulose “Onozuka” RS (in method S) in the first digestion step, which indicated that cellulase RS was more effective than cellulysin. Method S dramatically shortened the time required for obtaining high yields and high-quality GCPs. Moreover, according to the GCP yields, hydroponic plants were more effective than substrate-cultured plants. Conclusions In this paper, protocols for large-scale preparation of GCPs and mesophyll cell protoplasts were described, followed by some success examples of their use in biochemical and molecular approaches such as reverse-transcription polymerase chain reaction, real-time polymerase chain reaction and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The method was proved to be a more efficient GCP-isolating method, capable of providing high yields with better quality in less time.
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