Plant protoplasts provide an enabling technology to underpin aspects of development, physiology, and genetics. Reliable procedures are available to isolate and culture protoplasts from monocotyledons and dicotyledons. Several parameters influence the totipotency of protoplasts and their derived cells, particularly the source tissue, culture medium, and environmental factors. Novel approaches to maximize the efficiency of protoplast-to-plant systems include techniques already established for animal and microbial cells, such as electrostimulation and exposure of protoplasts to surfactants and artificial respiratory gas carriers, especially perfluorochemicals and hemoglobin. Somatic hybridization by protoplast fusion is undergoing a resurgence of interest, since it enables nuclear and cytoplasmic genomes to be combined at the interspecific and intergeneric levels without prior knowledge of gene location, or involvement of recombinant DNA technology. DNA uptake into protoplasts has applications in transient and stable transformation, including the generation of transplastomic plants of commercial importance in molecular pharming. Other applications of isolated protoplasts are in studies of membrane function, cell structure, and longer-term toxicological assessments. Despite the century that has elapsed since protoplasts were first isolated, they still make a significant contribution to many aspects of modern plant biotechnology.