The use of in vitro potato shoot cultures for protoplast isolation is desirable for consistent protoplast qualities. Ethylene build-up in the culture vessels causes problems for leaf growth (e.g. decreasing leaf surface, dry weight and chlorophyll) in shoot culture. Fifty to one hundred µM Silver thiosulfate (STS) produced a larger leaf area as a source of plant material for protoplast isolation. STS also decreased the internode length of potato shoots but increased dry weight and chlorophyll. Regenerated plants were obtained from cultured protoplasts in a series of media based on the MS medium. Protoplasts of Delaware showed a better response in cell division and colony formation in agarose-solidified culture medium. Regenerated plants showed some degrees of aneuploid but basically were similar to the original potato plants.