Protoplast culture in fruit tree species is limited, among other reasons, by the lack of reproducibility of previous results. In this work, we show that a precise definition, of the protoplast isolation and culture conditions, as well as the characterization of the physiological state of plant material are particularly important, since they greatly affected both yield and viability. The inclusion of pectolyase among the enzymes for cell wall digestion was critical. We got best results with a combination of Onozuka R-10, pectolyase and macerozyme. Yield increased during protoplast isolation when donor shoots were previously cultured in darkness for 7 days. When etiolated shoots were used, both yield and viability increased significantly when M38 solution was used both for pre-plasmolysis and enzyme digestion. In addition, maximum yield and viability were achieved when shoots were cultured in a medium with zeatin. Cefotaxime did not affected protoplast isolation but had a positive effect in protoplast culture, allowing cell division and cell aggregates of up to 10 cells