In this study, eight diploid potato clones with different characters were used for developing the protocals of isolation, culture and regeneration of protoplasts for the potato breeding program using protoplast fusion technique. In order to obtain highest frequency and good quality of protoplasts, different factors including enzyme solution, the duration of incubation, the age of in-vitro plants used for isolating mesophyll protoplasts following the protocal of Mulers et all (1992) was investigated