Abstract:Protoplasts were isolated from young leaves of two cultivars of pepino, Solanum muricatum L., and cultured on SCM, LCM and M-K8p media for 1–2 d respectively. Protoplasts became enlarged and produced cell wall, then initial division was observed. Division rate of 85 % in LCM medium was calculated one week after culturing. The plate efficiency of small clone (PE) was higher than that of P Ⅰ (47.9%) and P Ⅱ (62.7%). Ability of shoot formation in calli derived from protoplast culture as well as genetypes of pepino; was different among that in dilution media. The best result in shoot formation (30. 1%) was obtained in regeneration medium supplemented with IAA 0. 1 mg/L, and ZT 2.0 mg/L. Shoots were transferred onto MS+ IAA 0. 2 mg/L to complete rooting following which the plantlets were successfully grown in soil.