Polyploidy can be observed in citrus species which often enables better adaptation to environmental stresses. In this context, this paper aimed to develop stable tetraploid C35 citrange by in vitro colchicine treatments. Seed-derived explants were obtained by a germination experiment using several concentrations of GA(3). Culturing seeds of C35 without seed coat on MT basal medium with the addition of 4 mg l(-1)GA(3)resulted in the highest germination rate (84%). Various colchicine concentrations and exposure time were tested to induce polyploidization. Seed-derived explants of C35 citrange treated with 0.1% colchicine for 48 h demonstrated high rates of mutation for polyploidization and the highest tetraploid induction percentage (15%) was determined in this treatment. Higher colchicine concentrations and exposure time decreased the survival rates of the seedlings. Flow cytometry demonstrated that the nuclear genome size of tetraploids (1.598 pg/2 C) was significantly (P< .01) higher than diploid (0.794 pg/2 C) C35 seedlings. Recovered seedlings were identified with morphological and cytological variables, such as leaf area and stomata size. Generally, leaf area and stomata size significantly increased with the increasing ploidy level. The method used in this present study was relevant and an effective way to induce polyploidy in C35 citrange.