Fenugreek (Trigonella foenum-graecum) is a medicinal plant belonging to the family Fabaceae. The leaves and seeds of this plant have been used extensively throughout the world for a variety of purposes, such as anti-diabetes, help lower blood sugar and cholesterol, anti-cancer and antibacterial. The medicinal properties of fenugreek are due to the presence of steroidal compounds, alkaloids, flavonoids and essential oils. In fenugreek saponins such as diosegnin and yamogenin, and alkaloid trigonelline are of great value products. One of the plant breeding methods is the production of polyploidy. Accordingly, polyploidy induction in fenugreek was intended. For induction of polyploidy under greenhouse conditions, seedlings were treated with different colchicine concentrations (0, 1250, 2500 and 5000 micromolar) at 3, 4 and 5 hours. Moreover, induction of polyploidy in in vitro conditions on embryos was investigated using colchicine treatment at concentrations of (0, 75, 150, 300 and 600 micromolar) at 8, 12, 24 and 48 hours. The tetraploidy induction was succeeded under greenhouse conditions of the treated seedlings using 5000 micromolar colchicine for 4 hours. In vitro treatment of embryos using colchicine at concentration of 300 and 600 micromolar for 8 hours were also identified as suitable for tetraploidy induction. The tetraploid plants showed longer and wider stomata, thicker leaf and stem in comparison with diploid plants. The expression of two key genes (HMGR and STRL) involved in the diosegnin biosynthesis pathway in the leaves of diploid and tetraploid plants was also analyzed. The results showed an increase in the expression level of HMGR in the leaves of fenugreek tetraploid plants, while the expression level of the STRL was not significantly different compared to diploid plants. In this study, tetraploid induction was successfully accomplished in greenhouse and in vitro conditions, and the expression profile of the genes involved in the biosynthesis of dio
