Production of polyploid plants in fenugreek (Trigonella feonum-graecum L.) normally involves artificial chromosome doubling by colchicine treatment of seedlings or seeds. Since colchicine is a hazardous chemical, it is replacing by less toxic chemicals would be highly required. Additionally, an in vitro culture method such as embryo culture to shorten the polyploidization is desirable. Polyploidization can be an important approach to improve yield and content of secondary metabolites in fenugreek. In this study, five concentrations of colchicine with four intervals duration and four concentrations of trifluralin with three exposure time were applied to induce polyploid plants from embryo cultures. Both treatments resulted in successful tetraploid induction. The regenerated plants in trifluralin-supplemented media showed larger stomata size with lower densities of stomata compared with control plants. Tetraploid plants were successfully identified by chromosome counting and flow cytometry analysis. Embryo mortality rates were higher with longer term exposure and higher concentration of colchicine or trifluralin treatments. 600 µM colchicine and 500 µM trifluralin were caused the highest mortality rates. However, the survival rate of embryos treated with trifluralin was higher in comparison with colchicine. The effect of ploidy level on transcript expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and sterol-3-β-glucosyl transferase genes involving in the biosynthetic of diosgenin, revealed that 3-hydroxy-3-methylglutaryl-coenzyme A reductase was significantly elevated in the leaves of tetraploid plants. The results indicate that tetraploid induction by trifluralin-treated embryos is an efficient approach for polyploidisation of fenugreek and can be adopted into breeding programs.