Abstract: The objective of this experiment was to determine the appropriate colchicine dose levels to produce polyploids of Medicago sativa cv. Qingshui. Three different colchicine concentrations (0.05%, 0.10%, 0.20%) and three treatment durations (4 h, 8 h, 12 h) were used to induce the radicle of gemmiferous seeds and changes were observed in the root tip cell chromosome number and in inclusions in the lower epidermis of leaves. The results showed that colchicine can change chromosomes in the root tip cells of alfalfa seedlings, producing a variety of different mutant types (4x<2n<8x, 2n=8x and 2n>8x). The appropriate dose and treatment period of colchicine to induce a higher proportion of octaploid cells (2n=8x) is 0.1%+8h, with a mutation rate of 13.3%. Increasing levels of colchicine were associated with increases in the size (length, width, length/width) of guard cells and in the number of chloroplasts in the leaves’ lower epidermis, while the density of stomata decreased. The size of guard cells and number of chloroplasts were largest in the 0.1%+8h, 0.1+12h and 0.2%+4h treatments, where stomatal densities were also relatively the smallest. The largest mutagenic effects were observed under the 0.1%+8h treatment.