Abstract: The overall aim of this research was to identify the optimal mutation method(s) for breeding new alfalfa varieties to cultivate in alpine regions. In this study, we investigated the effects several mutagenesis treatments [⁶⁰COγ-ray irradiation, ultraviolet radiation, ethyl methyl sulfonate (EMS), and magnetic field-free space] on the microstructure and ultrastructure of the leaves of four alfalfa cultivars; Gongnong 1, Wega7F, WL319HQ, and Aohan. The mutagenesis treatments were as follows: ⁶⁰COγ-ray irradiation (150, 300, 450 Gy), ultraviolet radiation (30, 60, 90 min), ethyl methyl sulfonate [0.1%, 0.2%, 0.4% (v/v)], and magnetic field-free space (180 d). The microstructure and ultrastructure of the leaf were analyzed after these treatments. All of the mutagenesis treatments increased leaf thickness. The greatest increase was in Gongnong 1 under the 0.4% EMS (v/v) treatment, where the final leaf thickness was increased by 25.86 μm to 446.16 μm. The degree of leaf vein protuberance was decreased by the mutagenesis treatments. The degree of cell tightness in Gongnong 1 increased after ⁶⁰COγ-ray irradiation and ultraviolet radiation treatments. The cells in the leaves of wega7F were closer together after all the mutagenic treatments. The ultramicrostructure of the chloroplasts in the alfalfa leaves was also observed under different mutagenic treatments. Under lower doses or concentrations of mutagenic treatments [150 Gy ⁶⁰Co-γ, 30 min ultraviolet radiation, 0.1% (v/v) EMS], the chloroplasts were distorted with disrupted membranes, loose grana lamellae, disintegrated thylakoids, and increased numbers of lipid droplets. Under the highest doses or concentrations of mutagenic treatments [450 Gy ⁶⁰Co-γ, 90 min ultraviolet radiation, 0.4% (v/v) EMS, and 180 d magnetic field-free space] the chloroplasts became swollen, distorted, and severely damaged with fractured grana lamellae and disintegrated membrane systems. The numbers of plastids and starch grains in cells were increased under these treatments. Compared with the other treatments, the ⁶⁰COγ-ray irradiation and EMS treatments showed higher mutagenic efficiency, produced more mutant materials, and were simpler to operate. Therefore, we concluded that these were the best mutation methods. The 150 Gy ⁶⁰COγ-ray irradiation, 60 min ultraviolet radiation, and 0.4% EMS treatments were the mutagenic treatments that were most conducive to screening mutants.

Key words: mutagenesis, alfalfa, microstructure, ultrastructure, mutants