Determination of the EMS Lethal Dose (LD50) and Characterization of Induced Mutations in Industrial Hemp (Cannabis sativa L.)

Cannabis sativa is a multi-use crop with applications in food, fiber, construction, and medicinal industries. Cannabis plants with low Δ9-tetrahydrocannabinol (THC) concentrations (industrial hemp) have recently been legalized for industrial cultivation by multiple nations across the globe. This crop has been traditionally ignored and lags behind established crops like wheat, corn, and cotton in breeding and the development of novel traits. We employed chemical mutagenesis to develop novel traits in industrial hemp (Cannabis sativa L.). We used ethyl methanesulfonate (EMS) as the chemical mutagen and determined the lethal dose (LD50) to be 2% (v/v) for 5 h for the heat-tolerant (HT) (diploid, 2n) accession and 2% (v/v) for 8 h for the 4x (tetraploid, 2n = 4x = 40) accession. The variations in seed germination percentages among mutagen treatments were found to be highly significant (p < 0.05). The parental generation (M0) seeds were treated with the LD50 dosage of the mutagen to produce mutant M1 populations. The first mutant generation (M1) and second mutant generation (M2) plants displayed a wide array of phenotypes such as dwarf plants, twin seedlings, leaf variegation, fasciation, purple anthocyanins, hard/leathery leaf texture, trifoliate leaves, bushy stature, and unusual leaf shape, which is a testament to the effectiveness and accuracy of the LD50 dosage calibration results.