Alfalfa (Medicago sativa L.), an important forage crop, is critical to the livestock industry and to sustainable agriculture worldwide. Weeds is the main biological disaster of alfalfa production, and it is also one of the important factors restricting alfalfa production mechanization and simplified cultivation. Glyphosate is the most widely used herbicide worldwide, being used in almost 80% of the agricultural land planted with glyphosate-resistant (RR) crops. Among genetically modified crops, genetically engineered alfalfa ranks fifth in global planting area, following soybean, corn, cotton, and canola. Despite the efficacy of herbicides in weed control, their use in Chinese alfalfa production is severely limited. This is primarily due to a shortage of herbicide-resistant germplasm and the legal prohibition of genetically modified (GM) alfalfa cultivation. Consequently, developing herbicide-tolerant alfalfa through conventional breeding is of critical importance. Here, we developed a non-transgenic germplasm D1 with high-resistance to glyphosate herbicide. Genetic analysis confirmed the stable heritability of this trait, and D1 exhibits high resistance to glyphosate (Resistance Index = 5.0). Following herbicide application, chlorophyll content, plant height, and leaf area were measured. The D1 line showed significant differences in these parameters compared to the control (CK). Furthermore, our findings indicate that resistance is likely conferred by an increased copy number variation of the EPSPS gene in the D1 line. The study should facilitate the development of alfalfa varieties with resistance to glyphosate herbicides in future.

Alfalfa, Weeds, Glyphosate, Resistant