Alfalfa (Medicago sativa L.) exhibits high feeding value in agricultural and livestock production, with its above-ground parts determining yield and quality. Promoting the development of molecular breeding of alfalfa helps to clarify the function of MsARG4 in regulating its growth. Obtaining MsARG4-overexpressing and RNA interference-positive plants via genetic transformation, we compared their phenotypic, nutritional, physicochemical, and cellular structural characteristics with those of wild-type plants. The results showed that the plant height, stem diameter, number of branches, number of lateral branches, internode length, and crude protein content of overexpressing plants were significantly higher than those of wild-type and RNAi plants (p < 0.05). Furthermore, we observed that the cell area of OE lines was larger than that of WT and RNAi lines. The stem-to-leaf ratio of overexpressing plants in the branching and budding stages was significantly lower than in wild-type and RNAi plants (p < 0.05). Our study suggests that MsARG4 promotes alfalfa quality improvement and rapid stem growth.

Medicago sativa (alfalfa) establishment is often limited by soil salinity, especially during germination. Seed-associated microorganisms may influence plant performance, but cultivar-associated seed endophytic communities and their relationship with germination under salt stress remain unclear. We evaluated germination of five alfalfa cultivars under 0, 100, and 150 mM NaCl and applied a seed-derived endophytic microbial suspension from Saiwo to Gleat seeds under salt stress. Bacterial and fungal communities were characterized by 16S rRNA and ITS amplicon sequencing, and culturable bacteria were tested for plant growth-promoting traits under non-saline conditions. Saiwo showed higher germination at 100 mM NaCl, and the Saiwo seed-derived suspension significantly improved Gleat germination under salt stress. Pseudomonadota dominated bacterial communities, with Liberibacter as the most abundant genus in Saiwo and Pantoea enriched in Gleat and Vegron. Fungal communities were dominated by Ascomycota, with Fusarium, Alternaria, and Cladosporium being the main genera and Penicillium being enriched in Saiwo. The isolated strains, mainly Bacillus, Erwinia, Kocuria, Leclercia, and Pantoea, exhibited growth on Ashby N-free medium, siderophore production, and phosphate and potassium solubilization, but these traits were not evaluated under saline conditions. These results indicate that cultivar-associated seed endophytic communities are associated with germination under salt stress, and the Saiwo seed-derived suspension has potential for improving alfalfa germination in saline soils.