Breeding of a Multifoliolate Alfalfa Population Using CRISPR/Cas9-Generated Mutants and Evaluation of Agronomic Traits and Nutritive Value

Alfalfa (<i>Medicago sativa</i> L.) is a major forage legume worldwide. Developing multifoliolate germplasm has been explored as a strategy to improve forage nutritive value and support more efficient forage livestock production. Here, we developed a multifoliolate population, SJ-ML, using CRISPR/Cas9-generated palmate-like pentafoliate mutants as donor parents. Molecular and phenotypic analyses indicated a stable inheritance of the edited alleles and the multifoliolate trait in SJ-ML. SJ-ML was evaluated under solar greenhouse and field conditions, with the recipient cultivar 'Aohan' as the greenhouse control and the commercial cultivars 'Galaxie-Max' and 'GN5' as field controls. SJ-ML showed a greater leaf area and a higher leaf-to-stem ratio, without reductions in yield or plant height. Nutritive analyses indicated that SJ-ML had a higher crude protein content, relative feed value, digestible dry matter, and dry matter intake, while acid detergent fiber, neutral detergent fiber, and lignin were lower than those of the controls. Across regrowth stages, SJ-ML generally maintained a higher nutritive value than controls. These results support SJ-ML as a multifoliolate germplasm resource for improving nutritive value without a trade-off in agronomic traits, with potential relevance for sustainable agriculture through enhanced forage protein value and a reduced reliance on supplemental protein in some ration contexts.