DOI: 10.1016/j.plantsci.2025.112532
Alfalfa (Medicago sativa L.) is a globally major forage crop for livestock due to its superior nutritional value and palatability. As a premium forage species, alfalfa breeding has traditionally emphasized high yield. In this study, we designed guide RNA (gRNA) to target the coding sequence of the MsCLE3a gene, and constructed a CRISPR/Cas9 vector to generate knockouts. Five mutant lines were successfully isolated, displaying upright petioles and a compact ternately compound leaf phenotype during the seedling stage. Cryo-scanning electron microscopy (Cryo-SEM) was employed to analyze cell morphology on the abaxial leaf surface. The results indicated that the cells on the abaxial side of the Mscle3a mutants were significantly elongated compared to the wild type (WT). Transcriptomic analysis further uncovered the down-regulation of brassinosteroid (BR) biosynthesis genes in Mscle3a mutants, which might underlie the mechanisms regulating petiole angle. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a major reduction in the transcript levels of several key genes involved in BR signaling pathways in the Mscle3a mutants relative to WT. This transcriptional down-regulation was strongly associated with the observed alterations in leaf petiole angle (LPA) in the mutant lines. Our findings provide novel insights into the novel regulatory role of MsCLE3a in alfalfa development and pinpoint potential targets for manipulating LPA in crop breeding programs.