A broadly applicable somatic embryogenesis platform accelerates regeneration and genetic transformation in elite alfalfa

Alfalfa ( Medicago sativa L. ) is a globally important perennial forage crop, but molecular breeding is constrained by slow, genotype-dependent regeneration and transformation. This bottleneck is particularly pronounced in elite commercial cultivars, for which efficient protocols are scarce. A robust, broadly applicable regeneration platform is therefore essential for functional genomics, genome editing, and targeted improvement of agronomic traits. Using the elite cultivar ‘Zhongmu No. 1’, which has a high-quality reference genome, we systematically optimized explant type and stage-specific culture conditions across the entire regeneration pathway. Stem explants cultured on the optimized callus-induction medium CIM-MS1 showed markedly reduced browning, a 3.5-fold increase in callus induction, and callus formation 10–20 days earlier than under the reference condition, while maintaining stable embryogenic morphology. Transcriptome analysis of 15-day stem-derived calli associated this response with enhanced carbohydrate metabolism, cell-wall remodeling, and expression of embryogenesis-related regulators. Transfer of embryogenic calli to plant growth regulator-free N6 medium increased somatic embryo production by 1.3-fold and accelerated conversion to plantlets. Compared to a previously reported protocol, the integrated pipeline increased the frequency of transgene-positive plants 2.8-fold and shortened the transformation cycle by 2 to 3 months. The system supported both transgene overexpression and CRISPR/Cas9 editing and, importantly, enabled complete somatic embryo-mediated regeneration in all 25 tested cultivars from diverse geographic origins. We established an efficient, stage-integrated regeneration and transformation platform for elite alfalfa. Its novelty lies in combining axenic clonal explants, low-browning embryogenic callus induction, plant growth regulator-free somatic embryo development, and direct plantlet conversion into a single streamlined workflow. The broad regeneration response across 25 cultivars substantially extends the utility of the system beyond a single highly responsive genotype. This platform removes a major technical barrier to functional genomics and precision breeding in cultivated alfalfa.