An efficient gene delivery method utilizing the regenerative capacity of alfalfa ( Medicago sativa )

Alfalfa (Medicago sativa), an autotetraploid species, is a globally important forage crop. Traditional genetic transformation methods predominantly rely on leaf-derived callus, a process that is complex, inefficient, and particularly challenging to generate stable transgenic lines in polyploid plants. To address these limitations, we developed an efficient transformation system that bypasses callus induction by directly exploiting stem regeneration. Our protocol enables the generation of stable gene-edited alfalfa plants from shoot tips. It accommodates stem explants from various developmental stages and significantly reduces the time required. Moreover, this approach was effective across multiple alfalfa cultivars, and preliminary proof-of-concept experiments in Nicotiana tabacum and Solanum lycopersicum suggested potential transferability beyond alfalfa. This study represents a substantial advancement in perennial plant transformation and provides a robust and practical platform for optimizing stem regeneration-based genetic modification systems.