Multifaceted alfalfa improvement through FTO overexpression facilitated by an efficient MsPep1-based transformation platform

Abstract Alfalfa (Medicago sativa) as one of the most important leguminous forages globally, plays an irreplaceable role in the development of animal husbandry and ecological conservation. However, its low genetic transformation efficiency severely constrains molecular breeding progress. In this study, the application of elicitor peptides MsPep1 significantly increased the genetic transformation efficiency of alfalfa by 1.61 times and shortened the transformation cycle by 20 days. Using this improved transformation system, we investigated FTO, an m⁶A RNA demethylase gene that regulates plant growth and stress responses. In the greenhouse, overexpression of FTO in alfalfa significantly enhanced plant height, root length, the number of lateral roots, as well as drought and salt tolerance. Under field conditions, it substantially increased fresh weight per plant by 24.98%, relative feed value (RFV) by 10% and hundred-seed weight. Through transcriptome sequencing (RNA-seq), we inferred that the FTO gene promotes alfalfa growth by regulating auxin, optimizes forage quality by enhancing secondary metabolite synthesis and enhances salt-drought tolerance by activating oxidative stress pathways. FTO mediates large amounts of m6A demethylation in plant RNA, altering chromatin composition and transcriptional activation. In summary, these findings provide molecular breeding support for developing novel alfalfa cultivars with high yield, superior quality and strong stress resilience.