Development of High-efficiency Genome Editing System for Hairy Roots in <italic>Melilotus albus</italic>

INTRODUCTION: Melilotus albus is an excellent forage crop, suitable for crop rotation and soil and water conservation. However, the stable genetic transformation system and gene editing system of M. albus have not been reported, which limits its application in gene function analysis and the creation of new germplasm resources. RATIONALE: The plant can produce hairy roots after infection by Agrobacterium rhizogenes harboring Ri plasmids. MtLAP1 expression can activate the anthocyanin synthesis process and then produce purple/red anthocyanin accumulation in the transgenic hairy roots. RESULTS: This study verified that the 35S::MtLAP1 expression cassette can induce the accumulation of anthocyanins in regenerants, resulting in red color visible to the naked eye. When using the hypocotyls of M. albus as explants, the induction efficiency of hairy root was as high as 62%, and the positive rate was as high as 30.8%. In addition, the research results showed that the gene editing vector, which carried the 35S::MtLAP1 expression cassette and co-expressed the Cas9 and sgRNA modules driven by the constitutive strong promoter of Arabidopsis Ubiquitin-10, could achieve an editing efficiency of 42.5% targeted to PDS (Phytoene Desaturase) gene in M. albus. CONCLUSION: This study successfully established a rapid inducing and screening system for transgenic-positive hairy roots and developed an efficient genome-editing tool for M. albus, laying a foundation for functional gene studies and the development of high-quality new germplasm resources in this species. Hairy root induction, the-naked-eye screening system and MaPDS gene editing in Melilotus albus. *** P