Establishment of an efficient Agrobacterium-mediated transformation system and CRISPR/Cas9-mediated genome editing of the OvPDS1 gene in Oxalis vulcanicola 'Sunset Velvet'.

This study overcomes a key technical barrier by establishing transformation and enabling first CRISPR/Cas9 editing in Oxalis, providing a platform for functional genomics and breeding. The lack of an efficient genetic transformation system has considerably hindered functional genomics studies in Oxalis vulcanicola 'Sunset Velvet'. Here, we established a stable and efficient Agrobacterium-mediated transformation system using stem segments as explants. Key parameters, including pre-culture duration, infection time, Agrobacterium cell density, acetosyringone (AS) concentration, and co-cultivation period, were systematically optimized. Under optimal conditions, the highest transient β-glucuronidase (GUS) expression rate reached approximately 9.0%, and eight stable transgenic lines were successfully obtained. CRISPR/Cas9-mediated genome editing was achieved in Oxalis for the first time. Targeted mutagenesis of OvPDS1, a gene involved in carotenoid biosynthesis, resulted in an albino phenotype, and Sanger sequencing confirmed a base substitution at the target site. Although the editing efficiency was relatively low (0.5%), this result demonstrates the feasibility of genome editing in Oxalis. This study overcomes a major technical bottleneck and provides a robust platform for functional gene analysis, trait improvement, and molecular breeding in O. vulcanicola 'Sunset Velvet' and other non-model ornamental plants.