A robust and high-efficiency Rhizobium rhizogenes hairy root transformation platform for Vaccinium

Efficient transformation remains a major constraint to functional genomics and genome editing in Vaccinium, where stable transformation systems are often genotype-dependent and inefficient. Here, we establish a rapid and high-efficiency Rhizobium rhizogenes-mediated hairy root transformation platform optimized for the genus. Using the RUBY visual reporter, transformation efficiency reached 46.7% in leaf explants infected with strain Ar. A4 and cultured on half-strength Woody Plant Medium, with transgenic roots visible within 16 days post-co-cultivation. Comparative evaluation of six R. rhizogenes strains identified Ar. A4 and ATCC15834 as consistently superior across diverse Vaccinium germplasm representing different taxonomic sections, achieving up to 80% efficiency in selected genotypes. While conventional regeneration from transgenic roots was not successful, overexpression of developmental regulators enabled shoot formation with 7% efficiency, demonstrating a path toward stable plant recovery. This platform delivers a rapid, genotype-flexible system for gene validation, metabolic pathway analysis, and genome editing in Vaccinium, substantially expanding the molecular toolkit available for perennial fruit crop research and translational breeding.