A Universal Hairy Root Transformation Protocol for Evaluating Editing Efficiency in Dicot Plants

ABSTRACT Hairy root transformation has emerged as a promising method for assessing genome editing efficiency in dicot plants. However, the absence of a universally applicable protocol has posed a significant challenge. In our study, we addressed this challenge by investigating the impact of explant age and Agrobacterium rhizogenes strain. We employed five distinct Agrobacterium rhizogenes ( A . rhizogenes ) strains, namely K599, MSU440, Ar.1193, Ar.Qual and C58C1,to infect Arabidopsis seedlings at 2‐, 4‐, 6‐, 8‐ and 10‐day‐old stages, as well as seedlings of other dicot species at 1‐, 2‐ and 3‐day‐old stages. We successfully established a universal protocol, which utilizes explants derived from 2‐day‐old Arabidopsis seedlings and 1‐day‐old seedlings of other dicots, in conjunction with the A . rhizogenes strain K599. Leveraging this optimized system, we achieved reliable evaluation of gene‐editing vector efficiency in dicot plants prior to tissue culture, through the combined application of Sanger sequencing, PCR/restriction enzyme (PCR/RE) assay and deep sequencing techniques.