High‐efficiency genome‐editing, transgene evaluation, and antimicrobial efficacy testing using Citrus medica L. hairy roots

SUMMARY Huanglongbing (HLB) disease, associated with the fastidious bacterium Candidatus Liberibacter asiaticus ( C Las), has a significant impact on citrus production worldwide. Conventional biochemical and genetic evaluation studies to identify potential disease resistance strategies have been mainly hindered due to the inability to culture C Las in a defined medium and the general recalcitrance of Citrus cultivars (grapefruits and oranges) to Agrobacterium ‐mediated plant transformation. We previously demonstrated the utility of plant hairy roots to co‐cultivate C Las. In this study, we developed a hairy root transformation system using citron ( Citrus medica L.), which is highly amenable to Rhizobium ‐mediated hairy root transformation. The explant survival and hairy root transformation efficiencies were up to 100% and 73%, respectively, and transgenic roots can be attained in as little as 30–60 days. We demonstrate the utility of this citron‐based hairy root transformation for rapid CRISPR/Cas9‐mediated gene editing, transgene evaluation, and antimicrobial efficacy testing. The citron‐based hairy root transformation system will significantly help the research community to speed‐track the assessment of potential HLB disease resistance strategies.