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

## TL;DR

Researchers developed a fast and efficient system using citron hairy roots to study citrus greening disease and test resistance strategies.

## Contribution

A highly efficient citron-based hairy root transformation system for rapid CRISPR/Cas9 gene editing and antimicrobial testing in citrus.

## Key findings

- Citron hairy roots achieved 100% explant survival and 73% transformation efficiency.
- The system enables rapid transgene evaluation and CRISPR/Cas9 gene editing in 30–60 days.
- The platform supports antimicrobial efficacy testing against citrus greening disease.

## Abstract

Huanglongbing (HLB) disease, associated with the fastidious bacterium Candidatus Liberibacter asiaticus (CLas), 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 CLas 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 CLas. 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.

Huanglongbing (HLB) or citrus greening disease threatens global citrus production, yet progress toward effective resistance strategies has been limited by the causal pathogen's unculturable nature and the difficulty of transforming citrus plants. This study introduces a highly efficient citron‐based hairy root transformation system that enables rapid gene editing, transgene testing, and antimicrobial evaluation. By accelerating functional studies and resistance screening, this platform provides a practical tool to advance citrus disease research and supports the development of sustainable HLB management solutions worldwide.