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

Aditya Kulshreshtha·Manikandan Ramasamy·Sonia Irigoyen·Carmen S Padilla·Chi-Kuan Tu·Kinnie Laughlin·James Borneman·Kranthi K Mandadi
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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.

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CitrusPlant RootsPlants, Genetically ModifiedPlant DiseasesGene EditingTransformation, GeneticTransgenesDisease ResistanceLiberibacterCRISPR-Cas SystemsAnti-Infective AgentsRhizobiaceaeCandidatus Liberibacter asiaticus (CLas)Citrus medica L.Huanglongbing (HLB) diseaseNONEXPRESSOR OF PATHOGENESIS‐RELATED GENE 3 (NPR3)antimicrobial peptideJournal ArticleResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't

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Aditya Kulshreshtha, Manikandan Ramasamy, Sonia Irigoyen, Carmen S Padilla, Chi-Kuan Tu, Kinnie Laughlin, James Borneman, Kranthi K Mandadi. 2026. High-efficiency genome-editing, transgene evaluation, and antimicrobial efficacy testing using Citrus medica L. hairy roots.. https://doi.org/10.1111/tpj.70745

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