Substantial enhancement of Agrobacterium-mediated transgene-free genome editing via short-term chemical selection using citrus as a model plant

Abstract

Citrus production is threatened by biotic and abiotic stresses, particularly Huanglongbing (HLB), creating an urgent need for efficient engineering of citrus for disease resistance. Gene editing, especially transgene-free approaches, offers a promising alternative to traditional breeding, which is slow and constrained by citrus’ long juvenile phase. However, producing transgene-free, genome-edited citrus remains challenging. Here, we present a novel method to significantly enhance the efficiency of transgene-free gene editing in citrus using Agrobacterium-mediated transient expression of Cas9 and gRNAs. By treating Agrobacterium cells and citrus explants and applying a 3-day transient kanamycin selection, we achieved a 17-fold increase in transgene-free editing efficiency. The transient kanamycin-mediated suppression of shoot regeneration from non-Agrobacterium-infected cells not only improved the efficiency of identifying edited plants but also enhanced shoot regeneration efficiency from Agrobacterium-infected cells, regardless of whether these cells had stably incorporated T-DNA or not. This enhancement was likely due to reduced competition for space and nutrients from shoots regenerated from noninfected cells. In experiments targeting the phytoene desaturase (PDS) gene, transgene-free mutant shoot recovery increased from 0.017% to 0.291% of the total shoots produced. With an efficient screening method for gene-edited plants, the development of transgene-free gene-edited plants becomes relatively easy and practicable. These results suggest that this optimized protocol could be applicable to other perennial crops, offering a valuable tool for improving citrus varieties and other economically important plants.

Acknowledgements

This work was supported by the Citrus Research and Development Foundation (CRDF), USDA Crop Research Initiative Citrus Disease Research and Education (CDRE), and the University of Connecticut. We thank Prof. Yannick Jacob (Yale University) for sharing plasmid pYAO::hSpCas9-AtU6:sgRNA(CsPDS)-CaMV 35S:eGFP-NPTII. We thank Prof. Jianjun Chen (University of Florida) and Dr. Yongping Duan (U.S. Department of Agriculture, ARS) for providing bench and experiment materials and their kind guidance.

Author contributions

Study conception and design: Y.L. and Y.J.L; data collection: Y.J.L.; analysis and interpretation of results: Y.J.L., Y.L, and Z.R.L.; manuscript drafting: Y.J.L., Y.L, and Z.R.L. All authors reviewed and assessed the results, revised the manuscript, and approved the final version.

Data availability

All data included in the main text.

Conflict of interest statement

The authors declare no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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