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

Authors: LI, YANGJUN - University Of Connecticut; LIU, ZONGRANG - Retired ARS Employee; GMITTER JR, FREDERICK - University Of Florida; DENG, ZHANAO - University Of Florida; CHENG, BAOPING - Guangdong Academy Of Agricultural Sciences; Duan, Hui; LI, YI - University Of Connecticut

Submitted to: Horticulture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2025
Publication Date: 9/19/2025
Citation: Li, Y., Liu, Z., Gmitter Jr, F., Deng, Z., Cheng, B., Duan, H., Li, Y. 2025. Substantial enhancement of Agrobacterium-mediated transgene-free genome editing via short-term chemical selection using citrus as a model plant. Horticulture Research. 12(9). https://doi.org/10.1093/hr/uhaf153.
DOI: https://doi.org/10.1093/hr/uhaf153

Interpretive Summary: Gene editing technology provides a precise breeding tool, especially for trees with long juvenility, self-incompatibility, and complex genetic linkages. Agrobacterium-mediated T-DNA transfection is the primary method of plant editing, which inherently integrates transgenes into host genomes, raising environmental and food safety concerns. The development of a transgene-free editing method is therefore highly desirable to avoid introducing and integrating transgenes into plant genomes. In this study, we developed an efficient transgene-free editing method in citrus using a 3-day antibiotic selection to enhance transient transgene expression. This approach will expedite the fruit breeding process while mitigating concerns about transgene integration. Consequently, it holds significant promise for the genetic improvement of horticulturally important traits in fruit trees.

Technical Abstract: Target gene editing technology represents a powerful tool for improvement of agronomical traits but the incorporation of transgene into host genome often raises concerns because of potential uncertain impact on environment and food safety. In this study, we tested and applied a variety of parameters optimized for transient gene expression to Agrobacterium-mediated transformation in citrus, aiming to improve transgene-free editing efficiency, a major obstacle impeding gene editing-mediated improvement of horticulturally important traits in fruit trees including citrus trees. We showed that applying three-day kanamycin selection pressure early in the infection process significantly increased transgene-free editing efficiency. In addition, combining this transient selection with optimized transient expression treatments synergically enhanced the transgene-free editing efficiency by 17 times, representing a significant improvement. Such improvement has practical implications for the development of transgene-free cultivars with novel traits in citrus and other fruit crops using gene editing technology, potentially accelerating breeding programs and enhancing fruit quality and other traits such as biotic and abiotic resistance.