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.

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