Substantial enhancement of Agrobacterium-mediated transgene-free genome editing via short-term chemical selection...

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.