Improving gene editing efficiency has been a prominent research focus with the increasing application of CRISPR/Cas9 in crop genetic enhancement. In this study, we demonstrated that increasing exogenous auxin levels during in vitro tissue culture significantly enhances gene editing efficiency, leading to a higher frequency of functionally edited T₀ plants. While higher auxin levels promoted callus growth, it also delayed shoot initiation and slightly decreased shoot regeneration. Subsequent RNA-Seq analysis revealed significant alterations in the expression of plant developmental regulatory genes and chromatin remodeling genes at two plant regeneration stages. Further analysis using nuclei staining and Transposase-Accessible Chromatin using sequencing showed that excessive auxin resulted in a more relaxed chromatin structure in callus cells, thus enhancing the genomic DNA accessibility to Cas9. Additionally, the prolonged growth period of dedifferentiated callus cells and the delay in shoot initiation likely provided additional time for Cas9 to exert its function, explaining the improved gene editing efficiency due to excessive auxin application. To mitigate the inhibitory effects of excessive auxin on shoot regeneration, a ‘two-phase’ culture strategy was developed and validated using tomatoes, in which the explants were first cultured in media containing excessive auxin to promote calli growth and gene editing, then transferred to the media with lower auxin concentrations to promote the following shoots regeneration. Overall, our research has revealed novel aspects of auxin function in gene editing, offering new insights and a theoretical basis for future studies. Furthermore, the proposed culture method could accelerate the application of gene editing across various plant species.

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