CRISPR/Cas9-mediated genome editing has become a common technology for gene manipulation in plant gene research and crop improvement. Studies have developed technologies with Agrobacterium-mediated transient expression of genome-editing machinery to generate transgene-free plants with an edited locus. This study examined the combined effect of short-term (three-day) antibiotic selection and heat treatment (24 hours at 37 °C) on genome editing efficiency in two different systems. Both systems targeted the same two genes, PDS and MAR1, in tobacco. The first system employed developmental regulator genes (DRs) inducing shoot formation to select plants with transient foreign gene expression. The other relied on phytohormone-induced shoot formation. The DRs, including the cytokinin-producing ipt gene, induced tobacco shoot formation after Agrobacterium-mediated transient expression. The combined treatment and heat treatment alone reduced the shoot formation, but the three-day selection alone did not. Interestingly, the combined treatment elevated the percentage of transgene-free mutant shoots to 7.6% as compared to 1.2% with heat treatment alone in ipt-induced shoots. In the shoots induced by the externally added phytohormone, the percentages of transgene-free mutant shoots were 2.8%, 5%, and 7.7% after three-day antibiotic selection alone, heat treatment alone, and the combined treatment, respectively.
Genome editing, transgene-free, mutation, heat treatment, antibiotic selection
The study investigated the impact of combining three-day antibiotic selection and 24-hour heat treatment on CRISPR/Cas9-mediated genome editing in a model plant, tobacco. The combined treatment significantly increased the percentage of transgene-free mutant shoots compared with individual treatments, regardless of shoot induction modes: transient expression of morphogenic inducer or optimized phytohormones in a medium.