Development of in planta genome editing by transient expression of genome-editing tools in tomato

Two major processes are important for genome editing in plants: transformation by stable transfection, in which nucleic acids encoding genome-editing enzymes are introduced into plant cells and the regeneration of plant individuals from cells harboring mutations by genome-editing enzymes. The efficiency of transformation and regeneration by tissue culture varies across plant species, and is low in some practical crop species. In planta methods have been developed to exclude the need for tissue culture. However, few reports are available on methods that do not require stable transfection. Therefore, this study aimed to develop a new protocol for delivery genome editing tools that does not require transformation or tissue culture, by combining the in planta method with transient genome editing tools instead of stable transfection. Cas9, guide RNAs, and developmental regulators, which are factors involved in mitotic tissue induction, were transiently expressed by agroinfiltration of the stem tissue cut surfaces of tomatoes. New chimeric mutants, containing a mixture of cells with mutations introduced at or near the target sequence, were obtained. After examining conditions such as the concentration of Agrobacterium used for infection and post-infection treatment, we succeeded in obtaining chimeric mutants with an efficiency of 11.7%. In addition, most of the observed mutations were single base substitutions. These results indicate that the in planta method with transient expression of genome editing tools and induction of meristematic tissue can be used to introduce genome-edited mutations in tomatoes.