CRISPR/Cas9-Mediated Mutagenesis of Floral Termination Genes: A Way to Control Fruit Size

Tomato fruit weight and shape depend upon a complex regulatory network occurring during multiple stages throughout plant development. This network is initiated as early as in the floral meristem in which the maintenance of stem cells is terminated after the production of the last floral structure, the gynoecium. Hence, the process of floral meristem termination directly impacts the number of cells that are specified to form a carpel primordium, as well as the number of primordia. This will ultimately influence the number of fruit locules, and hence directly impact on the size of the developing fruit. Using CRISPR/Cas9-mediated mutagenesis we targeted two genes known to be involved in the floral termination process, namely Inhibitor of Meristem Activity (IMA) and SlKNUCKLES (SlKNU). Using a unique plasmid encoding kanamycin resistance, the Cas9 nuclease and two single guide RNAs, we generated quickly mutated tomato plants via Agrobacterium-mediated transformation,. First, we targeted the SlKNU coding sequence, inducing deletionsthat led to gene knock-out and resulted in multicarpellar fruit phenotype. Next, because of the expected lethality of the IMA knock-out in connection with its role in regulating cell division, we targeted the promoter regulatory box responsible for its expression in floral meristem. This resulted in the knock-down of IMA, and multicarpellar fruits were produced. Taken together, our data establish the efficiency of the CRISPR/Cas9-targeted mutagenesis technology to create partial and complete loss-of-function of key genes involved in floral termination resulting in multicarpellar fruit in tomato.