The FW2.2-like (FWL) genes encode cysteine-rich proteins with a placenta-specific 8 domain. They play roles in cell division and organ size control, response to rhizobium infection, and metal ion homeostasis in plants. Here, we target eight rice FWL genes using the CRISPR/Cas9 system delivered by Agrobacterium-mediated transformation. We successfully generate transgenic T₀ lines for 15 of the 16 targets. The targeted mutations are detected in the T₀ lines of all 15 targets and the average mutation rate is found to be 81.6%. Transfer DNA (T-DNA) truncation is a major reason for the failure of mutagenesis in T₀ plants. T-DNA segregation analysis reveals that the T-DNA inserts in transgenic plants can be easily eliminated in the T₁ generation. Of the 30 putative off-target sites examined, unintended mutations are detected in 13 sites. Phenotypic analysis reveals that tiller number and plant yield of OsFWL4 gene mutants are significantly greater than those of the wild type. Flag leaves of OsFWL4 gene mutants are wider than those of the wild type. The increase in leaf width of the mutants is caused by an increase in cell number. Additionally, grain length of OsFWL1 gene mutants is higher than that of the wild type. Our results suggest that transgene-free rice plants with targeted mutations can be produced in the T₁ generation using the Agrobacterium-mediated CRISPR/Cas9 system and that the OsFWL4 gene is a negative regulator of tiller number and plant yield.

Root system architecture plays a crucial role in water and nutrient acquisition in maize. Cytokinins, which can be irreversibly degraded by the cytokinin oxidase/dehydrogenase (CKX), are important hormones that regulate root development in plants. In this study, ZmCKX5 was resequenced in 285 inbred lines, 68 landraces, and 32 teosintes to identify the significant variants associated with root traits in maize. Sequence polymorphisms and nucleotide diversity revealed that ZmCKX5 might be selected during domestication and improvement processes. Marker–trait association analysis in inbred lines identified 12 variants of ZmCKX5 that were significantly associated with six root traits, including seed root number (SRN), lateral root length (LRL), total root area (RA), root length in 0 to 0.5 mm diameter class (RL005), total root volume (RV), and total root length (TRL). SNP-1195 explained the most (6.01%) phenotypic variation of SRN, and the frequency of this allele G increased from 6.25% and 1.47% in teosintes and landraces, respectively, to 17.39% in inbred lines. Another significant variant, SNP-1406, with a pleiotropic effect, is strongly associated with five root traits, with the frequency of T allele increased from 25.00% and 23.73% in teosintes and landraces, respectively, to 35.00% in inbred lines. These results indicate that ZmCKX5 may be involved in the development of the maize root system and that the significant variants can be used to develop functional markers to accelerate the improvement in the maize root system.