Cytokinin has important functions during plant growth and development;
hence, many researchers have extensively studied cytokinin biosynthesis and
degradation. Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes that
regulate oxidative cleavage to maintain cytokinin homeostasis. In rice, 11 OsCKX
genes have been identified to date; however, most of their functions remain
unknown. Here, we comprehensively analyzed the expression patterns of OsCKX
genes and their genetic relationships using RNA sequencing (RNA-seq) and
β-glucuronidase (GUS) staining. Using CRISPR/Cas9 technology, we constructed
nine osckx mutants to determine the OsCKX function in rice development. Results
revealed that each OsCKX gene has a unique expression pattern. Furthermore, the
single osckx and higher-order osckx4 osckx9 mutant lines showed functional
overlap and subfunctionalization. Mutant phenotypes associated with decreased
CKX activity exhibited changes in leaf and root growth, inflorescence
architecture, fertilization, and grain weight. Notably, we found that the osckx1
osckx2 and osckx4 osckx9 double mutants displayed contrasting phenotypic changes
in tiller number, culm diameter, and panicle size as compared to the wild-type
(WT). Moreover, we identified several genes that were significantly expressed in
osckx4 and osckx9 single and double mutant plants. Many differentially expressed
genes, such as OsPIN2, OsRR4, and OsNRT2.3, were found to be associated with
auxin, cytokinin, and nitrogen pathways. Therefore, our findings provide new
insights on the functions of OsCKX genes in rice growth, that may be used as a
foundation for future studies aimed at improving rice yield and initiating green
production.