Generation and characterization of rice OsCENH3 mutants for haploid induction.

CRISPR/Cas9-mediated modification of OsCENH3 induces aneuploidy but fails to trigger haploid formation in rice, underscoring limited efficiency and the need for strategy refinement. The centromeric histone H3 variant (CENH3) is essential for kinetochore assembly and accurate chromosome segregation during cell division. Alterations in CENH3 have been shown to trigger haploid induction in various plant species; however, its utility in rice remains largely unexplored. In this study, six OsCENH3 mutant lines were generated through CRISPR/Cas9-mediated genome editing, complemented by a GFP-tagged construct. To assess their haploid induction potential, both self pollination and outcrossing with the cultivar Nipponbare (NIP) were performed. Flow cytometry analysis revealed that none of the mutants produced true haploids. However, two aneuploid individuals (Het-C1-1-1 and Het-C1-5-1) were identified among the hybrid progeny derived from tail domain mutants C1-1 and C1-5, suggesting partial chromosome missegregation. These aneuploid plants exhibited reduced pollen viability, abnormal morphology, and compromised agronomic performance. In addition, significantly elevated rates of embryo/endosperm abortion were observed across different crosses, which far exceeded the frequency of aneuploid production, implying that early-stage chromosomal instability may result in embryo lethality. Collectively, while specific OsCENH3 mutations can induce limited chromosomal instability, their haploid induction efficiency remains low in rice. Further refinement of editing strategies and exploration of favorable genetic backgrounds will be essential for developing effective centromere-based haploid induction systems. This study provides a theoretical framework and technical reference for engineering haploid inducers in rice through centromere manipulation.