In planta haploid induction in maize and tomato through disruption of KOKOPELLI

Haploid induction is a key component of doubled haploid technology and an increasingly valuable tool for plant breeding, genome editing, and clonal seed production. While in planta haploid induction through haploid inducer lines offers significant advantages over in vitro approaches, its application remains limited in many crop species. Previously, disruption of the sperm cell-expressed KOKOPELLI (KPL) gene was shown to induce maternal haploids in Arabidopsis thaliana. Here, we report the creation of novel haploid inducer lines in two globally important crops, maize (Zea mays), a major staple food crop, and tomato (Solanum lycopersicum), a widely cultivated vegetable crop. Using targeted genome editing, we generated mutations in KPL orthologs and demonstrated that loss of KPL function confers haploid induction capacity, enabling the production of haploid seedlings in both species. These findings establish KPL as a conserved target to trigger haploid induction and expand the genetic toolbox available for haploid inducer development in crop species.