Targeted CENH3 protein depletion in egg cells enables highly efficient haploid induction

Doubled haploid technology is a transformative tool for accelerating plant breeding by enabling the rapid development of homozygous lines. While manipulation of the centromere-specific histone H3 variant, CENH3, has been shown to induce haploids in Arabidopsis and selected crop species, a broadly applicable approach remains elusive. The prevailing hypothesis is that CENH3 asymmetry between parental genomes during early embryonic development leads to the selective elimination of the parental chromosomes with reduced CENH3 and consequently weaker centromeres. We experimentally validate this hypothesis by depleting EYFP- or ALFA-tagged CENH3 using ubiquitin-mediated proteasomal degradation specifically in the egg cell prior to fertilization with wild-type pollen. This approach consistently generated paternal wild-type haploids with induction frequencies of up to 57% among progeny derived from egg cells containing the constructs for CENH3 depletion. We enhanced the system by incorporating a plant-derived E3 ubiquitin ligase for efficient CENH3 degradation and a fluorescent seed marker for rapid haploid identification. This approach also proved effective with in-locus ALFA-tagged CENH3 lines generated by gene targeting. Furthermore, the plant-derived E3 ubiquitin ligase successfully degraded CENH3 from tomato. Thus, this gametic CENH3-depletion system establishes a rational, modular framework for engineering haploid inducers and provides a potentially universal platform for haploid induction across diverse crop species.