Somatic genome doubling determines fertility in maize haploids of inbred A427

Doubled haploid (DH) technology underpins modern maize breeding, yet widespread male sterility in haploids necessitates toxic chemical chromosome doubling. Spontaneous haploid genome doubling (SHGD) offers a biological alternative, but its developmental dynamics and cellular basis remain unresolved. Here, using the high haploid male fertility (HMF) genotype A427, we integrate flow cytometry, stomatal phenotyping and whole-plant performance analyses across three independent experimental years involving 2,924 individual measurements to characterize the temporal progression of genome doubling. Fertile and sterile A427 haploids showed progressively divergent nuclear DNA-content profiles during vegetative development, with fertile plants exhibiting increasing enrichment of 2C and 4C nuclear fractions at later leaf stages. Stomatal length at the 5th leaf stage (L5) strongly discriminated between plants subsequently classified as male fertile and male sterile (apparent AUC = 0.886), providing a proof-of-concept for early, non-destructive selection. Diploidization at the 15th leaf stage (L15) correlated with vegetative vigor and reproductive architecture, linking cellular ploidy dynamics to whole-plant performance. These findings support a developmental association between somatic diploidization and male fertility in A427 and provide a framework for evaluating chemical-free selection strategies, while validation across independent populations, genetic backgrounds and environments will be required before broader breeding application.