Identification of Triploid Plants in Seed-Derived Progeny of Cultivated Olive

The large and hard olive pit adversely affects oil quality during traditional crushing, as seed- and pit-derived enzymes modify phenolic profiles and volatile compounds. Polyploid breeding offers a potential means to reduce pit size and improve processing traits, yet cultivated olive (Olea europaea L. subsp. europaea) is a strictly diploid species, and natural polyploids have not been previously documented. To evaluate the potential of triploids in olive improvement, we screened seed-derived progeny from multiple cultivars for polyploidy using flow cytometry and chromosome observation. One naturally occurring triploid seedling (‘Olive-3x’) was identified from a mixed lot of open-pollinated seeds. Whole-genome resequencing was used to develop 64 polymorphic InDel markers, and three markers indicated ‘Koroneiki’ as one putative parent of the triploid. Morphological and cytological analyses showed that the triploid exhibited typical polyploid characteristics, including thicker leaves and enlarged epidermal and palisade mesophyll cells compared with diploid controls. These findings provide the first evidence of a naturally occurring triploid in cultivated olive and show that triploids can arise within seed-derived progeny. The identified triploid plant and the developed markers offer useful resources for future studies on olive polyploidy and provide foundational resources for future research on olive polyploidy and cultivar improvement.