Intergeneric plants developed by protoplast fusion between two Citrus hybrids for producing high-quality fruit

• Somatic cell fusion of 'Shiranuhi' and 'Meiwa' was performed for citrus production. • Two types of fused cells were obtained—callus cells and direct somatic embryos. • Tetraploid plants from somatic embryos showed heterogeneous nuclear fusion. • Phylogenetic analysis confirmed chromosomal inheritance from both citrus types. • Securing tetraploid genetic resources for seedless citrus production. Citrus peels are rich in antioxidants, such as carotenoids. The genus Fortunella , including varieties, such as ‘Meiwa,’ allows for whole-fruit consumption, securing important genetic resources for producing high-quality and seedless citrus fruits by breeding and developing intergeneric fusion plants through protoplast fusion. Therefore, somatic cell fusion was performed between calli capable of somatic embryogenesis from 'Shiranuhi' and protoplasts from 'Meiwa' leaf tissues to generate plants for producing high-quality citrus fruit that can be consumed whole. Moreover, an interspecific fusion between 'Miyagawa Wase' and 'Sanguinello' was performed. Two fused cells were obtained, one proliferating solely as callus cells (type-I and type-II) and the other generating direct somatic embryos (type-III). Two types of fusion-presumed cells, proliferating only as callus cells, induced somatic embryos. The induced and directly formed somatic embryos developed into normal plants with shoots and roots. The type-I fused cells contained nuclei and cytoplasm derived from mesophyll cells, and type-II fused cells were tetraploid homozygotes obtained from callus cells capable of forming somatic embryos. The plants obtained from directly formed somatic embryos (type-III fused cells) were intergeneric nuclear-fusion plants, tetraploid with heterogeneous nuclear fusion, and somatic-cell-fused. Phylogenetic analysis revealed that all plants possessed the internal transcribed spacer region related to the chromosomal inheritance of 'Shiranuhi' and 'Meiwa'. Overall, a regeneration system based on cell types was utilized to select homotetraploid and heterotetraploid fusion plants among interspecies and intergeneric plants as genetic resources for producing seedless citrus fruits suitable for whole-fruit consumption.