Cybridization should be a way to induce phenotypical variations in highly heterozygous secondary citrus species or genotypes, without breaking their very complex multilocus structure by sexual recombination. It should have particular interest to manipulate the nucleo-cytoplasmic male sterility, to transfer tolerance traits with cytoplasmic determinisms and eventually to improve tolerance to oxidative stress. The present work is focused on the creation of new cybrids through three different approaches of protoplast fusion: (i) symmetric hybridization, (ii) donor-recipient protoplast fusion by UV irradiation of donor protoplasts, and (iii) cytoplasts + protoplast fusion. Sixteen combinations of symmetric protoplast fusion have produced cybrids. No cybrid was obtained after UV irradiation of donor protoplasts while three combinations of cytoplasts + protoplast fusion produced cybrids. Therefore, cytoplast fusion appears much more promising than the UV approach for targeted cybridization. The innovative plant material is currently propagated for further cultivar and rootstock selection and for basic researches on the impact of nucleo-cytoplasmic interaction on desirable traits.