Following differing periods of long-term (13 years and 6 years, respectively) subculture and selection, two types of calli of the same wheat (Triticum aestivum L.) cv. Jinan 177 were obtained. The one (named cha9) grew fast and easily formed cell suspensions that were non-regenerable, but the protoplasts possessed a high division capacity. The other (named 176) was regenerable, but the derived protoplasts grew slowly. These two types of protoplasts were mixed and fused with protoplasts of Italian ryegrass (Lolium multiflorum Lam.). The protoplast of Italian ryegrass was UV irradiated. Many vigorous green plants having a wheat-like morphology were obtained. Regenerated calli and plants were recognised as somatic hybrids by a combined analysis of cytological, isozyme and random amplified polymorphic DNA (RAPD) markers. All the hybrids analysed are highly asymmetric, carrying only a few gnetic markers from Italian ryegrass. RFLP and SSR genotyping, using mitochondria- and chloroplast-specific markers, revealed a random segregation and rearrangement of the mtDNA, with a pronounced bias towards wheat, and a similarly biased segregation of cpDNA. Possible reasons for donor genome exclusion and the biased organelle segregation are discussed.