Abstract Protoplasts have been isolated from non-differentiating ovular callus of Citrus sinensis and cultured in solid medium. Optimal plating density for cell division and colony formation was in the range of 10 5 cells per ml. Colony formation took place in about 4–9% of the protoplasts. Survival of protoplasts was reduced to about one half after exposure to 0.3% ethyl methanesulphonate (EMS) for one hour. Exposure of protoplasts to X-ray radiation resulted in half-survival at about 3400 R. Embryoid regeneration was promoted in callus from X-irradiated protoplasts.