Enucleation of protoplasts derived from suspension cultures of winged bean and from a crown gall cell line ofParthenocissus tricuspidata

Protoplasts derived from suspension cultures of the winged bean and a crown gall lineof Parthenocissus tricuspidata were enucleated by centrifugation on iso-osmotic discontinuous Percoli gradients and on discontinuous sucrose-mannitol density gradients. Enucleation was achieved according to the buoyant density of the protoplasts and preparations of both cytoplasts and miniprotoplasts were recovered. The Percoll gradients gave more satisfactory results than the sucrose-mannitol gradients. Enucleation was increased by raising the centrifugal force from 20 kg to 60 kg. The presence of nuclei was determined by the DNA specific probe 4′6-diamidino-2-phenylindole (DAPI). Membrane integrity, as measured by staining with fluorescein diacetate, showed that some 85 % of the cytoplasts were bounded by an intact plasma membrane.