Isolation and characterization of cytoplasts and miniprotoplasts derived from protoplasts of cultured cells

We have isolated and partially characterized subprotoplasts containing nuclei, i.e. miniprotoplasts, and enucleated subprotoplasts, i.e. cytoplasts, from freshly isolated protoplasts of cultured cells from Hyoscyamus muticus, Nicotiana tabacum and especially Zea mays. Protoplasts were fragmentated by centrifugation through discontinuous iso-osmotic density gradients containing colloidal silaca gel (Percoll), calcium chloride and mannitol. Using this method metabolically active miniprotoplasts and highly purified cytoplast fractions with less than 4% contamination with nucleated protoplasts were obtained. The cytoplasts prepared by our method are suitable for use in fusion experiments aimed at transferring nuclear and cytoplasmic genetic information separately.