Plant Protoplast Enucleation by Density Gradient Centrifugation

A number of different methods have been developed for the enucleation of cells, particularly mammalian cells. Centrifugation of cells adhering to a surface may lead to the nucleus being drawn out from the cytoplasm, resulting in the formation of an enucleated cell or cytoplast and a nucleus surrounded by a thin layer of cytoplasm bounded by a plasma membrane (mini-cell). In those cases where cells do not adhere sufficiently firmly to a surface, enucleation has been achieved by centrifugation in density gradients. This method takes advantage of the differing densities of the nucleus and the cytoplasm. When cells are centrifuged at high speed in a suitable density gradient, the nucleus and the cytoplasm tend to float in those regions of the gradient matching their own densities. If the distance between the regions with densities corresponding to that of the cytoplasm and that of the nucleus is relatively long, considerable stretching forces are generated. Under optimum conditions, this force is usually sufficient to enucleate a high percentage of the cells. The use of density gradients and high-speed centrifugation for generating cytoplasts and mini-cells is much less explored in plant than in mammalian cells.