FLOW CYTOMETRIC QUANTIFICATION OF ELECTROPORATION-MEDIATED UPTAKE OF MACROMOLECULES INTO PLANT-PROTOPLASTS

Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplast s. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasm a membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to t he presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast popul ations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and tra nsient gene expression following plasmid uptake. Thus, simultaneous el ectroporation of protoplasts with foreign DNA and FITC-dextran followe d by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.