The use of fluorescent dyes in the microinjection of alfalfa protoplasts

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Abstract
A major obstacle in the development of microinjection technology for plant protoplasts has been the poor resolution of the intracellular compartments by conventional light microscopy. This was overcome through the use of fluorescent stains. The nuclei of living alfalfa (Medicago sativa) protoplasts were stained specifically with both Hoechst 33258 and 4'',6-diamidino-2-phenylindole. Mitochondria were stained specifically with 3,3''-diethyloxadicarbocyanine iodide and rhodamine 123. Cytoplasm was stained selectively with fluorescein diacetate. Under the specific conditions employed, the stains did not retard or suppress DNA synthesis, mitosis, cell division, or microcolony formation. Intranuclear microinjection did not significantly inhibit cell division in protoplasts stained with Hoechst 33258. Furthermore, the pattern of protein synthesis remained constant during the early stages of protoplast culture and was not perturbed by staining. These combined results demonstrated the suitability of alfalfa protoplasts stained with fluorescent dyes as hosts for microinjection.