Surface labelling of plant protoplasts with fluorochromes for discrimination of heterokaryons by microscopy and flow cytometry

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Abstract

Surface labelling of plant protoplasts was tested for use in mass fusion systems and heterokaryon detection. Parameters have been established for biotinylation and subsequent incubation with avidin-coupled fluorochromes. The procedure is rapid (less than 3 hours) and does not affect viability. Fusion responses were the same as with unlabelled protoplasts. From a range of fluorochromes tested, fluorescein and phycoerythrin proved best suited for detection experiments with protoplasts of both suspension and leaf origin. With this standard combination of labels, as applied in experiments with animal cells, heterokaryons from fused plant protoplasts could clearly be discriminated from other protoplasts by means of fluorescence microscopy or flow cytometry with a single combination of filters and wavelengths.

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