Color Differential Staining of Nor-Associated Heterochromatic Segments Using Acridine Orange

A new staining technique has been evaluated for detecting heterochromatic segments accompanying nucleolus organizing regions (NORs). This technique essentially consists of C-banding followed by acridine orange staining. When the technique was applied to five species of plants, the NOR-associated heterochromatic segments (NOR H-segments) were differentiated from other segments of the chromosomes as regions emitting yellowish green fluorescence. An incubation of at least 30 min in hot 2 × SSC was required to make the NOR H-segments emit yellowish green fluorescence in Nothoicordum fragrans. Fluorescence on other heterocnromatic segments varied from reddish orange to bright yellow; euchromatic segments emitted orange or yellowish orange fluorescence. The technique permits identification of NOR H-segments throughout mitosis based on the characteristic fluorescent color.