Methyl Green-Pyronin Staining in Germinated Fruit Tree Pollen: Methods to Enhance Stainability of the Nuclei and Chromosomes

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Brachet's method for the identification of tissue DNA and RNA is very specific because the nature of the stained material is confirmed by the enzymatic extraction. However, the small sized nuclei of pollen of fruit trees (Malus, Persica, Corylus) lose their affinity for methyl green after heating for 2 hours at 37°C with RNase or distilled water (as control). RNA cannot be extracted with cold perchloric acid, because of the low specificity of this extraction.
In contrast, treatment with glacial acetic acid or acetic anhydride for 18 hrs, following proper alcoholic dehydration, enhances the nuclear stainability. The recommended fixative is Navashin's, with increased concentration of acetic acid up to 30% (to remove mitochondria), for 3 hrs. After thorough washing and bleaching with mixture of equal parts saturated aqueous ammonium oxalate and 3% H₂O₂ the slides are incubated with either enzyme or distilled water at 37°C for 2 hrs and treated with glacial acetic acid or acetic anhydride. Staining with methyl green (B. D. H.) 0.3% (w/v) and pyronin (Merck) 0.25% (w/v) in acetate buffer at pH 4.7, is performed at 37°C for 2 hrs. Differentiation is achieved by dipping in ice cold distilled water followed by a 5-40 min rinse in two changes of t-butyl alcohol.
The slides are mounted in Euparal. The acetic anhydride treatment was shown to block the amino groups of basic proteins which, otherwise, would interfere with the DNA staining by methyl green.