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Quantitative cytochemistry ofβ-galactosidase in normal and enzyme deficient (gal) pollen ofBrassica campestris: Application of the indigogenic method

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Summary

The available cytochemical methods for localization of β-galactosidase have been evaluated using pollen grains ofBrassica campestris. β-Galactosidase-deficient pollen (gal), served as a control. Azo dye methods involving naphthyl substrates showed high and nonspecific background staining to the exine. The indigogenic method, employing 5-bromo-4-chloro-3-indoxyl β-d-galactoside (X-gal) as the enzyme substrate, gave specific opaque-blue final reaction pproduct, while mutant pollen grains remained colourless. Final reaction product formation was blocked byd-galactono-1,4-lactone, thus demonstrating the specificity of the enzyme reaction. Using microspectrophotometry, the absorbance of the final reaction product was found to be a linear function of incubation time and section thickness in cryostat sections up to 8 μm thick and was only slightly reduced by glutaraldehyde prefixation. The validity of the indigogenic method for quantitative analysis was confirmed by using an enzyme-containing polyacrylamide gel model system and enzyme-coupled Sepharose 4B beads. Cellular sites of enzymic activity have been determined using plastic sections: final reaction product occurred in the intine wal layer and peripheral cytoplasm

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  1. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3052, Parkville, Victoria, Australia

    M. B. Singh & R. B. Knox

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  1. M. B. Singh
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Singh, M.B., Knox, R.B. Quantitative cytochemistry ofβ-galactosidase in normal and enzyme deficient (gal) pollen ofBrassica campestris: Application of the indigogenic method. Histochem J 16, 1273–1296 (1984). https://doi.org/10.1007/BF01003726

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  • DOI: https://doi.org/10.1007/BF01003726

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