Quantitative cytochemistry of beta-galactosidase in normal and enzyme deficient (gal) pollen of Brassica campestris: application of the indigogenic method.

Abstract

The available cytochemical methods for localization of beta-galactosidase have been evaluated using pollen grains of Brassica campestris. beta-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 beta-D-galactoside (X-gal) as the enzyme substrate, gave specific opaque-blue final reaction product, while mutant pollen grains remained colourless. Final reaction product formation was blocked by D-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 micron 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 wall layer and peripheral cytoplasm.