<i>In‐situ</i> enzyme histochemistry on plastic‐embedded plant material. The development of an artefact‐free β‐glucuronidase assay

Abstract

Summary An in‐situ enzyme histochemical method is described that preserves the tissue and cell structure as well as the enzyme activities. Flower buds at different developmental stages from wild‐type and transgenic Brassica napus plants, the latter containing the GUS gene under the control of a tapetum‐specific promoter, were used as starting material. The method is based on the following principles: processing of the tissue on crushed ice, no fixation but a pretreatment with spermidine, partial dehydration with acetone, and a final embedding in a water‐miscible glycol methacrylate resin at 5°C. This method was used to set up a sensitive p‐glucuronidase histochemical assay with a high resolution. A succinate dehydrogenase assay was included as a control for tissue and cell viability as well as a standard for the relative metabolic activities between different tissues and cell types.