Substrate Infiltration and Histological Fixatives Affect the In Situ Localisation of β‐Glucuronidase Activity in Transgenic Tissues

Abstract

AbstractThe β‐glucuronidase (GUS) gene is a widely used reporter gene in transgenic research. This study shows that although histochemical localisation of GUS activity may be very specific, differences in incubation conditions and tissue status can lead to artificial localisations that are independent of gene activity. The objective of the current studies was to evaluate the factors that affect the in‐situ localisation of β‐glucuronidase using transgenic tobacco plants as model tissues. The aspects considered include tissue size as well as and the addition of surfactants, vacuum infiltration and chemical fixatives. Transgenic tobacco plants exhibited variable staining patterns dependent on the size of tissue assayed and the treatments that affected the infiltration of substrate. A gradient of blue staining was observed in larger tissue pieces (10 mm2), where staining in central areas was light blue in contrast to edges, which stained deep indigo. More intense staining was associated with peripheral cell layers and regions adjacent to leaf veins. Thinner tissue strips incubated under similar conditions exhibited intense and even X‐Gluc staining. Addition of Triton X‐100 (1%) surfactant and vacuum infiltration (2 min) produced considerably quicker and more uniform staining (intense and consistent indigo blue colour) of the examined tissue after a 4 to 6‐h incubation. Chemical fixation of tissues before GUS assay resulted in quantitative and histochemical differences in enzyme activity that were dependent on the fixative type and duration. Quantitative measurements using the MUG fluorometric assay showed that Histochoice™ provided the highest retention of GUS activity, maintaining more than 80 and 50% of the activity after fixation for 15 and 30 min, respectively. Activity in decreasing order was obtained with paraformaldehyde, glutaraldehyde, ethanol and FAA. GUS activity was affected not only by the type of fixative, but also by the duration of fixation with longer fixation producing lower GUS activity. From the experiments performed it can be concluded that those treatments that enhance substrate penetration, i.e., the addition of surfactant and vacuum infiltration, improve the consistency and speed of X‐Gluc staining.