Anatomy and cytochemistry of abscission zones by microscopical examination of nitrocellulose tissue prints

Abstract

Tissue printing on nitrocellulose membranes has been used to localize extensin in soybean seeds and as a matrix for histochemical localization of enzymes in bean abscission zones. Pressing hand cut sections of abscission zones into nitrocellulose membranes results in a tissue print which is seen as a three dimensional image when viewed with side illumination and shows the characteristic morphology and anatomical organization of the tissues in these segments. Changes in the cells of the abscission layer can be observed on the tissue prints as the process of abscission proceeds, and treatment of the tissue prints with antibodies to abscission specific enzymes identifies the cells in the abscission layer.Ten day old explants of Phaseolus vulgaris c.v. red kidney were induced to abscise by removing the leaf blades. Tissue prints were made on dry nitrocellulose membranes (0.45 μm pore size) at zero time, 24, 48 and 53 h and observed using either transmitted or side illumination at 10X magnification. Some tissue prints were incubated with rabbit 9.5 cellulase antibody followed by exposure to an alkaline phosphatase conjugated-goat antirabbit antibody. Color was then developed using standard alkaline phosphatase substrates. The 9.5 cellulase is an abscission specific form of the enzyme.