The use of ultracryotomy to localize sites of activity of endogenous and exogenous enzymes in plant cells

Abstract

During the recent years ultracryotomy revealed as a technique suitable for the study of plant cells. The main advantages are that the tissues support the minimum of chemical modifications and that the sections retain enzymatic activities. On an other hand, since the sections are devoided of plastic embedding, problems of penetration are eliminated. The sections are hydrophilic and directly accessible to reactives Therefore it seems possible to perform a wide range of enzymatic attacks at this level.Both aspects were investigated for different cases previously studied with conventional methods and for which some technical problems remained unsolved. For example, the attack of wall components is difficult to perform, even when the specimens are embedded in more or less hydrophilic resins.Attempts were made on adult leaves of spinach and mung bean hypocotyls prepared for ultracryotomy according to Tokuyasu. The specimens were prefixed (lh, 4°C) in 1 - 3% glutaraldehyde, infused (10-20 min, 4°C) in 1. 5 M sucrose, frozen in liquid nitrogen or supercooled nitrogen and sectioned at - 80°C on a Sorvall MT2 microtome fitted with a cryokit FTS.