Use of polyethylene glycol as an alternative to optimal cutting temperature medium in freeze sectioning for plant histochemical studies.

Abstract

Plant anatomical and histochemical studies are concerned with the structural organization of tissues as well as localization of various metabolites and enzyme activity inside cells and tissues. Traditionally, rotary microtomes are used for paraffin and resin-embedded samples which provide excellent preservation of tissue morphology but removes enzymes, lipid components, and various specialized metabolites. Freeze sectioning apparently remained unexplored in plant histology because of the presence of rigid cell walls and highly vacuolated cytoplasm in plant tissues. In this study, we have described a simple cryostat-based sectioning technique using polyethylene glycol (PEG) as embedding medium after glycerol infiltration that protects the plant tissues from freezing and thawing damage. We have also compared the suitability of inexpensive aqueous PEG solution as compared to commercially available optimal cutting temperature (OCT) medium and obtained identical microscopic images. Diverse plant organs from different genera were sectioned to check the application of this method in plant anatomical studies. In all the cases, cross sections were shown to be well preserved similar to paraffin-embedded plant tissues. In addition, histochemical analyses showed retention of metabolites and even enzymes in the tissues, which can make this method an alternate choice in cryo-microtomy replacing the expensive OCT medium.