A new and simple cryosectioning protocol for pollen analysis under light microscopy

Conventional plant microtechniques typically involve fixation, dehydration, and embedding in acrylic or epoxy resins followed by room temperature microtomy. Some cryomethods have been used for pollen grain analysis under light microscopy. However, these protocols do not allow for obtaining sections with similar or better quality compared to resin embedding methods. In this paper, a simple cryoprotocol for use in pollen studies under light microscopy is presented. Aldehyde-fixed anthers were cryoprotected in an aqueous sucrose solution and frozen in liquid nitrogen. A simple and inexpensive water-based gelatin–sucrose solution was used to support samples for frozen sectioning. With this new method, 1 μm thick sections were cut in a cryostat at -30 °C. The pollen grain sections showed cell structures with excellent preservation. Furthermore, different sublayers of the intine can be visualised with unprecedented clarity. The pollen cryostat sections have a higher level of quality than those of the resin embedding protocols at the same thickness. The main advantage to the method is the absence of artefacts, such as shrinkage and lipid extraction, caused by solvent dehydration and resin inclusion. Since the sections remain hydrated and free of embedding resins, the stain action is faster and more effective.