Grass leaf stomata preparation techniques for scanning electron microscopy

Abstract

Studies on gramineous stomatal characteristics, such as stomatal density and aperture size, relate these traits to ecological concerns, e.g. drought tolerance and production. Techniques for studying stomata have been utilized in the past using light microscopy. In a comparative study of stomatal apertures, it is imperative that guard cell closure not occur and die cells retain as closely as possible their natural state at the time of collection. Two species utilized in seeding roadsides and pastures, Dactylis glomerata L. and Elymus canadensis L., were processed with four different techniques for the scanning electron microscope. Abaxial and adaxial surfaces, respectively, were examined.Leaf tissue was harvested from clonal greenhouse-grown plants and separated into four groups. One was air dried under ambient conditions. The second group was placed into a standard fixative. Following fixation, the samples were dehydrated through a standard series of ethanol and critical point dried. The third group was prepared the same as group 2, except the leaves were transferred to tetramethylsilane for two ten minute exchanges following ethanol dehydration.