Large Plant Samples: How to Process for GMA Embedding?

Abstract

It is often necessary to process large plant samples for light microscopy studies, but due to structural characteristics of plant tissues, especially intercellular spaces, large vacuoles, and phenolic substances, results are often unsatisfactory. When large samples are embedded in glycol methacrylate (GMA), their core may not polymerize, remaining soft and moist and making it difficult to cut microtome sections. This situation has been erroneously interpreted as the result of poor infiltration, when the soft core of these samples is actually the result of incomplete polymerization. While GMA is in fact present inside samples, unsatisfactory polymerization results from rapid external polymerization that does not allow sufficient hardener to reach the sample core, while the relatively large volume of GMA inside the tissue block also dilutes the hardener. In this chapter we propose a new method for processing large plant specimens that avoids these problems by: (1) slowing the polymerization process through cooling in order to permit the penetration of hardener into the sample core and (2) increasing the hardener:GMA ratio to aid polymerization of the sample core.

Stenospermy and seed development in the “Brazilian seedless” variety of sugar apple (Annona squamosa)

“…To prepare permanent slides, the material was dehydrated in a graded ethanol series and embedded in methacrylate (Leica ® ), according to the protocol of Paiva et al (2011). Serial longitudinal sections and cross-sections (6 µm thick) were prepared using a rotary microtome.…”

Leaf morphoanatomy of the medicinal <em>Maytenus imbricata</em> (Celastraceae): an ecological approach

“…A voucher specimen (MCTB Messias, 2565) was deposited at the OUPR Herbarium (Universidade Federal de Ouro Preto, Minas Gerais, Brazil). Samples of the median region of the leaf blade and the petiole were fixed in Karnovsky solution (Karnovsky 1965) for 12 hours, dehydrated in a graded ethanol series (Jensen 1962), and cold-embedded (Paiva et al 2011) in hydroxyethyl-methacrylate resin (Leica Microsystem Inc., Heidenbeg, Germany). Cross sections of the median rib, transverse and paradermal sections of the internerval region, and cross-sections of the margin and the petiole were obtained using a rotary microtome (Atago, Tokyo, Japan), stained with Toluidine blue pH 4.7, rutenium red (O'Brien et al 1964, modified), basic Fuchsin and Astra blue and mounted in acrylic resin (Itacril, Itaquaquecetuba, Brazil).…”

IMPACT OF PACLOBUTRAZOL ON THE GROWTH AND DEVELOPMENT OF NURSERY GROWN CLONAL OIL PALM (Elaeis guineensis Jacq.)

“…Plant samples of each treatment were fixed in glutaraldehydeparaformaldehyde-caffeine, tissue was dehydrated using an ethanol series (Elmar and Frank, 2010;Paiva et al, 2011;Kok et al, 2015), before infiltration and embedding in Resin LKB kit (Technovit 7100). Sections were cut using a rotary microtome (Leica system), and then stained with periodic acid, Schiff's reagent in dark, and 80% (w/v) of naphtol blueblack solution.…”