Small and hard seeds: a practical and inexpensive method to improve embedding techniques for light microscopy

Abstract

The traditional techniques employed for embedding plant samples are not suitable for the proper processing of small seeds with hard seed coats. Usually, these seeds are broken off from blocks during microtomy, which limits the technical success of this procedure. In this study, Melastomataceae seeds of 101 species were treated prior to embedding in historesin according to three treatments: (1) control, using the standard procedure; (2) fixation and subsequent softening with Franklin solution, glycerin and heating in a water bath; and (3) softening with Franklin solution and subsequent fixation (glycerin and heating in a water bath were also included). For Melastomataceae species, the second treatment provides the best results, and we were able to produce very good sections of the entire seed. Small hard seeds, similar to those found in Melastomataceae, are better embedded when they are softened after subjected to fixation. A combination of softening techniques is necessary to improve the embedding process and to obtain high-quality sections of the embedded samples. In this study, we established a practical, slightly toxic and inexpensive methodology to ensure good preparations for light microscopy that can be applied to a wide range of subjects related to seed science.

A new seed coat water-impermeability mechanism in Chaetostoma armatum (Melastomataceae): evolutionary and biogeographical implications of physiophysical dormancy

“…The samples were infiltrated in the refrigerator for 1 week and embedded in (2-hydroxyethyl)-methacrylate (Leica™) according to Paiva et al (2011). The mature seeds were fixed and softened according to Ribeiro and Oliveira (2014) using Franklin solution (Franklin, 1945), glycerin and heating in a water bath; the embedding was performed as previously described (Paiva et al ., 2011). Longitudinal and transverse sections (6 μm thickness) were stained with toluidine blue 0.05%, pH 4.7 (modified from O'Brien et al , 1964) and mounted in Entellan™.…”

Does seed coat structure modulate gut-passage effects on seed germination? Examples from Miconieae DC. (Melastomataceae)

“…The manually extracted and gut-passed seeds were fixed in formaldehyde-acetic acid-50% ethanol (Johansen, 1940) for 48 h and exposed to a vacuum. A sample of seeds was processed for light microscopy according to Ribeiro and Oliveira (2014), embedded in (2-hydroxyethyl)-methacrylate (Leica™, Heidelberg, Germany) and processed in a freezer to delay polymerization, as indicated by Paiva et al (2011). Eight-micrometre-thick transverse and longitudinal sections were obtained in a rotary microtome (Zeiss Hyrax M40, Microm GmbH, Walldorf, Germany).…”

Apomixia como estratégia reprodutiva em Microlicia D. Don (Microlicieae, Melastomataceae)

“…As amostras foram lavadas em tampão fosfato de sódio 0.1M, pH 7.2, desidratadas em série etanólica, estocadas em etanol a 70% (v/v) e posteriormente incluídas em resina plástica (Historesin, Leica) seguindo as especificações do fabricante. Antes da inclusão, as sementes foram hidratadas em série etanólica decrescente e amolecidas em solução de ácido acético glacial e peróxido de hidrogênio a 30% (1:1 v/v) em estufa à 60ºC por cerca de 12 horas(Ribeiro & Oliveira 2014). Em seguida, as sementes foram lavadas em água destilada e desidratadas em série etanólica para inclusão em resina plástica.…”