Preparation of Bryophyte Herbarium Specimens for the SEM Using Aerosol OT Solution in Combination with FDA Rapid Dehydration

Abstract

Aerosol OT solution as a softener for herbarium specimens of bryophytes in combination with FDA (formaldehyde-dimethyl-acetal) rapid dehydration was tested for use in SEM preparation. Even the extremely fragile tissues of the gametophyte-sporophyte junction in liverworts and mosses (especially the sporophyte and gametophyte placental cells) show excellent preservation of anatomical and micromorphological structures after this treatment. The technique is proposed when living material is lacking. In the past, Aerosol OT has been used as a softening agent for reconstruction of herbarium specimens of different plant groups including seed plants and ferns (Ayensu 1967; Peterson et al. 1978). Its neutral chemical reaction and the efficiency in penetration of dried tissues without disruption of cells are clear advantages when compared to softening agents such as KOH or NaOH where boiling is often needed (Bruhl & Ashford 1986; Cunningham 1969; Peterson et al. 1978). Other methods need a rather long rehydration time (Furness 1995). Dehydration for electron microscopy (TEM or SEM) can be done by amyl acetate series (Horton 1982; Vitt 1983), but mostly a graded series of ethanol is used where changes every 30 to 60 minutes are necessary. But even 100% ethanol contains substantial amounts of water, from which it can be separated only by time consuming fractional distillation (Boyde et al. 1981; Boyde & Tamarin 1984). In addition to physical dehydration it is also possible to dehydrate material by use of chemicals that react with the water. Acidified dimethoxypropane (DMP) is reported for use in TEM (Muller & Jacks 1975; Postek & Tucker 1976), whereas dimethoxymethane (FDA = formaldehyde-dimethyl-acetal) is proved a useful agent for rapid chemical dehydration for SEM (Gerstberger & Leins 1978). In plant systematic research and structural botany, it is often necessary to utilize herbarium specimens, due to the lack of living material. Therefore we have tested a new combination of Aerosol OT solution followed by FDA-dehydration. Investigated objects were gametophyte-sporophyte junctions of bryophyte herbarium specimens. MATERIALS AND METHODS Material examined.-Brachythecium velutinumn (Hedw.) B.S.G. (Brachytheciaceae): GERMANY. SACHSEN. ElbhtiFIGURES 1-4. SEM micrographs of longitudinal razor-sectioned gametophyte-sporophyte junctions in mosses. 1-2. Brvumn elegans. 1. Elongate foot with surrounding gametophytic stern tissue, including vaginula. 2. Detail of the sporophytic transfer cells and gametophytic cells (notice not only the sporophytic cells, with wall ingrowths, but also the gametophytic cells are not collapsed). 3. Brachvthecium velutinum. Short side branch with elongate foot and surrounding gametophytic stem tissue, main axis on right. 4. Drepanocladus uncinatus. Foot tip. Wall ingrowths visible in the outermost sporophytic cell layer (notice the thin-walled gametophytic cells are not collapsed). Scale bars: 1, 3 = 250 rpm; 2, 4 = 25 pm. Abbreviations: C--collar, F-foot, G-gametophyte, IS--intraplacental space, S-sporophyte, Se--Seta, V-vaginula, W-wall ingrowths. 0007-2745/96/385-389$0.65/0 This content downloaded from 157.55.39.78 on Mon, 20 Jun 2016 07:25:33 UTC All use subject to http://about.jstor.org/terms 386 THE BRYOLOGIST [VOL. 99