Biology Reference
In-Depth Information
Bright-field and epifluorescence techniques can be useful to study nec-
tary structure. Autofluorescence of chlorophyll can be used to highlight the
distribution of chloroplasts in the nectary and subnectary parenchyma (see
Fig. 5). Phenolic compounds, the lignin of xylem vessels, and cuticles can be
located by autofluorescence. Details of the cuticle can be highlighted using
Auramine O. When the fluorescence is strong enough, UV and visible light
of an appropriate intensity can be used simultaneously. This makes it possi-
ble to observe samples treated with conventional stains and fluorochromes at
the same time.
As far as electron microscopy is concerned, the zinc iodide-osmium
tetroxide (ZIO) method is suitable for general impregnation of the en-
domembrane system of many plant, algal, and fungal tissues. It has also been
used for staining subcellular compartments of the nectary (Machado &
Gregorio, 2001), where it facilitated observation of membranes and helped
to elucidate the role of nectary regions and cytoplasmic organelles in nectar
secretion.
Conventional chemical fixation can damage cell components and any re-
sults from studies using this technique must be considered with caution,
especially when applied to highly dynamic systems such as those operating
during nectar secretion. To overcome such problems, the freeze-substitution
technique was recently applied to the study of nectary ultrastructure and nec-
tar secretion (Robards & Stark, 1988; Zhu et al., 1997; Zhu & Hu, 2002;
Stpiczyńska et al., 2005b). According to Zhu et al. (1997), the membranes of
organelles, vacuoles, and nuclei showed less shrinkage than with chemical
fixation. With this technique, Robards and Stark (1988) observed an open
extracytoplasmic space external to all the cells of the secretory hairs of
Abutilon
. According to these authors, the endomembrane system of nectar
secretory cells is not appreciably affected by chemical fixation.
