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  • 1
    ISSN: 1615-6102
    Keywords: Barley aleurone ; Fluorescein diacetate ; Propidium iodide ; Protoplasts ; Viability determination ; Vital stains
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The utility of numerous dyes for determining the viability of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts was studied. Protoplasts isolated from the barley aleurone layer synthesize and secrete α-amylase isozymes in response to treatment with gibberellic acid (GA) and Ca2+. These cells also undergo dramatic morphological changes which eventually result in cell death. To monitor the viability of protoplasts during incubation in GA and Ca2+, several types of fluorescent and nonfluorescent dyes were tested. Evans blue and methylene blue were selected as nonfluorescent dyes. Living cells exclude Evans blue, but dead cells and cell debris stain blue. Both living and dead cells take up methylene blue, but living cells reduce the dye to its colorless form whereas dead cells and cell debris stain blue. The relatively low extinction coefficient of these dyes sometimes makes it difficult to distinguish blue-stained cells against a background of blue dye. Several types of fluorescent dyes were tested for their ability to differentially stain dead or living cells. Tinopal CBS-X, for example, stains only dead cells, and its high extinction coefficient allows its ultraviolet fluorescence to be recorded even when preparations are simultaneously illuminated with visible light. To double-stain protoplasts, the most effective stain was a combination of fluorescein diacetate (FDA) and propidium iodide (PI). By employing a double-exposure method to record the fluorescence from cells stained with both FDA and PI, dead and living cells could be distinguished on the basis of fluorochromasia.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1615-6102
    Keywords: Calcium ; Endomembrane system ; Enzyme secretion ; Freeze fracture ; Gibberellic acid ; Protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Freeze-fracture electron microscopy was used to study changes in the endomembrane system of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts. Protoplasts were used for this study because their response to calcium and the plant hormone gibberellic acid (Ga3) can be monitored prior to rapid freezing of cells for electron microscopy. Protoplasts incubated in Ga3 plus Ca2+ secrete elevated levels of a-amylase relative to cells incubated in Ga3 or Ca2+ alone. The endoplasmic reticulum (ER) and Golgi apparatus of protoplasts incubated in Ga3 plus Ca2+ undergo changes that are well correlated with the synthesis and secretion of a-amylase. The ER, which appears as short, single sheets of membrane in Ca2+-and Ga3-treated protoplasts, exists as a series of long fenestrated stacks of membranes following incubation in Ga3 plus Ca2+. The Golgi apparatus is also more highly developed in protoplasts treated with Ga3 plus Ca2+. This organelle is larger and has more vesicles associated with its periphery in protoplasts that actively secrete a-amylase. Evidence that the Golgi apparatus participates in a-amylase secretion is also provided by experiments with the ionophore monensin, which causes pronounced swelling of Golgi cisternae and inhibits the secretion of a-amylase. We interpret these observations as showing that the ER and Golgi apparatus of barley aleurone participate in the intracellular transport and secretion of a-amylase. The plasmalemma (PF face) of barley aleurone protoplasts shows a high density of intramembranous particles (IMPs) which, in general, are evenly distributed. Occasionally, ordered arrays of IMPs are observed, possibly resulting fro m osmotic stress. after 48 hours the plasmalemma of some Ga3-treated protoplasts show particle-free areas considered to be indications of senescence.
    Type of Medium: Electronic Resource
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