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Electron microscopy and X-ray microanalysis as tools for fine localization of the β-glucuronidase activity in transgenic plants harbouring the GUS reporter gene

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Summary

The GUS reporter gene encoding β-glucuronidase is very useful in various domains of plant genetic engineering. A method for ultrastructural detection of its activity was developed using 35S-GUS transgenic tobacco root tips. Short glutaraldehyde prefixation at 4°C preserved up to 70% enzyme activity and was followed by brief incubation in X-Glu, strong postfixations, then quick dehydration at low temperature before resin embedding. In these conditions, transgenic cells were well preserved and displayed electron dense indigo precipitates with a crystalline structure as shown by electron diffraction. Due to other dense structures in the tissues, controls of the nature of the reaction product (diX-indigo) were necessary. A first control was carried out by means of X-ray microanalysis in order to check the presence of bromine. Other controls, including incubated non-transformed tissues, non-incubated or boiled transgenic roots as well as transgenic samples incubated with the specific β-glucuronidase inhibitor, D-saccharic acid-1,4-lactone, were also carried out. The discussion points out the potential uses but also the limits of the method, non-specific localizations of the diX-indigo microcrystals being possible.

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Abbreviations

X-Glu:

5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid

diX-indigo:

5,5′-dibromo-4,4′-dichloro-indigo

MUG:

methyl umbelliferyl glucuronide

4-MU:

4-methyl umbelliferone

EDTA:

ethylene diamine tetraacetic acid disodium salt

PB:

phosphate buffer

CaMV:

cauliflower mosaic virus

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  1. Laboratoire C. E. M. V., Bâtiment N2 (Boîte 150), Université Pierre et Marie Curie, 4 place Jussieu, F-75252, Paris Cedex 05, France

    J. -C. Caissard, J. Rembur & Dominique Chriqui

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  1. J. -C. Caissard
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  2. J. Rembur
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  3. Dominique Chriqui
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Caissard, J.C., Rembur, J. & Chriqui, D. Electron microscopy and X-ray microanalysis as tools for fine localization of the β-glucuronidase activity in transgenic plants harbouring the GUS reporter gene. Protoplasma 170, 68–76 (1992). https://doi.org/10.1007/BF01384458

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