ISSN:
1615-6102
Keywords:
β-Glucuronidase
;
Cytoenzymology
;
CaMV 35S-GUS
;
Transgenic tobacco
;
X-ray microanalysis
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01384458
Permalink