Agriculture Reference
In-Depth Information
10.4 Detection Method for Transformation
A transgenic plant is a plant in which one or more desired genes of foreign origin
have been artificially transferred and inserted into the host genome, instead of the
plant acquiring them naturally by crossbreeding or natural recombination events.
The origin of transgene can be from the same genus, from a different genus within
the same family, or even from a different kingdom (e.g., genetically modified Bt
cotton, which produces the natural insecticide, contains a gene from a bacterium
Bacillus thuringiensis ). The development in the field of biotechnology is moving at
a rapid pace, more transgenic varieties are emerging, and even the area under
cultivation of transgenic is increasing day by day. Genetically modified crops can
be identified either by detecting the insertion of a foreign gene at DNA level, or by
the detection of mRNA transcribed from the newly inserted gene, or by the
detection of a resulting protein, metabolite, or phenotype expressed by the foreign
gene. The polymerase chain reaction (PCR method) helps in the detection of the
inserted DNA, and immunological assays help in detecting the protein after their
expression in the resultant phenotype. Although much progress in the development
of genetic analysis methods is satisfactory, like those in PCR based, still other
analytical technologies are emerging to provide solutions to current technical issues
in the GM sample analysis. Emerging methods include the use of mass spectrom-
etry, chromatography, near-infrared spectroscopy, microfabricated devices, and, in
particular, DNA chip technology (microarrays) for GM sample analysis. The
PCR-based method has been found to be more reliable for the detection of GM
crop by the regulatory authorities.
10.4.1 Histochemical GUS Assay
GUS histochemical reporter gene assay is useful for the detection of transgenic
plants in plant molecular study; GUS fusion constructs are previously described
(Duchesne and Charest 1991 ; Sudan et al. 2006 ). GUS assay is available depending
on the type of substrate used, and the best substrate currently available for GUS
assay and demonstration of histochemical localization of
-glucuronidase activity
in tissues and cells is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc). This
substrate works by giving a blue precipitate at the site of an enzyme activity. The
product formed by glucuronidase action on X-Gluc is not colored itself. Instead, the
indoxyl derivative produced by the enzymatic action must undergo an oxidative
dimerization to form the insoluble and colored compound. Dimerization is enthused
by atmospheric oxygen and can be also enhanced by using an oxidation catalyst
such as a K+ ferricyanide/ferrocyanide mixture. Lacking a catalyst, the results are
often good, but one can have concern about the possibility of apparent localization
of glucuronidase. The procedure is as follows: (1) Take fresh leaves directly from
β
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