10.6.2 Criteria to Consider: Whether My Plant Is Transgenic
Successful transgene introduction in plants can be confirmed in a number of different ways
(see the following chapter). Validation of transformation is based on either the presence of
foreign DNA in the plant genome or the expression of the transgene in the form of a new
enzyme or protein. Few of these validation methods are reliable on their own; often analysis
at a number of different levels is required. Some considerations for the main methods that
are used to confirm the transgenic nature of transgenic plants are described below.
10.6.2.1. Resistance Genes. One of the most common methods for false confirmation
of transgene expression is to evaluate plant tissues and seedlings for resistance to herbicides
(any compound that is toxic to plant tissues). Although herbicide resistance genes are
almost always used as a selective agent, the levels of herbicide used for selection are
often at the lower end of toxicity. This means that there is the possibility of allowing
escapes, which may not contain the transgene but could still survive in the presence of
the herbicide. It is rare that transformation experiments give rise to plant tissue and
plants that either grow unaffected or die in the presence of the herbicide. In most cases,
the recovered tissues show some yellowing or browning, indicating slight toxicity
effects. The ability for plant tissue to survive in the presence of toxic agents depends on
the density and vigor of the plant tissue, the medium used for growth of the target cells,
and the stability of the selective agent. Some selection systems that have been thoroughly
worked out and optimized may be very trustworthy. However, growth of tissues or seed-
lings on selective media is not enough to confirm the presence and expression of a herbicide
resistance transgene.
10.6.2.2. Marker Genes. Expression of marker genes results in the direct or indirect
formation of a product that can be either chemically analyzed or visually confirmed.
The most common marker genes are those that can be visualized. The presence of the
b-glucuronidase (GUS) enzyme encoded by the uidAgene is analyzed by placing
the plant tissue in the presence of an artificial substrate that is broken down by the
enzyme to yield a blue product. When the GUS enzyme is present, the tissues expressing
the transgene will turn blue. Often, the blue product is difficult to see in green plant tissues.
The chlorophyll can be removed from the tissue after treatment, for clarification. If the
solution containing the artificial substrate is incorrectly modified or the plant tissue is incu-
bated for too long, everything can turn blue, leading to false-positive results.
Another commonly used marker gene encodes the green fluorescent protein (GFP),
which emits a fluorescent green light, if the tissue expressing the gene is illuminated
with UV or high intensity blue light (Figs 10.5, 10.9, 10.10, 10.12). Special instrumentation
is needed to detect GFP, and filter sets are required. If black lights or UV lamps are used
without filter sets, detection of this fluorescent protein is impossible unless the
amounts of GFP protein are quite high (Fig. 10.5). The main problem in detecting GFP
in plants is the presence of other plant compounds that either interfere with detection or
fluoresce themselves. For example, chlorophyll fluoresces bright red under UV or strong
blue light. Waxes, materials in leaf hairs or trichomes, and even dirt on the leaves can flu-
oresce a similarly to GFP, and some filter sets can make everything resemble GFP
expression. The presence of the appropriate color for these marker genes must be carefully
268 TRANSGENIC PLANT PRODUCTION