Principles of Plant Genetics and Breeding

236 CHAPTER 14
the non-destructive, visual identification of transgenic
cells by standard fluorescence microscopy.
The search for new selection systems
Antibiotic selection systems have been severely criticized
by activists for being environmentally risky.
Consequently, scientists are searching for new marker
systems with no perceived or real adverse environmental
consequences (so-called benign markers). Some of
these selection systems (called positive selection) are
based on metabolic pathways. An example is the mannose
selection system, where the phosphomannose
isomerase gene (pmi) is used as a selectable marker, and
mannose as the selecting agent. Transgenic cells have
a metabolic advantage over untransformed cells and
hence grow and develop better. Various approaches
are also used to select transformation events without
markers. These include cotransformation, site-specific
recombination, and use of transposable elements.
Confirmation of transformation
After selecting transformation events and culturing
cells into transgenic plants, it is necessary to confirm the
success of transformation in the putative transgenic
plants. Several methods are used, including phenotypic
assays, PCR analysis, enzyme assays, and Southern or
western blots. When A. rhizogenes is used, the transgenic
roots are very hairy.
Transgene integration into the host genome
Once transferred into a host cell, transgenes integrate
into the genome at sites located randomly throughout
the genome, but predominantly in transcriptionally
active regions. Further, as previously discussed, multiple
copies of the transgene may be incorporated into one
cell. The number of insertions at a locus may vary
between one and five, a factor that is influenced by
the methodology (e.g., the preparation and physical
status of the plasmid DNA (coiled, linear)), and the
concentration and amount of DNA delivered. However,
Agrobacterium-mediated transformation generally
produces fewer insertions per locus.
Transgene expression in transgenic plants
Once integrated into the genome, the success of the
project is determined not only by the stability of transformation,
but also by the desirability of expression.
In genetic engineering research, promoters are the
“engines” that drive DNA expression by determining the
level of transcription of a selectable coding sequence.
Sources of promoters include viruses, bacteria, and plants.
Promoters may be grouped into three major classes.
1 Constitutive promoters. As previously indicated,
some genes are “turned on” continuously, while
others are turned on as needed. In other words,
structural genes are transcribed either continuously
(constitutive expression) or periodically as the
gene product is required (regulated expression).
Constitutive promoters have high affinity for RNA
polymerase and consequently promote frequent transcription
of the adjacent region. This category of
promoters is often used to drive the expression of the
selectable marker gene for identification of transgenic
tissues in vitro. A widely used constitutive promoter
is derived from the cauliflower mosaic virus, and is
called CaMV 35S.
2 Tissue-specific and developmentally regulated
promoters. Certain genes are expressed only in
certain tissues (even though all cells are equally
genetically endowed). In animals, milk is expressed
only in the mammary glands. Similarly, some genes
need to be expressed only at certain developmental
stages, and hence need to be regulated for such
specific roles. In plants, genes for grain quality, for
example, should be targeted for expression in the
endosperm. Promoters for targeted gene expression
are available for plant tissues such as endosperm
(e.g., for storage protein), anther tapetum (e.g., for
engineering male sterility), embryo (e.g., for engineering
grain quality traits), and phloem (e.g., for
engineering pest resistance to sucking insects). Some
promoters are responsive to environmental stimuli
(e.g., light). Some genes need to be expressed only at
certain developmental stages, and hence need to be
regulated for such specific roles. The choice of a promoter
is made according to the trait being improved.
3 Inducible promoters. Some promoters are designed
to drive the expression of genes in response to injury.
For example, a transgene may be developed to trigger
the expression of a gene in response to pathogenic
invasion or wounding. Wound-induced promoters
have been isolated in species such as rice (e.g., the rice
basic chitinase, RC24, which drives the expression of
the uidA gene in wounded roots and stems).
Stability of transgene expression
There is no guarantee that once stably transferred
and expressed that the condition will be permanent.