Agriculture Reference
In-Depth Information
standing of the targeted trait. At the basic end of
the research spectrum are reverse genetic experi-
ments to confi rm the identity and characterize the
DNA sequences of wheat genes that control or
modulate specifi c traits. These approaches rely on
data provided by classical genetics and genomics
approaches. At the applied end of the spectrum,
biotechnology uses genes already well-character-
ized and/or expected to modify a trait, and aims
to either change existing wheat gene expression
levels or add genes that are new to wheat. One of
the great strengths of the biotechnology approach
is that one experiment can yield insights into
gene-controlled processes and produce plants
with new and useful properties that can be propa-
gated. In the following sections, we give several
examples of each of these applications, but fi rst
we discuss some general experimental design
considerations.
tion in all or nearly all tissue types at nearly
all stages of development under a wide variety
of environmental conditions. The most com-
monly used promoters for selection genes in
wheat are those from the maize (
Zea mays
L.)
ubiquitin (
Ubi1)
(Christensen et al., 1992; Stoger
et al., 1999b; Rooke et al., 2000) and rice actin
(
Act1)
genes (Zhang et al., 1991), and the 35
S
gene promoter from
Caulifl ower mosaic virus
(Becker et al., 1994). The
Ubi1
and
Act1
promot-
ers include the fi rst introns of their respective
genes. In these and many other genes, the fi rst
intron increases the levels of gene expression
relative to those of the promoter alone (Rasco-
Gaunt et al., 2003; Bourdon et al., 2004). The
native 35
S
promoter does not have an intron, so
a fi rst intron from a cereal gene, such as maize
ADH1
, is often included in transformation con-
structs to ensure high expression levels in wheat
(Rasco-Gaunt et al., 2003; Bourdon et al., 2004).
The use of constitutive promoters for selection
genes also gives the experimenter an easily
assayed phenotype—herbicide or antibiotic resis-
tance—with which to follow transgene inheri-
tance. The activity of the
Ubi1
promoter in
several different transgenic wheat tissues is visu-
alized using the GUS reporter gene in Color
Plate 33a-d.
For the genes of interest or candidate genes,
promoter choice can vary widely, depending on
the aims of the experimenter. By judicious selec-
tion of promoters, the experimenter can limit
transgene expression to only tissues or conditions
where it is needed. This conserves resources for
the transformed plant and ensures that the trans-
gene-encoded mRNAs and proteins do not have
unintended effects in nontarget tissues. For
example, applications aimed at changing wheat
seed properties can employ endosperm-specifi c
promoters, such as those for genes that encode a
high-molecular-weight glutenin subunit (HMW-
GS) (Lamacchia et al., 2001) or the puroindoline
proteins (Wiley et al., 2007). The activity of a
HMW-GS gene promoter in the endosperm of
transformed wheat plants segregating for a
HMW-GS::GUS transgene is shown in Color
Plate 33e. Transgenes aimed at controlling patho-
gens can employ promoters induced by infection.
Promoters
As described previously and depicted in Fig. 18.1,
a typical transformation experiment includes at
least two expression units that need to function in
plant cells: the selection gene and one or two
experimental or candidate genes. An important
consideration in designing transformation experi-
ments is the choice of promoter to drive expres-
sion of the transgene(s). With recombinant DNA
methods, the experimenter can fuse any promoter
to any coding sequence
in vitro
, thereby specify-
ing when and where a protein will be made in the
plant. Novel fusions of gene sequences made
in
vitro
are designated with double colons separating
their sections, that is, promoter::coding sequence::
transcription termination signal sequence. The
different segments can be from natural genes or
made synthetically.
Selection genes must be active during the
initial stages of cell proliferation and/or differ-
entiation in tissue culture and, if they are to be
used to identify transformants after regeneration,
they must be active in at least some tissues of
regenerated plants. In nearly all cases, selection
genes used in wheat transformation experiments
thus far have been controlled by constitutive
promoters, which drive high levels of transcrip-
