Agriculture Reference
In-Depth Information
the same site(s) in the genome (Pellegrineschi
et al., 2001; Rasco-Gaunt et al., 2001; Wright
et al., 2001; Permingeat et al., 2003). For
Agro-
bacterium
transformation, both gene cassettes
are placed on a single plasmid between the left
and right border sequences of the transferred
(T-) DNA (Fig. 18.1b). Unless there are rear-
rangements after the transfer to plant cells, all the
genes on a single T-DNA integrate together.
Integration into the host cell chromosomes
occurs sometime after entry of the DNA into the
cell. An informative experiment was reported by
Lonsdale et al. (1998), who followed expression
patterns of DNA introduced into wheat callus
cells by biolistics. The experiment employed a
luciferase gene, which encodes an enzyme that
emits light during conversion of its substrate to
its product (Ow et al., 1986). Transient luciferase
gene expression was visible in the wheat calli as
light emissions in many different cells shortly
after bombardment (similar to the GFP expres-
sion shown in Color Plate 32c). The emissions
peaked at 48 hours, after which they declined,
becoming undetectable 10 to 20 days after bom-
bardment. Around 30 days after bombardment, a
few new foci of gene expression could be seen;
presumably these were the descendents of single
cells that had stably incorporated the luciferase
gene. This experiment suggests that integration
can occur days or even weeks after the DNA is
introduced.
The process by which DNA integrates into the
host chromosomes is not well understood. Figure
18.1 shows the simplest structures that result
from biolistic (Fig. 18.1a) and
Agrobacterium
-
mediated (Fig. 18.1b) transformations. For
biolistics, using linear DNA to coat the particles
yields at least as many transformants as using
circular plasmid DNA (Uze et al., 1999; Yao
et al., 2006). Genes that originally are on differ-
ent circular plasmids usually end up co-inte-
grated at the same site (Fig. 18.1a bottom). For
example, Campbell et al. (2000) found that three
separate plasmids were cotransformed in 9 (36%)
of the 25 regenerated plants they assessed.
Cotransformation frequency for two separate
plasmids was 66% in 88 transgenic events studied
by Rasco-Gaunt et al. (2001) and 85% of 32
plants studied by Stoger et al. (1998). These
results suggest that, prior to integration, either
recombination occurs between circular plasmid
DNAs or the circles are linearized and then
ligated to one another by their ends. Kohli et al.
(2003) review the data on the integration struc-
tures that result from biolistic transformation
and discuss molecular mechanisms that could
account for them.
For
Agrobacterium
-mediated transformation,
the structure of the integrating DNA is better
understood (reviewed in McCullen and Binns
2006). In the presence of an inducer,
de novo
synthesis of the T-strand is initiated within the
bacterium, usually at single-stranded nicks, using
the T-DNA as template. There is evidence that
the T-strand synthesis is initiated at the right
border, proceeds in the 5' to 3' direction along the
T-DNA and usually terminates at the left border.
The displaced T-strand is coated with VirE2, a
single-stranded nucleic acid binding protein, and
the whole complex is transferred to plant cells,
where it integrates at one or two loci in the plant
genome (Fig. 18.1b).
Identifi cation of transformants
As can be seen by the transient expression of
GFP in Color Plate 32c, many target cells receive
and express DNA after transformation. However,
integration of the incoming DNA typically occurs
in only a few percent of these cells, necessitating
the use of selection methods to identify cells in
which the DNA persists and ultimately inte-
grates. Several selection systems have been suc-
cessful in favoring the preferential survival of
transformed cells during division and regenera-
tion (Table 18.1). Each system consists of a selec-
tive agent added to the media, used in conjunction
with a selection gene, usually from bacteria, that
confers an advantage to cells containing it on that
media. Most selection systems rely on a cytotoxic
agent, typically a herbicide or antibiotic. Cells
with the appropriate resistance genes either make
versions of the target that are insensitive to the
agent or detoxify the agent. Cells lacking the
resistance genes stop growing and eventually
die.
