Agriculture Reference
In-Depth Information
For all these reasons, it would be valuable to
eliminate selection genes from the fi nal trans-
formed plant. Several strategies have been
employed to achieve this goal. To increase the
likelihood of separate integration sites, the selec-
tion genes and genes of interest can be trans-
formed on separate DNAs in biolistic experiments
or on separate T-DNAs housed on the same or
different binary plasmids for Agrobacterium trans-
formation. For the latter, experiments in rice and
tobacco have shown that the two T-DNAs inte-
grate at unlinked loci in up to about a quarter of
Agrobacterium transformants (Komari et al.,
1996). There are a few reports that suggest that
use of “clean genes” without plasmid backbones
for biolistic transformation increases the likeli-
hood of obtaining transgenics with selection genes
that can be segregated from other transgenes (Yao
et al., 2006; Gadaleta et al., 2008b). Another
method to eliminate selection genes is to fl ank
them with target sites for site-specifi c recombi-
nases. Srivastava et al. (1999) demonstrated that
Ubi1 :: bar transgenes fl anked by lox sites were
excised from their integration loci when plants
containing them were crossed to another transfor-
mant that expressed the cre recombinase. Segre-
gation of the cre transgene in the following
generation resulted in plants with neither Ubi1 ::
bar nor cre transgenes. A third strategy is to use
the mannose/ pmi or another metabolic-advantage
selection system. The pmi gene is only useful in
tissue culture and confers no advantage for plant
growth and fertility during fi eld propagation. A
fourth strategy is to use transformation methods
and genotypes that are so effi cient that transfor-
mants can be directly identifi ed by DNA screen-
ing without the aid of selection (Zhang et al.,
2006).
formants of wheat, but much is known from
experiments in the model plant Arabidopsis. In
the absence of selection, Kim et al. (2007) recov-
ered Arabidopsis transformants in all regions of
the genome, implying that integration is com-
pletely random. The use of selection genes to
identify Arabidopsis transformants results in
recovery of transformants with integration sites
mainly in gene-rich regions of the genome, where
expression of the selection gene is favored (Alonso
et al., 2003).
The lack of integration site predictability means
that a given genome site cannot be targeted a
second time using either Agrobacterium or biolis-
tic transformation. Thus, when traits require
expression of several different genes to be mani-
fested or when multiple different traits are desired
in a single background, all genes must be included
in the initial transformation to have some chance
of being linked. Otherwise, the different trans-
genes will have to be brought together by cross-
ing. Assembling multiple desirable traits in a
single genotype is a problem that breeders rou-
tinely face. Ow (2005) proposed a strategy for
using site-specifi c recombination to stack multi-
ple transgenes in a single location, but the effi -
ciency of such a process relative to random
integration is not yet known.
Inheritance anomalies
Many wheat transgenes exhibit normal inheri-
tance upon selfi ng of regenerated plants. However,
there have been several reports that some events
resulting from biolistic transformation exhibit
poor transmission of transgenes, as manifested in
fewer than expected transgenic progeny (Cannell
et al., 1999; Campbell et al., 2000; Rasco-Gaunt
et al., 2001), and even complete loss of transgenes
between generations in a minority of transformed
lines (Srivastava et al., 1996; Stoger et al., 1998;
Iser et al., 1999). Some investigators report occa-
sional rearrangements within loci between gener-
ations (Srivastava et al., 1996), while others noted
a lack of rearrangement over fi ve generations
(Demeke et al., 1999). The mechanism(s) by
which some transgenes are lost or rearranged is
not yet known. Poor or absent transmission could
Integration location
Transgene loci produced by biolistic transforma-
tion of wheat have been detected in most parts of
chromosomes, including telomeric (Color Plate
34), subtelomeric, intercalary, and centromeric
regions (Abranches et al., 2000; Jackson et al.,
2001). Little information is available for the
chromosomal locations of Agrobacterium trans-
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