Agriculture Reference
In-Depth Information
selection methods (Huber et al., 2002) or two
screening methods (Doshi et al., 2007).
encoded phenotypes in such plants may give an
overestimate of transformation effi ciency, because
these methods can detect nonintegrated genes or
transient gene expression, respectively. This is
particularly important when plant tissues have
been incubated with
Agrobacterium
, which can
persist within regenerating plants and give false
positives in PCR screening.
For both bread and durum wheat, biolistic
transformation effi ciency varies greatly among
cultivars (Takumi and Shimada 1997; Iser et al.,
1999; Rasco-Gaunt et al., 2001; Varshney and
Altpeter 2001; Pellegrineschi et al., 2002a,b).
Other factors implicated in transformation effi -
ciency are donor plant health (Harvey et al.,
1999; Pellegrineschi et al., 2002a), donor plant
age (Pastori et al., 2001), the type of explant
(Cheng et al., 2003), and the selection system
used (Ortiz et al., 1996; Witrzens et al., 1998;
Cheng et al., 2003; Przetakiewicz et al., 2004).
DNA delivery is generally not a limiting factor
for stable transformation, and the conditions for
optimal transient expression are not necessarily
the same as those for the highest effi ciencies of
stable transformation, both for the biolistics
(Altpeter et al., 1996a) and
Agrobacterium
(Wu
et al., 2003) approaches. The highest reported
effi ciencies for bombardment were 20% when
the
pmi
selection method was used (Reed et al.,
2001; Wright et al., 2001) and 70% when a super-
transformable line of 'Bobwhite' was used (Pel-
legrineschi et al., 2002b). The effi ciency of the
fi rst
Agrobacterium
transformation was 1.1% to
1.6% (Cheng et al., 1997), but recent improve-
ments in the method have increased it to as high
as 10% with 3% to 5% being typical (Huber et
al., 2002; Cheng et al., 2003; Chugh and Khurana
2003a; Hu et al., 2003; Khanna and Daggard
2003; Wu et al., 2003).
Regeneration of fertile plants
The fourth process in plant transformation is
the regeneration of cells containing newly inte-
grated DNA into a fertile plant that can pass
the transgene to its progeny as part of its
chromosomes.
Shortly after
in vitro
culture, some of the cells
in the original explant begin to divide and form
callus tissue. It is at this stage that the DNA is
introduced. The callus cells continue dividing
and during 2 to 6 weeks of culture in the dark,
clusters of cells begin to differentiate into somatic
embryos. During this period after
Agrobacterium
transformation, the media include antibiotics to
inhibit bacterial growth. Transfer of the embryo-
genic calli to media with reduced auxin levels and
incubation under light/dark conditions induces
the somatic embryos to differentiate shoots
(Color Plate 32d). The regeneration media
includes the selection agent to favor transformed
shoots and, for
Agrobacterium
transformation,
antibiotics that inhibit bacterial growth. Shoots
are subsequently transferred to selection media
with compositions that favor formation of roots
(Color Plate 32d). The rooted plantlet can then
be transferred to a potting medium, and after 7
to 10 days of acclimation to lower humidity, to a
greenhouse.
Effi ciency of wheat transformation
The typical effi ciencies with which stable wheat
transformants are recovered vary from 1% to 5%.
Effi ciencies are usually reported as the percentage
of individual starting explants (e.g., cultured
wheat embryos) that give rise to transformed
plants (T
0
), although some authors report effi -
ciencies as the percentage of explants that give
rise to stably transformed callus lines. The number
of integrative transformants is most accurately
assessed by Southern blot analysis of DNA from
regenerated T
0
plants, or by inheritance of DNA
or transgene-encoded phenotypes. Use of PCR
on DNA from T
0
plants or assays for transgene-
APPLICATIONS OF WHEAT
TRANSFORMATION
Although the ultimate goal of wheat transforma-
tion is to improve the crop's agronomic and uti-
lization properties, the objectives of individual
experiments depend on current genetic under-
