Agriculture Reference
In-Depth Information
in regions (usually surfaces) that are accessible to
and viable after DNA introduction. In wheat, the
most commonly used targets for transformation
are scutellar cells from immature embryos that
are typically 10 to 15 days after anthesis (early
soft-dough stage, Feekes scale 11.2; Large 1954).
These cells can be transformed immediately upon
culture or, more commonly, after 2 to 6 days of
precultivation to form embryogenic calli (Weeks
et al., 1993, and many subsequent reports). Color
Plate 32a illustrates the processes for selection
and culture of immature embryos: searching
wheat plants for heads at the appropriate devel-
opmental stage, surface sterilization of immature
caryopses, and aseptic excision of embryos. For
some genotypes, it is important to prevent germi-
nation of the zygotic embryo by removing the
embryo axis before culturing the scutellar cells
(Nehra et al., 1994; Li et al., 2003a,b). For other
genotypes, the embryo is placed in contact with
the medium. In all cases, the media contain 2,4-
dichlorophenoxyacetic acid, an auxin that inhibits
germination and promotes dedifferentiation and
division of scutellar cells into embryogenic calli.
The culture media also contain carbon and
nitrogen sources for heterotrophic growth in the
dark.
Another explant shown to be useful for trans-
formation of many cultivars of both durum and
bread wheat is the immature infl orescence, from
which meristematic regions can be triggered to
proliferate in culture (Rasco-Gaunt and Barcelo
1999; Lamacchia et al., 2001). Other wheat
explants used for transformation include 5- to
7-month-old embryogenic callus cultures (Vasil
et al., 1992); protoplasts from embryogenic sus-
pension cultures (He et al., 1994); germinating
pollen tubes (Chong et al., 1998; Sawahel and
Hassan 2002); anther cultures (Brisibe et al.,
2000); shoot apical meristems (Ahmad et al.,
2002; Sticklen and Oraby 2005; Zhao et al., 2006);
leaf basal segments (Chugh and Khurana 2003b);
mature embryos (Supartana et al., 2006) and
calli derived from them (Patnaik et al., 2006).
The usefulness of these tissues and their amena-
bility to
in vitro
culture depends strongly on
genotype, primarily because of varietal differ-
ences in wound responses to methods of
DNA introduction (Parrott et al., 2002), and in
competence for regeneration into fertile plants
via a defi ned tissue culture regimen (Takumi and
Shimada 1997; Iser et al., 1999; Pellegrineschi et
al., 2002b).
DNA delivery methods and integration
There are many ways by which DNA has been
introduced into wheat cells, including bombard-
ment with DNA-coated metal particles termed
biolistics (Vasil et al., 1992, and many others
subsequently); cocultivation with
Agrobacterium
,
a bacterium with a natural ability to conjugate
DNA to plant cells (Cheng et al., 1997, and many
others subsequently); permeabilization by elec-
tric current (electroporation)(He et al., 1994; He
and Lazzeri 1998; Sorokin et al., 2000); vortexing
with silicon carbide fi bers or whiskers (Sawahel
and Saker 1997; Brisibe et al., 2000); combina-
tion of
Agrobacterium
and silicon fi ber wounding
(Singh and Chawla 1999); laser micropuncture
(Badr et al., 2005); uptake by germinating pollen
tubes (Chong et al., 1998, and others subse-
quently); and microinjection (Simmonds
et al., 1992; Ponya et al., 1999). Among these,
Agrobacterium
and biolistics have reproducibly
yielded transformed wheat plants in several
laboratories.
For
Agrobacterium
-mediated transformation,
bacteria cocultivated with immature embryos
(Color Plate 32b) transfer DNA into plant cells in
discrete, protein-coated units called T-DNAs,
which are delimited by left and right border
sequences (McCullen and Binns 2006). The
biolistics method consists of coating plasmid
DNA onto tungsten or gold particles and propel-
ling the coated particles through the cell wall with
the force of a pressure pulse of helium gas (Klein
et al., 1987), exploding gun powder (Sanford
1988), or electric discharge (McCabe and
Christou 1993). Color Plate 32c shows some of
the components of a biolistics transformation
experiment: the helium-driven commercially
available BioRad PDS 1000/He particle-delivery
device (Kikkert 1993), immature embryos on agar
