Biomedical Engineering Reference
In-Depth Information
within the T-DNA region, the
sacB
marker for
negative selection in
E. coli
, interrupted by a poly-
linker for cloning foreign DNA.
One of the most attractive uses of high-capacity
binary vectors is for the positional cloning of genes
identified by mutation. The ability to introduce large
segments of DNA into the plant genome effect-
ively bridges the gap between genetic mapping and
sequencing, allowing the position of mutant genes
to be narrowed down by complementation. Genomic
libraries have been established for several plant
species in BIBAC2 and TAC vectors (Hamilton
et al.
1999, Shibata & Lui 2000) and a number of novel
genes have been isolated (e.g. Sawa
et al.
1999,
Kubo & Kakimoto 2001).
of hairy-root cultures is the difficulty involved in
scale-up, since each culture comprises a heterogen-
eous mass of interconnected tissue, with highly
uneven distribution (reviewed by Giri & Narassu
2000).
Many of the principles explained in the context of
disarmed Ti plasmids are applicable to Ri plasmids.
A cointegrate vector system has been developed
( Jensen
et al.
1986) and applied to the study of
nodulation in transgenic legumes. Van Sluys
et al.
(1987) have exploited the fact that
Agrobacterium
containing both an Ri plasmid and a disarmed Ti
plasmid can frequently cotransfer both plasmids. The
Ri plasmid induces hairy-root disease in recipient
Arabidopsis
and carrot cells, serving as a transforma-
tion marker for the cotransferred recombinant T-
DNA and allowing regeneration of intact plants. No
drug-resistance marker on the T-DNA is necessary
with this plasmid combination.
Agrobacterium rhizogenes
and
Ri plasmids
Agrobacterium rhizogenes
causes hairy-root disease
in plants and this is induced by root-inducing (Ri)
plasmids, which are analogous to the Ti plasmids of
A. tumefaciens
. The Ri T-DNA includes genes homo-
logous to the
iaaM
(tryptophan 2-mono-oxygenase)
and
iaaH
(indoleacetamide hydrolase) genes of
A.
tumefaciens.
Four other genes present in the Ri T-DNA
are named
rol
for
root locus.
Two of these,
rolB
and
rolC
, encode P-glucosidases able to hydrolyse indole-
and cytokine-
N
-glucosides.
A. rhizogenes
therefore
appears to alter plant physiology by releasing free
hormones from inactive or less active conjugated
forms (Estruch
et al.
1991a,b).
Ri plasmids are of interest from the point of view of
vector development, because opine-producing root
tissue induced by Ri plasmids in a variety of dicots
can be regenerated into whole plants by manipula-
tion of phytohormones in the culture medium. Ri T-
DNA is transmitted sexually by these plants and
affects a variety of morphological and physiological
traits, but does not in general appear deleterious.
The Ri plasmids therefore appear to be already
equivalent to disarmed Ti plasmids (Tepfer 1984).
Transformed roots can also be maintained as hairy-
root cultures, which have the potential to produce
certain valuable secondary metabolites at higher
levels than suspension cultures and are much more
genetically stable (Hamil
et al.
1987, Signs & Flores
1990). The major limitation for the commercial use
Direct DNA transfer to plants
Protoplast transformation
Until comparatively recently, the limited host range
of
A. tumefaciens
precluded its use for the genetic
manipulation of a large number of plant species,
including most monocots. At first, the only alternat-
ive to
Agrobacterium
-mediated transformation was
the introduction of DNA into protoplasts. This pro-
cess has much in common with the transfection
of animal cells (Chapter 10). The protoplasts must
initially be persuaded to take up DNA from their
surroundings, after which the DNA integrates stably
into the genome in a proportion of these transfected
cells. Gene transfer across the protoplast membrane
is promoted by a number of chemicals, of which
polyethylene glycol has become the most widely
used, due to the availability of simple transformation
protocols (Negrutiu
et al.
1987). Alternatively, DNA
uptake may be induced by electroporation, which
has also become a favoured technique (Shillito
et al.
1985). As with animal cells, the introduction of a
selectable marker gene along with the transgene of
interest is required for the identification of stable
transformants. This can be achieved using plasmid
vectors carrying both the marker and the transgene
of interest, but the use of separate vectors also results
