Agriculture Reference
In-Depth Information
engineering with important implications for crop pro-
duction. One route by which progress in engineering
insect resistance in transgenic plants has been achieved
is by using the genes of
Bacillus thuringiensis
(
B.t.
) that
produce insect toxins (so called
B.t. toxins
).
B
.
t
.isa
bacterium that produces a crystalline protein during
sporulation, which, when cleaved to the mature toxin
peptide, produces paralysis of the mouthparts of spe-
cific insects and so leads to their death. Thus is provides
a useful and selective means of insect control.
Transgenic tomato, tobacco and cotton containing
the B.t. gene exhibited tolerance to caterpillar pests in
laboratory testing. The level of insect damage in field tri-
als has been similarly encouraging where tomato plants
with the B.t. gene suffered no significant damage while
non-tolerant lines were completely defoliated by insect
pests. However, an overall strategy is needed to avoid
evolution of resistance to the toxin in the insects that are
being controlled. Also recent work is investigating the
effects on, for instance, ladybirds feeding on the aphids
feeding on the transgenic plants.
Significant resistance to tobacco mosaic virus (TMV)
infection, termed 'coat protein-mediated protection'
has been achieved by expressing only the coat pro-
tein gene of virus in transgenic plants. This approach
has produced similar results in transgenic tomato and
potato, although in some other cases a similar approach
seems not to be as effective and it has been suggested
that other genes, such as viral replicase genes, might pro-
vide an effective mechanism to control virus infection
in plants.
used vector has been
A. tumefaciens
.
Agrobacterium
tumefaciens
is the casual agent of crown gall disease
and produces tumorous crown galls on infected
plants. The utility of this bacterium as a gene transfer
system was first recognized when it was demonstrated
that the crown galls were actually produced as a
result of the transfer and introgression of genes from
the bacterium into the genome of the host plant
cells. (
Note
: a natural process already in existence of
introducing genetic material.)
Physical or mechanical DNA delivery systems have
been developed, and have been particularly popular
for monocots. The most common, at least initially, of
these systems involved electroporation of protoplasts
but now particle bombardment is regarded as having
wide applicability. Particle bombardment involves the
DNA to be introduced being carried through the cell
wall on the surface of a small (0.5 to 5.0
m) piece of
metal (often gold) particles that have been accelerated
to speeds of one to several hundred metres per second.
Particle bombardment has been used for gene transfer
into a variety of target tissues including pollen cells,
apices and reproductive organs.
µ
•
A suitable construct has to be created that includes:
A promoter region that is recognized by the host.
These may be:
◦
Constitutive promoters
such as the 35S of
cauliflower mosaic virus and
nos
from the nopa-
line synthase gene which switch the gene on in all
tissue
◦
Tissue specific promoters
such as those from
α
-amylase (specific to the aleurone), patatin
(specific to tubers) and phaseollin (specific to
cotyledons)
Process of plant transformation
Before plant transformation can be used successfully in
a plant breeding programme and cultivars are developed
using recombinant DNA techniques, the following have
to be in place.
◦
Inducible promoters
such as those from alcohol
dehydrogenase I (induced by anaerobiosis); and
chlorophyll
a
/
b
binding protein (induced by light)
A transcript terminator at the 3
end of the gene.
•
•
A desirable gene must be available for insertion into
the target host plant. Therefore a DNA clone of a gene
that it is believed will confer a particular expression
of the trait of interest must be developed or provided
to the breeding programme.
It might also be noted that breeders may have to re-
design the gene of interest to use codons that are the
ones more preferred by plants.
Irrespective of the delivery (vector) system used to
transform the gene into plants, foreign DNA will be
inserted into relatively few cells. A means, therefore,
must be available to select, or at least significantly
•
There must be a suitable mechanism to transfer the
gene to the target plant. In dicots (and also now
increasingly cereals and grasses) the most commonly
