Agriculture Reference
In-Depth Information
genes, they become resistant to the
antibiotics, and those antibiotics can then
no longer be used to cure the disease. No
evidence was found for the spread of the
resistance genes and no generally accepted
scientii c arguments can be provided to
underpin the concern in using antibiotic-
resistance selection markers, but as a result
of an increasing worldwide concern, several
other selection markers have been developed
for the selection of transgenic plants. One of
them allows the transformed cells to
metabolize a substrate that the wild-type
cells cannot use as an energy source, and as
a result, the transformed cells will grow out
of the mass of non-transformed cells.
into the genome of a plant cell
As discussed earlier, gene exchange between
any organisms
becomes possible by genetic
transformation. h is powerful tool enables
plant breeders to broaden the genetic
variation from which they can select in order
to obtain new combinations of genes,
leading to improved/adapted plant varieties.
In other words, genetic transformation
expands the possibilities for breeders beyond
the limitations imposed by traditional cross-
breeding and selection (see Fig. 2.1).
For most of the plant species, genetic
transformation is carried out on tissue
explants, of which a fraction of the cells is
competent for regeneration to complete
(fertile) plants after the transformation
process. h ere are no universally applicable
protocols for plant tissue culture, because
experience has shown that protocols need to
be optimized for each genus, species,
cultivar, ecotype and dif erent tissues used
for transformation.
h e following criteria need to be fuli lled
in order to set up a successful transformation
platform, and the dif erent steps are
discussed in the dif erent paragraphs below:
1.
Delivery of DNA to the plant genome
without inl uencing cell viability negatively.
2.
Selection of the transformants, the
selectable marker gene and promoter.
3.
Regeneration of intact plants.
4.
Transmission of the transgenes into the
next generations in fertile plants or stable
maintenance and expression in vegetatively
propagated crops.
In summary, transformation aims to create
heritable changes in the plant as the result
of the uptake and the stable integration of
introduced DNA in one of the plant
chromosomes.
Dif erent approaches have been studied
and developed to achieve DNA transfer into
plants. A common feature is that in all cases
the foreign DNA needs to enter the plant
cell. h erefore, the DNA should i rst
penetrate the cell wall and the plasma
membrane before reaching the nucleus and
transgenes
A plethora of dif erent ready-to-use cloning
vectors for introducing the constructed
transgene is available. h ese vectors are
replicating in
Escherichia coli
and in
Agrobacterium
and already contain one of
the available selectable markers or a marker
for visual scoring of the presence of an
expressed transgene (Fig. 2.3c). In a
multicloning site or in a Gateway cassette, a
fully assembled transgene can be introduced.
Alternatively, these vectors contain the
cloning site in between the regulatory
elements, such that the coding sequence for
the trait of interest can be inserted between
regulation elements of choice. For specii c
goals such as for silencing a plant gene, a
fragment of gene sequence is cloned in sense
and antisense direction in between the
regulatory elements, such that on tran-
scription, a self-complementary transcript
is formed and double-stranded RNA is
generated.
Once the cloning vector with the DNA
fragment to be transferred to the plant and
carrying the (trans)gene and selectable
marker is assembled, this vector is
transferred to a disarmed
A. tumefaciens
strain containing the molecular machinery
for transferring this DNA segment to the
plant.
