Agriculture Reference
In-Depth Information
Figure 8.2
Regeneration Indian mustard (
Brassica juncea
) plantlets from callus tissues.
enrich, the cells that have been transformed. Com-
monly at present, this requires that the transforming
gene, or a gene that accompanies it, confers some selec-
tivity. The most usual selection agents up to now have
been:
antibiotics
(mainly kanamycin or hygromycin) or
herbicides
(i.e. glufosinate or glyphosate).
Transformation allows the insertion of foreign DNA
into the plant DNA of a few selectable cells. A method
must, of course, be available to obtain intact mature
plants from these single transformed cells. One of the
biggest barriers in transformation of a number crop
species is the inability to regenerate whole plants from
single cells
in vitro
. In many dicots, leaf disks are trans-
formed by infection with
A. tumefaciens
. Plantlets are
then regenerated by tissue culture methods from the leaf
disks. In many monocots, cultured cells, or embryos,
are transformed by a suitable DNA delivery system (e.g.
the particle gun) and intact plants are then regenerated
from transformed cells, again, in tissue culture. Other
methods that have had success are the transformation
of embryogenic cell cultures or protoplasts, followed by
regeneration of whole plants.
Once whole transformed plants have been produced
they need to be characterized. This may be achieved by
some or all of the following:
these techniques cannot indicate whether the gene is
successfully integrated and active
•
Southern blots to show the gene is present in the plant
genome, and/or to estimate number of gene copies
that have been inserted
•
Northern blots to detect mRNA from transgenes
•
Western blots or enzyme assays to show expression of
the trait in plant tissues
However, this is just the start since after such tests
have shown that transformed plants have the gene of
interest and that the gene is functional simply means it
is worth proceeding further. It then needs to be demon-
strated that the gene (and expression of the trait) is
stable, this generally means proving transmission of the
transgene through clonal or sexual generations in order
to show that any progeny will inherit and express the
gene. Subsequently, the testing begins to ascertain if the
expression of the gene has the desired effect on the phe-
notype, if any other characters are being affected directly
or by the transformation process and, of course, what
the actual field performance is.
Cautions and related issues
There have been a number of concerns that have arisen
over the past few years as the application plant trans-
formation technology has expanded and particularly as
•
Polymerize chain reaction (PCR) techniques can be
used to detect the presence of transgene, although
