Agriculture Reference
In-Depth Information
variety that will be commercialized as such.
As stated initially, this primary transformant
allows a new trait to be introduced in the
breeding programme. h erefore, it is neces-
sary to check that the integrated fragment is
stable over successive generations. h en, the
transgene insert can be bred into dif erent
varieties.
In the regulatory process associated with
the commercial release of a transgenic plant,
the transgenic plant as such, and the
transgene locus in particular, needs to be
fully characterized (see Fig. 2.3). Commonly
used methods for molecular characterization
are PCR, Southern blot analysis and sequenc-
ing. h ese methods allow the amplii cation
and sequencing of the inserted fragment
and also the DNA region of the plant genome
l anking the inserted fragment. h ese
sequence data allow checking whether the
DNA fragments that are to be inserted are
indeed inserted in the expected con-
i guration. In other words, it allows checking
for deletions, insertions, repeats and
mutations that could have occurred during
the integration process. Based on the
sequence data, a search is made for putative
new open reading frames and whether these
potentially newly formed open reading
frames show homology to known toxic or
allergenic proteins or peptides.
With Southern blotting, the structure of
the inserted DNA can be unravelled. h is
analysis allows searching for multiple
insertions, direct and indirect repeats of the
transferred DNA fragment. Southern
blotting is also used to scan the whole
transgenic genome with a probe of the
transferred DNA for potential secondary
inserted small fragments. Also, with a probe
of vector DNA, the absence of vector
backbone sequences is controlled, as these
vector backbone sequences may contain an
antibiotic-resistance gene and prokaryotic
origins of replication, which is unnecessary
and not wanted.
In the past, Northern blotting and at
present quantitative PCR are used to
measure the transgene-derived mRNA
accumulation levels in dif erent parts of the
transgenic plant. Western blotting and
ELISA are used to check whether translation
into proteins occurs. Western blotting also
allows the size of the recombinant protein
produced to be checked. For some transgene-
encoded enzymes, functional assays are also
available to check accumulation and the
specii c activity.
Risk evaluation for food/feed use and
environmental safety of GM plants is much
broader than characterizing the transgene
insert at the molecular level. Also, the
impact on the nutritional value and the
potential toxic and allergenic ef ects are
analysed and evaluated (see Chapters 3-8).
In the case of plants of the so-called i rst
generation (plants with input traits), the
evaluation is based on studies of substantial
equivalence with the original plant (see
Chapters 5 and 6). In these studies, the
choice of reference material with which the
GMO is compared is very important. h e
primary transformant could be compared
with the wild-type plant from which it is
derived, but also a null segregant progeny
plant of the transformant can be used as a
reference/negative control. However, in real
life these comparative studies are carried
out on material that is harvested from
plants that are the result of crossing and
backcrossing the primary transgenic plant
with a plant with a genetic background that
has a potential economic value. In that case,
the original plant is not the right
comparator. h e ideal comparator is the
plant with a genetic background that is as
identical as possible to the genetic
background of the GMO plant that is being
studied. Ideally, it is the isogenic material of
the GMO plant that is the subject of the
comparative analysis. When this material is
not available, an alternative is to compare
the dif erent parameters in the function of
the genetic variation that is at that moment
representative of the genetic background at
the basis of the commercialized GM
varieties.
