Agriculture Reference
In-Depth Information
dissemination of antibiotic-resistance genes.
Moreover, they found that the use of
antibiotic-resistance markers in transgenic
plants did not add a substantial additional
risk. Nevertheless, there is general agree-
ment that antibiotic-resistance genes should
be removed from transgenic plants on
commercialization.
h erefore, to end up i nally with
antibiotic-resistant, marker-free GMOs,
either an extra step is used to eliminate the
selectable marker gene from the inserted
DNA locus, or screening for DNA integration
is performed by using PCR on many
regenerated shoots in the absence of
selection.
To remove the selectable marker, a
technically simple approach is the use of
co-transformation with two separate
T-DNAs, of which one contains the transgene
of interest and the second contains the
selectable marker. In the case of unlinked
integrations of both T-DNAs, the marker
gene and the transgene of interest will
segregate in the next generation, and thus
marker-free GMO segregants can be
selected. Another approach to remove the
antibiotic selectable marker makes use of
site-specii c recombinases. When the
selectable marker is l anked by the specii c
recognition sequences, transient expression
of the site-specii c recombinase will result in
the removal of the DNA fragment containing
the marker gene from the integrated T-DNA
fragment. Best known is the Cre/Lox system.
However, it remains easier not to have to
use a selection marker. In experimental
conditions where the transformation fre-
quency is sui ciently high, screening for
uptake of DNA in the plant cells is feasible
by using PCR. In this function, shoots need
to be regenerated, allowing part of each
individual plant to be sampled in order to
extract genomic DNA for PCR analysis.
Plants for which a positive signal is obtained
in the PCR reaction have integrated the gene
of interest. However, this does not guarantee
expression of the trait. h erefore, the
transgene mRNA or the transgene-encoded
protein can be quantii ed.
For the herbicide-resistance marker, the
major concerns focus on gene l ow, develop-
ment of weediness and the potential toxic or
allergenic ef ects. Concerning gene l ow and
weediness, one should distinguish the
impact on the environment on the one hand
and agricultural management on the other
hand. In case a functional herbicide-
resistance gene is transferred to a wild
relative, or if seeds from the herbicide-
resistant crop are spread in nature, these
plants will not have a competitive advantage
over the natural population, as the selective
agent is not used in nature and is thus not a
problem. It is dif erent in an agricultural
context. Dif erent management systems are
available to cope with weeds, which can be
wild species that have become resistant
because of gene transfer or they can be
volunteers being, for example, the result of a
previous culture. h e concern about toxicity
and allergenicity has rather to do with the
use of the herbicide than with the trans-
formation event or the expression of the
transgene.
of an elite event
Once a set of transgenic plants has been
selected, the identii cation of the best
performing transgenic plant has to be
started, referred to as the elite event. h is is
a step-by-step process, and depending on
the goal, the trait, the experience and the
available infrastructure, the order of these
various steps can be dif erent. In general,
however, the criteria screened are the
integration pattern, the expression proi le
and localization of the insertion in the
genome.
To be useful for commercial application,
i rst the expression levels should be
sui cient in order to obtain the desired
phenotype. Second, a simple integration
pattern is desired, because complex
integration patterns will make the breeding
work with the selected event more
complicated and might in some particular
cases also lead to unstable expression in
successive generations.
It is important to mention here that the
primary transgenic plant will not be the