Agriculture Reference
In-Depth Information
case of co-transformation, i.e. when two
gene constructs are mixed for particle gun
bombardment or when two A. tumefaciens
strains, each containing a dif erent gene
construct, are used, there is a high chance
that both gene constructs are inserted in the
same locus of the genome of the acceptor
plant. h is has consequences, as in such a
case the two inserted traits will co-segregate
during crossing experiments (see Section
2.2.10).
h e advantage of using A. tumefaciens for
transgene DNA delivery is that most of the
integrated transgenes are present as intact
constructs and, as predicted, inserted as a
linear fragment from left to right T-DNA
end. Another advantage is that most
transformed cells contain only one or a
limited number of integrated T-DNA copies.
In the case of particle bombardment, more
severe rearrangements occur both in the
acceptor genome as well as in the trans-
ferred DNA segment. Moreover, frequently,
multiple copies of scrambled DNA fragments
of the transgene segment are observed in
tandem arrays at the locus of insertion. As it
has been shown that this can have an impact
on the expression of the transgene, and on
the stability of expression in subsequent
generations (because the repeated tandem
arrays become silenced), the A. tumefaciens
transfer method is, in general, the preferred
one.
non-transformed cells (see Section 2.2.3).
When a selectable marker is linked to the
gene of interest, cells that are transformed
have a selective advantage over the non-
transformed cells when grown on a medium
containing the selection agent. h e selection
agent can be an antibiotic when the
selectable marker is an antibiotic-resistance
marker (such as the nptII gene or the hpt
gene) or a herbicide when it is a herbicide-
resistance gene such as biolaphos acetyl-
transferase ( bar ). h e result of the selection
is that cells that do not integrate or do not
express the selectable marker will not
survive on the medium to which the select-
able agent is added. Without a selection
marker, the small number of plant cells that
have integrated the foreign DNA would be
lost within the huge population of regenerat-
ing wild-type cells. Another claimed
advantage is that the negative impact of
position ef ects is immediately counter-
selected since only the transformants that
express the selection marker well will
survive the selection, and as the selection
marker and the trait of interest are linked
and integrated together in the genome,
there is an indirect selection for good
expression of the linked transgene. However,
this is not always the case, and stable
expression of the transgene should be
controlled in several subsequent generations.
h ere are two major concerns raised by
some stakeholders on the use of antibiotic-
resistance markers: it might af ect the
therapeutic ei cacy of the clinical use of
these antibiotics and it might increase the
potential horizontal gene transfer from the
transgenic plant into intestinal and soil
bacteria. h e European Food Safety
Authority (EFSA) evaluated these concerns
and came to the conclusion that, according
to information currently available, adverse
ef ects on human health and the environ-
ment resulting from the transfer of the two
antibiotic resistance marker genes, nptII and
hpt , from GM plants to bacteria, associated
with the use of GM plants, are unlikely. h e
EFSA also stated that mainly the presence of
antibiotics in the environment and the
frequent use of antibiotics were the key
factors in driving the selection and
2.2.7 Identifi cation/selection of
transformants
Most transformation methods make use of
tissue culture to propagate plant cells from a
tissue explant with regenerative properties.
All the cells of the tissue explants are, in one
step, subjected to the transformation
process. h e result is that a fraction of these
cells will have taken up the foreign DNA, and
only a fraction of these cells will also
integrate the foreign DNA within the nuclear
genome.
In order to identify the plant cells that
have integrated the foreign gene, a selectable
marker is used or a marker that allows
transformed cells to be distinguished from
 
 
Search WWH ::




Custom Search