Agriculture Reference
In-Depth Information
integrating into the nuclear genome. In
other words, the main goal of each of these
methods is to transport the new gene(s) and
deliver them into the nucleus of a cell
without killing it.
Currently, there are two major techniques
for transferring foreign DNA into an
organism:
h e i rst method is based on the indirect
physical transfer of foreign genes into
target plant cells. h e method used
mostly is particle bombardment
(biolistics). DNA is bound to tiny particles
of gold or tungsten, which are sub-
sequently shot into plant tissue or single
plant cells under high pressure. h e
accelerated particles penetrate both the
cell wall and the membranes. h e DNA
separates from the metal and is
integrated into the plant genome inside
the nucleus. Endogenously present DNA
repair mechanisms play an important
role in the stable integration of the
foreign DNA into the genome. h is
method has been applied successfully for
many cultivated crops, especially mono-
cots like wheat or maize, for which
transformation using
A. tumefaciens
has
been less successful. h e major dis-
advantage of this procedure is that
serious damage can be done to the
cellular tissue.
h e second method is making use of the
'machinery' of
A. tumefaciens
, which has
the capacity to transfer DNA and proteins
into the plant cell.
A. tumefaciens
is a
soil bacterium and a natural plant
parasite. To create a suitable environ-
ment for themselves, these
Agro bacteria
insert part of their genes into plant
hosts, resulting in crown galls, which are
a proliferation of plant cells near the soil
level. h e genetic information for
tumour growth is encoded by the
oncogenes that are transferred and
located on the T-DNA, while the genetic
information for the machinery to trans-
fer this T-DNA, encoded in the
vir
genes,
is found on a mobile, circular plasmid,
the Ti plasmid (Fig. 2.3a). h e T-DNA is
delineated by a left border (LB) and a
right border (RB), consisting of a 25 bp
repeat sequence, and only the DNA
segment in between the two border
sequences is transferred to the plant cell.
When
A. tumefaciens
attaches to a plant
cell, it transfers this T-DNA from the
bacterium through a cytoplasmatic
bridge to the plant cell.
When
A. tumefaciens
is used for plant
transformation, tumour induction is, of
course, not wanted and therefore the
bacterial oncogenes on the T-DNA are
removed. h e obtained
A. tumefaciens
strain
is called disarmed, but still contains the
virulence genes for DNA transfer. In such an
A. tumefaciens
strain, a replicating plasmid
vector carrying whatever DNA fragment in
between the LB and the RB can be introduced,
and this strain will then transfer the new
recombinant T-DNA to the plant cell (Fig.
2.3b).
h e bacterium is thus providing a
transport system, enabling transfer of
foreign genes into plants. h is method
works especially well for dicotyledonous
plants like potatoes, tomatoes and tobacco,
but was originally less successful in crops
like wheat and maize. However, recently,
much progress on
A. tumefaciens
-mediated
transformation
has been made
for monocot
crops, and currently it has become the
standard method. Several factors inl uence
the
A. tumefaciens
-mediated transformation.
h e most important factors are the plant
genotype and the explant type used as the
target tissue for co-cultivation to be
transformed. But also the
A. tumefaciens
strain, the binary T-DNA vector, the
inoculation and co-culture conditions and
the tissue culture/regeneration medium
used inl uence the success rate.
2.2.6 Integration of the transgene
Irrespective of the transfer method, the
introduced foreign DNA needs to be
integrated in the genome of the acceptor
plant cell. h is is always a non-targeted
process, meaning that the transferred DNA
