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1.2 Transformation methods and genetic elements introduced into
Numerous methods have been developed that are used to introduce
and integrate 'foreign' DNA into plant cells, leading to transformed
plant phenotypes. Only those methods used in the transformation
of approved agricultural crops will be briefly described below.
These are methodologies based on (i) biological vectors, (ii)
physical or (iii) chemical methods (occasionally used in combination
with electroporation). For a more detailed description of the
methodology, including protocols, the reader is referred to Potrykus
and Spangenberg (1995).
A transformation system should allow for (Niederhauser et al., 1996):
- Stable integration into the host genome without structural
alterations of the foreign DNA.
- Integration of a distinct number of copies of the transforming
DNA (usually 1).
- Stability of the new phenotype over several generations.
- Eventual tissue- and development-specific regulation of the
Points one and three are affected primarily by the choice within
the first generation of transformants and by long-term selection
for a transgenic marker. The fourth feature depends on the choice
of the promoter regulating the transcription of the transgene,
but possibly also on the presence of targeting sequences that
are directing the gene product to certain organelles (e.g. chloroplast
transit peptide sequences). The currently used transformation
methods do not allow for a precise prediction of the number of
copies of the transforming DNA that will be integrated into the
plant cell genome. Conventional back-crossing of the transformants
with the untransformed phenotype is frequently one technique for
reducing the number of copies of the transforming DNA (per haploid
genome) to one or a few. Structural integrity of the introduced
DNA and the precision with which the boundaries of the sequences
which will ultimately integrate into the host genome can be predicted
is partially dependent on the choice (if there is one) of the
transformation system (see below).
Figure 2: Prevalence of transformation methods used for
approved genetically engineered agricultural crops (28 genetically
distinct products in total; see later sections). At least 18 out
of the 28 products (64 %) have been transformed with Agrobacterium,
6 products with physical methods (e.g. particle gun) and 2 with
either a chemical method (e. g. using polyethyleneglycol, PEG)
or by electroporation. No data were available (nda) on the transformation
system used for 2 of the 28 genetically distinct products.
Among the array of genetically engineered plants which have currently
been approved, the transformation method of choice has been the
use of modified plasmids of Agrobacterium (Figure 2). This
is most often a binary vector system derived from Agrobacterium
tumefaciens, where one vector contains the genes to be transferred
and the other harbours genes (vir genes, not transferred)
encoding the necessary functions for transfer to occur (McBride
and Summerfelt, 1990). The system is based on a 'disarmed' Ti-plasmid
with genes responsible for the crown gall disease being removed.
The foreign DNA is confined by the right and left border
sequences ( 25 basepairs each); these are the only elements from
Agrobacterium transferred together with the T-DNA. This
method ensures that a defined region of the presented DNA is precisely
transferred to the new host genome. As mentioned earlier, several
copies of this DNA may integrate at the same or at distinct sites
in the plant chromosome.
Other transformation methods are based on physical and chemical
principles. According to one method, DNA fragments are bound to
the surface of minute metal particles and shot into plant cells
using specially developed devices. The chemical methods make use
of polyethyleneglycol (PEG) or CaCl2 to facilitate the entrance
of foreign DNA through the plant cell wall. Electroporation represents
another transformation method. Plants transformed using electroporation,
chemical or physical methods generally carry copies of the entire
DNA fragments presented. These plants may thus contain copies
of the antibiotic resistance genes used for the propagation of
the respective constructs in bacteria, if such has not been prevented
by removing the respective genes through restriction enzymes prior
to transformation. Frequently, some sections of the presented
plasmid sequences are not transferred using these methods. Therefore,
the boundaries of the transferred DNA will be predicted with less
precision using these methods than with Agrobacterium-mediated
Figure 3: (a) Schematic representation of gene cassettes,
consisting of a promoter (P), a structural gene ('coding region')
and a terminator (T); (b) frequently, two (or more) cassettes
are transferred together and integrated into the host genome (horizontally
bars) at one or several sites.
Enzymes are the products of the majority of transgenes introduced
into the currently approved genetically engineered agricultural
crops. The expression of these enzymes has conferred novel traits
to the respective plants. Proteins without an enzymatic activity,
such as viral coat proteins or the Bt-toxin (-endotoxin from Bacillus
thuringiensis), or antisense constructs have also been expressed.
Efficient expression of structural genes is assured only when
they are controlled by plant-derived promoters or by other promoters
that are active in plant cells such as the cauliflower mosaic
virus 35S promoter. Terminator sequences also have to originate
from plant sources or from plant pests such as the cauliflower
mosaic virus or Agrobacterium.
Direct selection for the many of the actual trait genes (e.g.
those conferring delayed fruit ripening) is not possible. Therefore
selectable marker genes, such as genes allowing growth in the
presence of antibiotics or herbicides, are often co-transformed
with the actual trait gene(s) together with appropriate regulatory
sequences (Figure 3b). The final number of 'foreign' gene cassettes
that are present in a transgenic crop may be as high as 4 or 5
due to the presence of multiple trait genes and marker gene(s)
(Table 3: Approved genetically modified crops in the United States)