These genetically modified grey poplars express the gshl gene from
Escherichia coli which codes for a γ-glutamylcysteine synthetase.
γ-glutamylcysteine synthase plays an important role in the
synthesis of glutathione (glutamyl-cysteinyl-glycine, GSH) insofar
as it specifically catalyses the ligation of glutamic acid and
cysteine to form glutamylcysteine under energy consumption. Other
γ-ECS substrates are not known. Glutathione is formed in a second
step, in which glycine is bound to the dipeptide by glutathione
synthetase. Increased levels of glutathione are synthesised in
these plants compared with control plants.
Glutathione performs several functions in plant cells. As an
antioxidant, it protects plant cells against oxidation, acts as a
reserve for organic sulphur and as a precursor for phytochelatins
[(γ-glutamylcysteine)n-glycine; n=2-11] it contributes to the
detoxification of xenobiotics and heavy metals. This is brought
about by the formation of stable complexes with metal ions from the
thiol group of the cysteine residues of phytochelatins, which are
pumped under ATP consumption into the plant cell vacuoles where
they are stored.
To transform the grey poplar the start codon of the endogenous gshI
gene from E. coli was modified from TTG to ATG in the p70gshl
construct. This causes an amino acid exchange from leucine to
methionine. Expression of the gshl gene takes place under the
control of the 35S promoter from the cauliflower mosaic virus
(CaMV) with a double enhancer region and the CaMV 35S termination
signal. Southern blot analysis demonstrated that two copies of the
gshl gene were transferred into the genome of the ggs11
Results of studies on the ggs11 transformant showed that the
transfer of the gshl gene leads to increased expression of active
γ-ECS in the cytosol and that increased levels of glutathione are
synthesised in these plants compared with control plants. In
contrast, in comparison to the wild type there was no increase in
either the total protein content of the examined leaves from the
transgenic plant or in the level of glutathione reductase activity
recorded. Phenotypic differences between the GM grey poplar and the
wild type were not observed.
γ-glutamylcysteine synthetase (γ-ECS) is found in all plants.
Therefore the enzyme is not expected to have toxic properties.
An increased production of glutathione is considered essential if
the plant to perform its intended function, i.e. the improved
uptake of heavy metals. The formation of phytochelatins necessary
for this may alter the source-sink distribution of the individual
components (in particular cysteine and glutamic acid). This process
will be the focus of studies to be conducted within the scope of
the deliberate release (field trial). This is not expected to have
harmful effects on human health or the environment.
The ggs11 plants produce more glutathione in the stem and in old
and young leaves than the non-transformed wild type. In the roots
of both plant groups no differences in glutathione synthesis were
found. Results of studies on absorption of cadmium from the soil
solution revealed that in comparison to the non-transgenic control
plants, only the young leaves of the ggs11 plants exhibited a
significantly higher Cd content. In the roots, stem and old leaves,
as well as in the whole plants, the Cd concentrations did not
differ between the GM plants and the non-GM control plants. To
date, it is not known to what extent the transgenic plants differ
from non-transgenic grey poplars and other plants that grow in the
area of the release site with regard to their capacity to absorb
additional pollutants and to store these in different plant organs.
This question is to be examined within the scope of the proposed
deliberate release (field trial).
The modified grey poplar plants were selected using the nptII-gene
product NPT. Hence the plants must be considered to be resistant
against antibiotics like neomycin and kanamycin.
The genetically modified plants are not intended for use in the
production of foodstuffs or animal feed.
Grey poplar has the capacity to form adventitious shoots (stolons,
root suckers) from its roots. Any residual material with the
potential to re-sprout shall be inactivated by applying herbicide.
During the release period, the trial sites are to be monitored for
the emergence of stolons. The fields are to be monitored and
treated with herbicide for a period of two years after completion
of the trials; any emerging root suckers are to be killed off with
herbicides. During and after the trials, monitoring is to include a
15 m area surrounding the release sites. The possibility that the
grey poplar root system might also grow beyond the area of the
release site during the course of the trial and that suckers could
develop from these roots is thus taken into account.
In studies and observations carried out to date on the
morphological characteristics of the GM grey poplar plants in
question, both under greenhouse conditions and within the framework
of a currently ongoing field trial, the applicant reports that no
differences between the transgenic and the non-transgenic plants
were found. Evidence of increased vitality or fertility of the
transgenic poplars which would promote the persistence or
invasiveness of the GM plants has not been found. Root parts that
may remain in the ground following trial completion and post-trial
monitoring are not expected to result in the persistence of the GM
plants. Therefore, the risk of the GM grey poplar persisting in the
environment or establishing new plants in this manner is extremely
The genetic modification transferred to the plants basically
confers a selective advantage under the conditions of heavy metal
contamination expected at the release sites. However, taking into
account the measures planned during the trial (trial duration,
prevention of flowering) and following completion of the project
(post-trial monitoring), the GM grey poplars are not expected to
have the ability to establish on these sites.
For these reasons neither the establishment nor the uncontrolled
persistence of these GM plants is to be expected.
Grey poplar is dioecious. The GM plants intended for release
originate from the female grey poplar clone INRA 717 1-B4. Pollen
formation and dispersal can thus be ruled out. The pollen of
Populus species is dispersed by wind. However, if the trial plants
were to reach the flowering stage, the possibility of in-crossing
and the development of fruit on the GM grey poplars could not be
According to the applicant, the GM poplars for the proposed
deliberate release have been propagated vegetatively since the
summer of 2002. By the time they are to be planted out (spring
2003) they will have reached a stage of development that roughly
corresponds to one-year-old trees. Generally speaking, grey poplars
reach the generative stage after about 7 to 15 years; under
conditions of stress it appears that flowering may occur earlier.
The traits transferred give no reason to expect a significant
reduction in the time taken for the transgenic plants to reach
The applicant plans to conclude the trial after three years, i.e.
before the generative stage has been reached. Furthermore, the
applicant plans to monitor the trees for emerging flower buds prior
to leaf development in the spring, and to remove any occurring
flower buds before anthesis in order to prevent any sexual exchange
with other plants in the environment. Corresponding measures are
described in the regulations set down in provision II.6 of the
decision on this notification.
Experience shows that in trees which flower prior to leaf
development the formation of flower buds can be reliably identified
before the flowers open. As a result, the pollen-mediated transfer
of genetic information from other plants to the GM poplars is not
expected to occur.
At the end of the trial, the GM grey poplars are to be removed from
the soil and burned in an incinerator equipped with the appropriate
filters, together with the leaves that have accumulated and been
collected over the period of the deliberate release, except in the
case that parts of these trees are needed for further analysis. The
approval authority expects this to be done in an environmentally