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Notification report


General information

Notification Number
B/DK/07/05

Member State to which the notification was sent
Denmark

Date of acknowledgement from the Member State Competent Authority
13/06/2007

Title of the Project
Further development of genetically modified Arabidopsis
thaliana (Thale cress) for the localisation of explosive
components in soil


Proposed period of release:
01/08/2007 to 31/12/2007

Name of the Institute(s) or Company(ies)
Aresa Biodetection ApS, Daugther company of Aresa A/S;


3. Is the same GMPt release planned elsewhere in the Community?
Not known

Has the same GMPt been notified elsewhere by the same notifier?
No

Genetically modified plant

Complete name of the recipient or parental plant(s)
Common NameFamily NameGenusSpeciesSubspeciesCultivar/breeding line
thale cressbrassicaceaearabidopsisarabidopsis thaliana

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
The genetically modified plants of Arabidopsis thaliana
(different ecotypes) capable of changing colour from green to
red when growing within close proximity to explosives,
contain the following components:

1. tt4 mutation
2. ga1-3 mutation
3. plasmid containing MYB transcription factors
4. plasmid containing the CHS gene regulated
by the presence of explosives

Ad. 1. The tt4 mutation was introduced to the Aresa plants by
traditional crossing. The tt4 mutant lacks the first enzyme
required for red pigment formation (anthocyanin).

Ad. 2. The ga1-3 mutation was introduced into the Aresa
plants by traditional crossing. The ga1-3 mutant is blocked in
the biosynthetic pathway responsible for formation of the
growth hormone gibberellinic acid.

Ad. 3. Aresa has transformed Thale cress with a plasmid
containing the transcription factors MYB75 and MYB90. These
transcription factors are responsible for high production of red
pigments.

Ad. 4. The last element in constructing the final landmine
detection plants was transformation of the plant with a
plasmid containing the CHS gene that is expressed from an
element (promoter) regulated by explosives.

The components listed above constitute a system ensuring
that the plants change colour from green to red when they
grow in the presence of explosives (Ad. 3 and Ad. 4) to
ensure that the plants do not change colour in response to
other external or internal signals or components (Ad. 1).
Furthermore, the lack of the growth hormone gibberellinic
acid reduces the risk of spreading genetically modified plants
to the surrounding environment in selected plant lines, which
are to be tested in the field trial (Ad. 2).

The underlying idea of applying the tt4 mutation is the fact
that this mutant cannot produce red pigments (caused by
external factors such as stressed growth conditions). The
ability of forming red pigments is reintroduced to the plants
by introduction of the CHS gene –in connection with an
element responsive to explosives. In this way the plants can
only produce the CHS enzyme if the plants are growing in the
presence of explosives in the soil. To obtain highest possible
production of red pigments (and avoid biosynthetic bottle
necks), the MYB transcription factors are constitutively
expressed.

The bar gene responsible for resistance towards the herbicide
Basta and the luciferase (Luc) gene from firefly have both
been applied as selection marker genes for introduction of the
genetic modifications.

The release of genetically modified A. thaliana include, a number of
different plant lines.


Genetic modification

3. Type of genetic modification:
Insertion;

In case of insertion of genetic material, give the source and intended function of each constituent fragment of the region to be inserted:
-35S-promoter from Cauliflower-Mosaic-Virus (CaMV) for
expression of the transcription factors MYB75 (At1g56650)and MYB90 (At1g66390) from A. thaliana (also designated as PAP1 og PAP2, respectively)

-35S-promoter from Cauliflower-Mosaic-Virus (CaMV) for
expression of the luciferase gene (ff-W-luc) from firefly used
as selection marker

-35S-promoter from Cauliflower-Mosaic-Virus (CaMV) for
expression of the Bar gene selection marker from
Streptomyces hygoscopicus

- E9-terminater sequence from peas (rbcS)

-The CHS-gene (from A. thaliana), which is responsible for one
of the initial anthocyanin biosynthetic steps, being expressed
from an NO2 induced promoter (from A. thaliana)


6. Brief description of the method used for the genetic modification:
-The methods applied for introduction of the genetic
modifications are based on Sambrook et al. 1989 (Molecular
cloning a laboratory manual. Second Edition. Cold Spring
Habour Laboratory Press).

-Transformation of plant lines with vectors containing the
desired insert was done using Agrobacterium (Clough and
Bent (1998) The Plant Journal 16: 735-743).


7. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination:
Not relevant.

Experimental Release

1. Purpose of the release:
Aresa strives to make the plant based biosensor technology
operational for humanitarian de-mining.. The purpose with the
release therefore contains the following elements:

- Test of the GM Thale Cress in an open air test on soil
containing TNT (explosive) granulate

- Test release of the genetically modified Thale cress plants for
the detection of land mines buried in different depths (both
anti-tank mines and anti-personnel mines)

- Determination of the optimal methodology for ground
preparation before sowing the seeds

- Determination of the optimal method and procedures for seed
spreading


2. Geographical location of the site:
The release will take place at Jægerspris Kasserne,
Hovedgaden 71, 3630 Jægerspris, which is a military area
administered by the Danish Army.


3. Size of the site (m2):
The site is approximately 43 meters times 60 meters (=2580
m2), and Aresa expects to sow on approximately 1000 m2,
since paths will be marked and established on part of the
area.


4. Relevant data regarding previous releases carried out with the same GM-plant, if any, specifically related to the potential environmental and human health impacts from the release:
In connection with all releases (contained and open) control
has been performed to observe whether an undesired
dissemination or genetically modified plants or crossing to
other plants has occurred. To date, neither dissemination nor
out-crossing has occurred.

2005
In 2005, Aresa Biodetection established an experimental setup to
test for plants that changed colour when they were growing in soil
that contained explosives. The experimental setup was approved for
the confined use of genetically modified organisms (GMO) by the
Danish Working Environment Authority and with the National Forest
and Nature Agency as advisors on the gene technology. The
purpose of the test was to identify the specific plant lines that had
the optimal sensitivity to change colour when the plants were
growing on top of real mines placed in the ground. Furthermore, the
purpose of the tests carried out in 2005 was to verify the results
and demonstrate them to a third party. The expectations for the
test were fulfilled.

2006
On March 17, 2006 Aresa submitted an application to the National
Forest and Nature Agency (Skov-og Naturstyrelsen), regarding
open air field tests of the selected ecotype of Arabidopsis Thaliana
(col-0). A requested supplement to this application was submitted
by May 3rd. By July 3rd, 2006 this application was approved by the
Minister of the Environment. (B/DK/06/01)

Subsequently, from July – September, 2006, Aresa Biodetection
performed the open air testing covered by the application. The tests
were performed on an area at Aflandshage, protected and controlled
by the Danish Military.
The overall purpose was to further test the ability of the selected
ecotype of Arabidopsis Thaliana (col-0) to change colour under
conditions for growth, that were as realistic as possible, in relation
to mine-infected areas.
The main conclusions from this test were as follows:

• The col-0 ecotype of Arabidopsis thaliana is not robust enough to
be applied as the plant for land mine detection. It does not obtain
a satisfactory size/biomass for this purpose
• The current promoter initiating the formation of red colour in the
modified plants when in the presence of explosives is also
induced by light. When exposed to strong sun light, the plants
turn red, whether or not they are within the defined proximity to
a landmine. This may lead to a lot of false positive results, and
can not be accepted.
As a consequence of the above two findings Aresa BioDetection is
developing new candidates for the mine plant. These plants must be
more robust and only turn red when there are explosives in the
ground within the defined proximity.
The new candidates will be more robust ecotypes of Arabidopsis
Thaliana (Thale Cress).

A detailed report about the GMO field test from 2006 has been
submitted to the Forest and Nature Agency on January 15, 2007.
(Notification number B/DK/06/01)


Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
The rate of survival of the genetically modified plants is
expected to be reduced compared to the native ecotype. The
genetically modified plants contain the tt4 mutation, which
causes a decreased ability of the plants to survive as a result
of a weak seed coat due to the lack of pigments within the
seed (Debeaujon et al. (2000) Plant Physiology 122: 403-
413).


Brief description of any measures taken for the management of risks:
Control of risk of dissemination
In the growth tests that are performed the growth of the
plants will be stopped before flowering. The plants will be
observed with 1-2 day intervals during the period where the
flower formation begins in the rosette. The growth will be
stopped when the formation of flower buds begins. Growth
will be stopped by spraying with a herbicide containing
glyphosat. This means that neither pollen nor seeds will be
formed, and therefore there is no risk of dissemination from
these.

Elimination of seed bank
The risk of establishing a seed bank of transgenic seeds of
thale cress is minimal. After growth of the individual plants
has been terminated, these are sprayed with a herbicide
containing glyphosat. When growth of all plants has been
terminated the upper layer of the whole growth area will be
steamed. This is done using special equipment that is
described in the full application. This treatment is expected to
destroy all seeds to a depth of at least 5 cm. After steaming
soil samples will be taken (cubes) 5 x 5 x 5 cm The soil
samples must be cold-stratified, or similar (for example, by
placing in cold storage at 2-5°C for 2-4 weeks) and
subsequently sown in a 1-cm thick layer in trial boxes that are
covered and kept damp up to 10-20°C. If the Arabidopsis
seeds sprout in the tests or directly in the trial fields, the soil
from the trial sites must be finished by steaming once more
and another control for sprouting be performed.

It is to be emphasized that the plants of Aresa do not contain
genes conferring antibiotic resistance, but the plants do
contain the Bar resistance gene from Streptomyces
hygoscopicus. Thus, the plants of Aresa are able to grow
despite the presence of the herbicide Basta.

The test area is surrounded by a 2 m metal fence. This will
block the access of larger animals to the area. From the plots,
that are used for growth tests, there is a 6 m buffer zone
towards the fence. The 1 m closest to the fence will be
vegetation-free. The other 5 m will have growth of grass. Just
outside the fence a 1 m vegetation-free zone will be
established.

Monitoration of test site
During the period where there is growth of GM plants the test
area will be monitored at least once a week.
During the period where that plants are forming flower buds
the area will be monitored each or every second day.

When all growth tests have been terminated, all the areas
that have been used for growth tests will be steamed as
described above. This steaming process is expected to
eliminate the seed bank. The elimination of seed bank will be
verified by taking samples from the ground as mentioned
above and also it will be observed whether germination has
been observed after one month.

At every control visit the observations made will be put in the
log-book which is on the site

When considering the consequences and the underlying
probabilities of undesired risks to take place, the overall risk
of the releasing the GM plants of Aresa on the designated
area of the Danish Army, is regarded as being minimal.


Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
With the very limited growth period of the Arabidopsis
thaliana plants it is not expected that we will gain data
appropriate for this point.


Final report
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European Commission administrative information

Consent given by the Member State Competent Authority:
Not known