Date of publication: April 28,
2006
Source:
http://gmoinfo.jrc.it/gmp_browse_geninf.asp
Notification number:
B/DK/06/01
Member State:Denmark
Date of Acknowledgement:17/03/2006
Title of the Project: Transgenic Arabidopsis thaliana for
detection of explosives in the soil
Proposed period of release From:01/06/2006
To:01/11/2006
Name of the Institute(s) or Company(ies): Aresa
Biodetection ApS;
3. Is the same GMPt release planned elsewhere in the
Community?
No
4 - Has the same GMPt been notified elsewhere by the same
notifier?
No
Genetically
modified plant
1. Complete name of the
recipient or parental plant(s)
|
Common Name
|
Family Name
|
Genus |
Species
|
Subspecies
|
Cultivar/breeding line
|
| thale
cress |
brassicaceae |
arabidopsis |
arabidopsis thaliana |
|
Col-0
|
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
(ecotype Col-0) able to change colour from green to red when
growing near by 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 responds 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 at Pionergården,
Amager, includes, for the majority of the designated area, the
plant line designated RD68. Apart from this plant line, the
release will also include 7 other genetically modified plant
lines to be grown in minor test boxes of approximately 1 m2. 4
of these plant lines lack the growth hormone gibberellinic acid.
Genetic
modification
3. Type of genetic
modification:
Insertion;
4. 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--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 in 2006. The purpose with
the release therefore contains the following elements:
-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)
-Test release of the genetically modified Thale cress plants for
the detection of unexploded ordnances (UXO’s) buried in
different depths
-Determination of the optimal methodology for ground preparation
before sowing the seeds
-Determination of the optimal method and procedures for seed
spreading
-The release of A. thaliana in respect of evaluating the risk
assessment
2. Geographical location of the site:
The release will take place at Pionergården 251, 2791 Dragør,
which is military land administered by the Danish Army.
3. Size of the site (m2):
The site is approximately 44 meters times 48 meters, and
Aresa expects to sow on approximately 1600 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:
The genetically modified plants of A. thaliana have been
released under contained conditions (test boxes covered with net
protection) at Pionergården in 2005. Based on these growth
experiments, no seeds have been observed outside the test boxes
so far. Observation of the area around the test boxes will
proceed during the growth season of 2006.
Environmental
Impact and Risk Management
Summary of the potential
environmental impact from the release of the GMPts:
In respect of the ability of A. thaliana to reproduce in
general, it should be emphasized that the plant is a
self-pollinating plant. Thus, the actual fertilization of the
ovarium by the stamina occurs in a closed environment. In case
of outcrossing to take place, pollen needs to be transferred to
an A. thaliana (or a closely related genus) ready for
pollination. The prime cause of outcrossing is assumed to be
catalysed by thrips (Hoffmann et al. (2003) Plant Biol. 5:
491-494). The rate of outcrossing of A. thaliana has been
reported to be in the range of 0.3-5.98% in different studies.
The risk of outcrossing with wildly growing A. thaliana and
related species is assumed to be greatly reduced, since the site
of the release is surrounded by a 4 meter wide paved road. This
assumption is based on references where the distance of gene
transfer between transgenic plants and wildtype plants is
estimated to 0.5 meter (Tan et al. (2005). Plant Biol 7:
405-410).
In case of an outcrossing event to take place, the traits
introduced into the plants developed by Aresa will be
transferred to the progeny. As a consequence of such an
outcrossing event between the plants of Aresa and native growing
plants, the progeny (F1 generation) will grow up as a red plant
without the presence of explosives (F1 is heterozygote in all
loci). The technical explanation of this color formation is that
outcrossing will result in elevation of the tt4 mutation, since
a single copy of the CHS gene is sufficient to enable red
pigment biosynthesis. Hence, by inspection of the area it is
possible to identify progenies of outcrossing events growing in
the wild, which may potentially contain foreign genetic material
such as the resistance gene toward the herbicide basta.
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 rate of survival of the genetically modified plants is
expected to be reduced compared to the native ecotype for a
couple of reasons. The genetically modified plants are developed
in an ecotype of Columbia, which is not expected to have the
same ability to survive under the growth conditions of Denmark.
Furthermore, 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).
The risk of establishing a seed bank of transgenic Arabidopsis
seeds is expected to be lower than the natural occurring Danish
ecotype. The plants of Aresa contain the tt4 mutation, which has
been demonstrated to cause an even higher rate of germination
compared to Col-0. Col-0 is selected as a preferred laboratory
plant line partly due to its high rate of germination and its
low need for dormancy. Furthermore plants carrying the tt4
mutation produce seeds with lower survival rate in soil due to
their more permeable and weaker seed coat.
A risk of the release with potential ‘indirect’ consequences
could be if plants are eaten by animals. It is assumed that no
toxic consequences are related to eating the plants, since the
plants are related to cabbage and the mustard plant. It is also
assumed that there are no toxic consequences from eating the red
plants with excessive anthocyanin levels, since large amounts of
anthocyanin is already an integrated part of human food
consumption (cabbage, lettuce, fruit, wine e.g.). It has
actually been reported that consumption of anthocyanin and
proanthocyanin has a life prolonging effect on fruit flyes
(Mylnikov et al. (2005) J. Agric. Food Chem. 53: 7728-7733). In
light of the release, the probability of larger animals
accessing the area is regarded as being strongly limited, since
the whole area around Pionergården is fenced up to app. 2 meter
in height.
Brief description of any measures taken for the management of
risks:
From risk assessment point of view, the field trial area has
been selected, since the field trial area is surrounded by a 4
meter paved road in addition to banks of earth exerting a
barrier to the surrounding environment.
Control procedures are put in place to observe potential
outcrossing events between the genetically modified plants and
plants growing in the wild, as well as growth of the genetically
modified plants outside the field trial area.
The weakly inspection of the area before, during, and after the
release will contribute to reduce the risk of genetically
modified plants growing outside the test site. In general the
genetically modified plants of A. thaliana can be distinguished
from the natural occurring Danish ecotype by comparison of the
phenotypic traits, since the genetically modified plants are
constructed in the ecotype of Columbia (Col-0). All individuals
of A. thaliana observed in the area around the test site will be
harvested and analysed by the following procedure:
1. Leaves of the individuals will be analysed using a luciferase
camera for the identification of transgenic plants containing
the Luc gene
2. Luciferase positive plants are analysed using PCR to detect
the presence of the bar gene
3. All harvested plant material will be treated and destructed
as being GM material
Furthermore, it should be noted that red individuals, which are
luciferase positive and containing the bar gene, strongly
indicate that an outcrossing event has taken place.
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 very small. This risk
will be handled with responsibility in the light of the
facilities at the site for the release, as well as due to the
control procedures to be followed over the years to come.
Summary of foreseen field trial studies focused to gain new
data on environmental and human health impact from the release:
In respect of the environment Aresa will gain new data on
potential outcrossing events with closely related plants to
Arabidopsis, if any, as well as the impact of introducing
male-sterility into the plants that are not able to produce the
growth hormone gibberellinic acid.
Final report
-
European
Commission administrative information
Consent given by the Competent
Authority: Not Known |
