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Notification report


General information

Notification Number
B/DK/06/01

Member State to which the notification was sent
Denmark

Date of acknowledgement from the Member State Competent Authority
17/03/2006

Title of the Project
Transgenic Arabidopsis thaliana for detection of explosives in the soil.

Proposed period of release:
01/06/2006 to 01/11/2006

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?
No

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 thalianaCol-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;

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 Member State Competent Authority:
Yes
30/06/2006 00:00:00
Remarks: