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Notification report


General information

Notification Number
B/DE/05/168

Member State to which the notification was sent
Germany

Date of acknowledgement from the Member State Competent Authority
20/12/2005

Title of the Project
On the biological security of genetically modified cereals: Effects of transgenic plants on beneficial fungal microorganisms

Proposed period of release:
01/03/2006 to 30/09/2008

Name of the Institute(s) or Company(ies)
Justus-Liebig-University, The President, Ludwigstraße 23, D-35390 Giessen, Germany;


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
barleypoaceaehordeumhordeum vulgarevulgarespring barley/Golden Promise, Baronesse

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
Two lines of GMPts, pYW210-9-(4001-4360) and pJH271-Beta-Glu-307, have been notified for release into the environment.

pYW210-9-(4001-4360) was transformed with a DNA sequence encoding an endochitinase (cThEn42(GC)) of the soilborne fungus Trichoderma harzianum, which is able to hydrolyze chitin of cell walls of Rhizoctonia solani AG-8. It was shown to reduce growth of the phytopathogenic fungi Rhizoctonia solani AG-8 and Rhizoctonia oryzae in vitro. The gene sequence of this fungal endochitinase used for plant transformation has been codon-optimized to a G+C content of 65,1% to allow its expression in barley. The bar gene (phosphinothricin acetyltransferase) of the soilborne bacterium Streptomyces hygroscopius is a marker during transformation and mediates resistance against the herbicide Bialaphos (glufosinate ammonium). Either gene is under control of the Ubi-1 promoter of the ubiquitin gene of maize and, therefore, each gene is expressed constitutively in all plant parts.

pJH271-Beta-Glu-307 was transformed with a DNA sequence encoding a (1,3-1,4)-ß-glucanase, which has been generated by intragenic recombination of two (1,3-1,4)-ß-glucanases of the bacteria Bacillus amyloliquefaciens and Bacillus macerans. The gene is under control of the hor-3 promoter of the D-hordein gene (hor3-1) of barley and, therefore, is expressed during seed development. Here, the (1,3-1,4)-ß-glucanase hydrolyzes (1,3-1,4)-ß-glucans and, consequently, mobilizes endosperm starch and protein to support initial seedling growth. The gene sequence of the microbial (1,3-1,4)-ß-glucanase used for plant transformation has been codon-optimized to a G+C content of 63% to allow its expression in barley. The GMPt expresses two marker genes, (i) a synthetic green fluorescent protein (sGFP) of Aequorea victoria and (ii) the bar gene (phosphinothricin acetyltransferase) of the soilborne bacterium Streptomyces hygroscopius. sGFP mediates green fluorescence to transformed cells when excited at a certain wavelength of light and is under control of the CaMV35S promoter of the cauliflower mosaic virus. The bar gene is a marker during transformation and mediates resistance against the herbicide Bialaphos (glufosinate ammonium). The bar gene is under control of the Ubi-1 promoter of the ubiquitin gene of maize. Due to used promoters, either marker gene is constitutively expressed in all plant parts.


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:
pYW210-9-(4001-4360)
The endochitinase (cThEn42(GC)) of the soilborne fungus Trichoderma harzianum is known to hydrolyze fungal cell walls and, therefore, is expected to enhance plant resistance against fungal diseases.
The bar gene (phosphinothricin acetyltransferase) of the soilborne bacterium Streptomyces hygroscopius is a selectable marker during transformation and mediates resistance against the herbicide Bialaphos (glufosinate ammonium).
Both genes are under control of the Ubi-1 promoter of the ubiquitin gene of maize and, therefore, are expressed constitutively in all plant parts.

pJH271-Beta-Glu-307
Hybrid (1,3-1,4)-ß-glucanases of the bacteria Bacillus amyloliquefaciens and Bacillus macerans are under control of the hor-3 promoter of the D-hordein gene (hor3-1) of barley and, therefore, are expressed during seed development. The gene mediates an enhanced hydrolization of (1,3-1,4)-ß-glucans and, consequently, mobilization of endosperm starch and protein to support initial seedling growth. In addition, glucans, but not (1,3-1,4)-ß-glucans, are components of fungal cell walls and might be hydrolyzed by the recombinant protein.
Synthetic green fluorescent protein (sGFP) of Aequorea victoria mediates green fluorescence to transformed cells when excited at a certain wavelength of light. The sGFP gene is under control of the CaMV35S promoter of the cauliflower mosaic virus. Therefore, the gene is constitutively expressed in all plant parts.
The bar gene (phosphinothricin acetyltransferase) of the soilborne bacterium Streptomyces hygroscopius is used as marker during transformation and mediates resistance against the herbicide Bialaphos (glufosinate ammonium. The bar gene is under control of the Ubi-1 promoter of the ubiquitin gene of maize. Therefore, the gene is constitutively expressed in all plant parts.


6. Brief description of the method used for the genetic modification:
Immature embryos were transformed by cocultivation with Agrobacterium tumefaciens strain AGL-1 harboring the constructs pYW210 or pJH271. After cocultivation, immature embryos were transferred to callus inducing medium containing the herbicide Bialaphos. Subsequently, transformed calli were transferred to shoot inducing- and root inducing media to obtain the genetically transformed plants.

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

Experimental Release

1. Purpose of the release:
The purpose of the release is (i) to investigate the influence of the recombinant enzymes on GMPts-Glomus intraradices interactions, considering G. intraradices as an agricultural relevant representative of mutualistic soilborne fungi, and (ii) to perform epidemiological examinations regarding the colonization of the GMPts by naturally occurring parasitic fungi. The extent of mutualistic and parasitic infestation on GMPt will be evaluated.

2. Geographical location of the site:
Field (Flur)/cadastral parcel (Flurstück) 15/75/2, Alter Steinbacher Weg 44, 35394 Giessen, District (Landkreis) Giessen, Hessen, Germany

3. Size of the site (m2):
12 m2 of genetically modified plants

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:
Both transgenic lines, pYW210-9-(4001-4360) and pJH271-Beta-Glu-307, have been notified for release and have been released in 2005 at various field stations at the Washington State University, Pullman (USA)(Notification numbers 05-035-13n (Th42-2) for pYW210-9-(4001-4360)and 05-035-14n (di 04-029-02n) for pJH271-Beta-Glu-307)in order to propagate and to backcross both transgenic lines with high yielding cultivars. Indicated notification numbers have been given by the “Animal and Plant Health Inspection Service – APHIS”, Riverdale (Maryland), USA.
Due to the nature of these releases neither positive nor negative effects of the transgenes on target or non-target organisms have been recorded. GMPts and respective parent cultivars were comparably colonized by aphids and their beneficial antagonists, GMPts showed the same characteristics regarding flowering, pollination and seed development and GMPts did not differ in survivability and straw decomposition in the field compared to their respective parent cultivars.
All inserted marker genes have been tested in feeding experiments and did not show any harmful impact on fed organisms. The substrates ((1,3-1,4)-ß-glucans and chitin) which are hydrolyzed by the respective recombinant enzymes ((1,3-1,4)-ß-glucanase and endochitinase) do not exist in mammalian organisms.
Albeit chitin is found in the cuticula of insects, it is not present in the outermost layers of the integument and, in inner layers, is tightly bound to proteins. Therefore, any chitinases (transgenic or plant chitinases) have no or limited access to chitin of insects. The portion of chitin present in peritrophic matrices lining up the gut epithelium of insects is rather low (3-13%) and not expected to be affected by the endochitinase since especially herbivoric insects are not impaired by plant chitinases. Consistently, the above described GMPts, pYW210-9-(4001-4360) and pJH271-Beta-Glu-307, were similary infested by aphids as compared to their respective parental cultivars when grown in the field at the Washington State University, Pullman (USA). The experiences in releasing both transgenic lines made by scientifically educated staff of the CAHNRS of the Washington State University, Pullman (USA), base on qualitative observations.
All transgenes expressed in both transgenic lines do not show any homology to known toxins and allergens.

All observations and experiences made, combined with the knowledge on the recombinant enzymes and the distribution of the respective substrates, do not indicate any direct or indirect impact on the environment or human health from the release.
The impact of the GMPts on arbuscular mycorrhizal fungi as mutualistic organisms with agricultural relevance can not be excluded. For that reason, one main focus of the release is the monitoring of GMPts-Glomus intraradices interactions.


Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
There is no scientific reason to assume that the traits introduced into either transgenic line could directly or indirectly confer increased advantages in natural environments or that a significant environmental benefit is expected. None of the traits introduced into either transgenic line has any impact on traits that are related to distribution, pollination, reproduction or survivability of barley in natural environments. Due to domestication, the cultivation and distribution of barley is entirely depending on anthropogenic agricultural practices. Taken together, the potential environmental impact from the described GMPts have to be regarded as similar to that from conventional barley cultivars.

pYW210-9-(4001-4360)
The introduced endochitinase (cThEn42(GC)) is expected to enhance plant resistance against fungal diseases because of its ability to hydrolyze chitin of fungal cell walls. In case that GMPts harboring the recombinant endochitinase are more resistant to fungal pathogens, these plants are expected to gain an increased selective advantage in agricultural environments when confronted with a high disease pressure. Apart from fungi, chitin is a component of inner layers of the cuticula of insects. Other parts of insects which are potentially accessible to transgenic or natural occurring chitinases are tightly bound to proteins and, hence, are not expected to be hydrolyzed by chitinases.
The bar gene (phosphinothricin acetyltransferase) mediates resistance against the herbicide Bialaphos (glufosinate ammonium) and is not expected to confer an increased selective advantage in natural environments.

pJH271-Beta-Glu-307
The introduced (1,3-1,4)-β-glucanase is expressed during seed development and mediates an enhanced hydrolyzation of (1,3-1,4)-β-glucans in germinating seeds. It is not known whether the recombinant enzyme is able to hydrolyze glucans of fungal cell walls, thereby mediating resistance to fungal pathogens. In case, pJH271-Beta-Glu-307 is more resistant to fungal pathogens, these plants are expected to gain an increased selective advantage in agricultural environments confronted with a high disease-pressure. However, the introduced (1,3-1,4)-β-glucanase is spatio-temporally expressed during seed development and the enzyme possesses an high substrate specificity to (1,3-1,4)-β-glucans of the seed endosperm and aleurone.
The introduced synthetic green fluorescent protein (sGFP) of Aequorea victoria mediates green fluorescence to transformed cells when excited at a certain wavelength of light and does not possess any physiological function in the GMPts.
The bar gene (phosphinothricin acetyltransferase) mediates resistance against the herbicide Bialaphos (glufosinate ammonium) and is not expected to confer an increased selective advantage in natural environments.


Brief description of any measures taken for the management of risks:
Barley is a self-pollinating plant with a limited ability to cross-pollination. Cross-pollination is a rare event and diminishes by distance between two plants. In order to prevent any cross-pollination, transgenic field plots are framed by a conventional barley variety (width: 5 m) that serves as a pollen trap. This barley is surrounded by a band without plants (width: 5 m), which is finally framed by dicotyledonous plants (width: 25 m).
Procedures performed to limit seed and pollen dispersal:
• Potential pollen acceptors (wild barley, Elymus sp., cereals) occurring within the site of the field trial will be eliminated throughout the ongoing field experiments. To this end, the field site will be regularly observed for respective plants.
• Plots of transgenic and non-transgenic plants will be harvested by hand to reduce the dispersal of seeds. Harvested seeds will be stored in certified S1 gene laboratories.
• Plants of the pollen trap barley culture will be harvested by a combine harvester and harvested seeds will be destroyed.
• Plants growing within the site of field trials that are not forwarded to analyses will be destroyed by application of a non-selective herbicide. Dead plant material will be chopped and covered with soil.
• The field will be observed for volunteer plants growing for at least one year after finishing all experiments. In case volunteer plants have been identified, the observation will be automatically extended for another year.


Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
The field trials are designed to investigate the mycorrhization of either transgenic line and the respective parent cultivars by the arbuscular mycorrhizal fungus Glomus intraradices. Therefore, one half of the plot sites will be inoculated with the mutualistic G. intraradices prior to planting transgenic and non-transgenic seeds. Mycorrhization as an important agricultural aspect in crop production will be monitored in randomly selected plant roots.
A second aspect will follow the occurrence of fungal diseases on selected transgenic and non-transgenic plants. The aim is to record whether transgenic or non-transgenic plants are preferably infected by naturally occurring fungal pathogens.


Final report


European Commission administrative information

Consent given by the Member State Competent Authority:
Yes
03/04/2006 00:00:00
Remarks: