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Notification report


General information

Notification Number
B/DK/06/02

Member State to which the notification was sent
Denmark

Date of acknowledgement from the Member State Competent Authority
06/06/2006

Title of the Project
High Fructan Ryegrass

Proposed period of release:
01/08/2006 to 01/11/2008

Name of the Institute(s) or Company(ies)
DLF- TRIFOLIUM A/S, P.O. Box 59
Ny Oestergade 9
DK-4000 Roskilde
Denmark;


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
ryegrassgramineaeloliumlolium perennenF6

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
Transgenic lines of perennial ryegrass with increased fructan content were generated through the constitutive expression of two heterologous fructan-fructosyltransferase genes Ac1-SST and Ac6G-FFT from onion (Allium cepa). The High-fructan lines show up to 3 fold increase in fructan content (%/dry weight) in the aerial parts. In addition to the fructan biosynthesis genes, the transgenic lines contain an herbicide selection gene (Basta).

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:
Fructan coding regions:
The high fructan content is obtained via the introduction of two heterologous fructosyltransferase coding sequences from onion (Allium cepa): Ac1-SST, encoding a sucrose:sucrose 1-fructosyltransferase, and Ac6G-FFT, encoding a fructan:fructan 6G-fructosyltransferase. The introduced coding sequences of both genes consist of the unmodified wild type nucleotide sequence. The two fructosyltransferase genes code for the enzymes catalyzing the first steps in the biosynthesis of fructan. SST catalyses the synthesis of a trisaccharide and a glucose from two sucrose molecules. FFT uses the trisaccharide formed by SST for further chain elongation of the fructan polymer. Through the chain elongation by FFT a degree of polymerization higher than three is achieved.
Transformation was done by means of biolistic co-transformation with 3 linear DNA cassettes containing [promoter-gene of interest-terminator] elements only, thereby avoiding introduction of bacterial backbone DNA.

The linear gene cassettes were prepared through digestion of plasmids K41, K42 and P41 with restriction enzymes followed by agarose gel electrophoresis. K41 and K42 contain the AcSST1 coding region and the Ac6G-FFT coding region, respectively. Plasmid P41 contains the coding region of the phosphinothricin acetyltransferase gene (Bar) from Streptomyces hygroscopicus, giving resistance against the plant herbicide Basta, as well as two lox recombination sites, which can be recognized by the Cre recombinase. Perennial ryegrass callus was transformed with a mixture of the three linear gene cassettes. Transgenic plants were regenerated from callus cells selected on bialaphos containing medium, and analyzed for presence and transcription of the AcSST1 and Ac6G-FFT genes by PCR and RT-PCR.

Genetic Elements contained in the linear genne cassettes:

Ac1-SST unit (K41)
p- rAct1: Rice actin1 gene promoter to drive constitutive Ac1-SST1 expression.

rAct1 intron: Intron from the rice actin gene to enhance Ac1-SST expression.

Ac1-SST: The coding region for the onion fructosyltransferase 1-SST to initiate the first step of fructan biosynthesis.

Rice RBCS term: The 3' nontranslated region of ribulose-1,5-bisphosphate carboxylase-oxygenase coding sequence from rice which directs the polyadenylation of the mRNA.



Ac6G-FFT unit (K42)
p- rAct1: Rice actin1 gene promoter to drive constitutive Ac6G-FFT expression.

rAct1 intron: Intron from the rice actin gene to enhance Ac6G-FFT expression.

Ac6G-FFT: The coding region for the onion fructosyltransferase 6G-FFT to elongate the fructan chain.

Rice RBCS term: The 3' nontranslated region of ribulose-1,5-bisphosphate carboxylase-oxygenase coding sequence from rice which directs the polyadenylation of the mRNA.



Selection unit (P41)
loxP: Recombination site recognized by Cre recombinase.

p-mubi1: Maize ubiquitin gene promoter to drive bar expression

mubi1 intron: Maize ubiquitin gene first intron, to enhance bar expression

bar: phosphinothricin acetyltransferase coding region isolated from Streptomyces hygroscopicus

NOS 3': NOS 3' 3' untranslated region of the nopaline synthase (NOS) coding sequence, terminates transcription and directs polyadenylation

loxP: Recombination site recognized by Cre recombinase.


6. Brief description of the method used for the genetic modification:
Transgenic plants were generated by biolistic transformation. Co-transformation with the herbicide (Bialaphos, Basta) selection gene allowed the selection of transformed cells.

Experimental Release

1. Purpose of the release:
The purpose of this experimental release is to test the performance of high fructan containing grasses under field growth conditions. Studies of the High-fructan transgenic lines will include: Growth/biomass, phenotype, fructan accumulation during growth season, other quality parameters as well as susceptibility against fungal diseases and insects, abiotic stress tolerance and winter hardiness. The transgenic plants will not be allowed to form stems and flowers, thus eliminating the spread of genes by pollen or seeds.

2. Geographical location of the site:
The DLF-TRIFOLIUM property Bakkegaarden 1 km north of Store Heddinge, in the south-east part of Sealand, Denmark.

3. Size of the site (m2):
Size of the site 56 m2. Area with transgenic lines 36 m2.

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:
No previous field trials.

Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
Carbohydrate limitation, and hence energy shortage, in ruminant animals mainly fed with fresh forage, hay and silage is the main reason for inefficient nitrogen use with the consequence of sub-optimal meat and milk productivity. The High-fructan ryegrass is expected to have a positive environmental impact by improving the nitrogen use efficiency and thereby reducing the release of ammonia into the environment through the manure of the animals.

Grasses produce fructans, and ryegrass contains endogenous genes encoding enzymes in the fructan biosynthesis pathway, including SST and FFT genes. Thus, the introduced enzymes do not comprise an entirely new metabolic pathway in ryegrass and the product, fructan is already being produced in the plant. As homologous genes and their products are already present in plants which form part of the habitat, no adverse effects of the presence of
the introduced fructosyltransferase genes in the transgenic plants are expected.

Uncontrolled intake of forage grass with high sugar content, including fructans, has been shown to increase the risk of development of laminitis in horses. Leaf material from the experimental release will not be fed to horses.

Published laboratory studies indicate that high fructan content may increase the plant cells tolerance against abiotic stress (drought and cold) albeit such correlation has not yet been confirmed at whole plant level under natural growth conditions. Initial drought tests performed on the High-fructan ryegrass lines did not show indications of increased drought tolerance. In greenhouse, the High-fructan lines show no visible phenotypic changes compared to the controls with respect to growth phenotype, generation time, reproduction and seed set.

The High-fructan ryegrass is non-contained. Since ryegrass is an out-breeding species, pollen mediated out-crossing to related wild and cultivated grasses may happen. In this release however, strict control measures will ensure that all reproductive structures (stems) are removed before flowering, thereby avoiding spread of transgenes by pollen or seeds.

In conclusion, no adverse effects on non-target organisms or natural environments are expected from the field release:
• The release site is small (area with transgenic lines 36 m2) and protected by a fence.
• Specific control measures will ensure that no plants are flowering during the entire release period (See pt. E below).
• The introduced genes are not expected to confer an increased selective advantage in the natural environment.
• The proteins produced by the introduced genes are not expected to have toxic or allergenic properties.


Brief description of any measures taken for the management of risks:
Strict handling and control procedures will ensure that none of the transgenic ryegrass plants are allowed to produce reproductive structures (stems, flowers) during the entire trial period. Once a week during the whole growth season all plant will be checked for the development of stems, and all emerging stems will be removed and destroyed immediately. The transgenic plot will be surrounded by a 4 m fallow (bare ground) border to detect and stop potential vegetative spread outside the trial plot. Any plant material removed from the field site will be treated as a regulated article. A fence surrounding the field trial will reduce animal access to the transgenic plots.

After finishing the trail (November 2008), all plants will be removed from the site and destroyed. During the next two growth seasons (2009 and 2010), the release site will be monitored every three weeks. All grasses will be removed from the site and destroyed.


Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
Not planned at this stage.

Final report
-

European Commission administrative information

Consent given by the Member State Competent Authority:
Yes
15/09/2006 00:00:00
Remarks: