Date of publication: June 14,
2006
Source:
http://gmoinfo.jrc.it/gmp_browse_geninf.asp
Notification number:
B/DK/06/02
Member State:Denmark
Date of Acknowledgement:06/06/2006
Title of the Project: High Fructan Ryegrass
Proposed period of release From:01/08/2006
To:01/11/2008
Name of the Institute(s) or Company(ies): DLF-
TRIFOLIUM A/S;
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
|
|
ryegrass |
gramineae |
lolium
|
lolium
perennen |
|
F6
|
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;
4. 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 Competent
Authority: Not Known |
