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Notification report


General information

Notification Number
B/FR/07/06/01

Member State to which the notification was sent
France

Date of acknowledgement from the Member State Competent Authority
02/05/2007

Title of the Project
Field trial of genetically modified poplars for wood properties and bio energy production. Agronomical and environmental assessment (extension of notifications B/FR/03.06.01 and B/FR/99.02.15)

Proposed period of release:
28/07/2007 to 31/12/2012

Name of the Institute(s) or Company(ies)
INRA, ;


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
hybrid aspensalicaceaepopuluspopulus alba x populus tremulaINRA #717-1B4

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
The genetically modified poplars exhibit modified lignin (a major constituent of wood) due to the decreased activity of an enzyme of the lignin biosynthetic pathway. Depending on the transgenic line, the altered enzyme is:
- COMT (Caffeic acid O-methyl transferase): 2 transgenic lines ASCOMT2B and ASCOMT10B
- CAD (Cinnamyl alcool deshydrogénase): 3 transgenic lines ASCAD21, ASCAD52 and SCAD1
- CCR (Cinnamoyl coenzymeA reductase): 2 transgenic lines WT52-3 and WT62-13
- CCoAOMT (Caffeoyl coenzymeA O-methyl transferase): 3 transgenic lines 101, 416 and 429
The down-regulation has been obtained either by antisense strategy (ASCOMT2B, ASCOMT10B, ASCAD21, ASCAD52, WT62-13, 101) or by co-suppression (416, 429, SCAD1 and WT52-3). The enzyme residual activity varies between 10 to 40 % and is not necessarily uniform within the plant. Consequently, the quality or/and quantity of lignin is modified. These modifications and the consequences on some wood properties have been described in several publications (Baucher et al., 1996, van Doorsselaere et al., 1995 ; Meyermans et al., 2000 ; Lapierre et al., 1999 ; Pilate et al., 2002 ; Lapierre et al., 2004).
In addition, all transgenic lines have also integrated a selection gene (either nptII or hpt) that confers an antibiotic resistance. This antibiotic resistance has been used during in vitro culture steps to screen for genetically modified cells: transgenic lines modified for CAD and COMT are kanamycin resistant, whereas transgenic lines modified for CCR and CCoAOMT are hygromycin resistant.


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:
The inserted genetic material is the T-DNA from the Ti plasmid of Agrobacterium tumefaciens harbouring the gene of interest (for lignin modification) and the gene for selection (antibiotic resistance). The gene of interest is one among four poplar genes coding for one among four enzymes of the monolignol biosynthetic pathway. Monolignols are the elementary units of the lignin polymer. The coding sequence of any of these 4 genes is inserted in sense or antisense orientation between i) the promoter of the cauliflower mosaic virus (CaMV) in a simple copy (p35S) or duplicated (p70) and ii) a terminator sequence, either from the T7 gene from the T-DNA (3’T7) or from the gene coding for the CaMV 35S RNA (pA35S). The antisense insertion aims to turn off the expression of the corresponding endogenous gene: The mRNA of the antisense gene interferes with the corresponding endogenous mRNA that results in a strong reduction in the production of the endogenous protein. A sense insertion leads in a few trangenic lines (this is the case for the sense transgenic lines included in this application) to a similar effect, i.e. a reduction in the activity of the target enzyme, through another mechanism named co-suppression.
The four poplar genes listed below derive from cDNA sequences isolated from i) a leaf cDNA library from the AFOCEL #064 clone (cv Hunnegen), an hybrid Populus trichocarpa x Populus deltoïdes, (for CAD and COMT cDNA) or ii) a xylem cDNA library from the Populus trichocarpa “Trichobel“ clone (for CCoAOMT and CCR cDNA).

i) COMT (Caffeic acid O-methyl transferase): a 900 pb fragment spanning the 3’ région of the COMT cDNA (Accession : AAF60951 ; Dumas et al. 1992), inserted in antisense orientation. Le corresponding chimeric gene (p35-AS-COMT-p3’T7) once inserted in the pGSJ780A binary vector generates the pGSJ780A/AS-COMT transformation vector.

ii) CAD (Cinnamyl alcool deshydrogenase): CAD (accession Z19568 ; van Doorsselaere et al. 1995) inserted in sense or antisense orientation. The corresponding chimeric genes (p35-S-CAD-p3’T7 and p35-AS-CAD-p3’T7) once introduced in the pGSJ780A binary vector generate respectively the pGSJ780A/S-CAD and pGSJ780A/AS-CAD transformation vectors.

iii) CCR (Cinnamoyl coenzymeA reductase): the full-length cDNA coding for CCR (accession AJ224986 ; Leplé et al., 1998) inserted in sense or antisense orientation. The corresponding chimeric genes (p70-S-CCR-pA35S and p70-AS-CCR-pA35S) once introduced in the pBIBHygro binary vector generate respectively the pBIBHygro/S-CCR and pBIBHygro/AS-CCR transformation vectors.

iv) CCoAOMT (Caffeoyl coenzymeA O-methyl transferase): the full-length cDNA coding for CCoAOMT (accession AJ224894 ; Meyermans et al., 2000) inserted in sense or antisense orientation. The corresponding chimeric genes (p70-S-CCoAOMT-pA35S and p70-AS-CCoAOMT-pA35S) once introduced in the pBIBHygro binary vector generate respectively the pBIBHygro/S-CCoAOMT and pBIBHygro/AS-CCoAOMT transformation vectors.

For the p70-S-CCoAOMT-pA35S, p70-AS-CCoAOMT-pA35S, p70-S-CCR-pA35S and p70-AS-CCR-pA35S constructs, the selection gene is the hygromycine B phosphotransferase (hpt) gene fused to the promoter of the nopaline synthase gene (pNOS) from Tn7 and to the terminator of the gene 7 from the T-DNA (pAg7),
For the p35-AS-COMT-p3’T7, p35-S-CAD-p3’T7 et p35-AS-CAD-p3’T7 constructs, the selection gene is the neomycine phosphotransferase (nptII) gene fused to the promoter of the nopaline synthase gene (pNOS) from Tn7 and to the terminator of the octopine synthase gene (3’OCS).


6. Brief description of the method used for the genetic modification:
The method used for the genetic transformation is based on Agrobacterium tumefaciens cocultivation of excised internodes from in vitro grown poplar plantlets (Leplé et al., 1992). After this cocultivation step where the gene transfer takes place, the transformed cells are selected using a positive screen (based on antibiotic resistance) and induced to regenerate a whole plant.

7. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination:
Hybrid aspen can disseminate vegetatively through the production of suckers from superficial roots. Pollen and seed are disseminated by the wind, possibly on rather long distance. The seed is very small and devoid of albumen: for this reason the seed viability in the wild is rather short (between 2 and 4 weeks). In fact, seed regeneration is not often observed as ecological conditions necessary to seed germination and plantlet development are seldom met: naked soil, no competition at all with any other species, full light, permanent humidity, but not in excess…

Experimental Release

1. Purpose of the release:
There are two main goals for this release.
As already specified, the genetically modified poplars are modified for the content and/or quality of lignin. Lignin is very important for both tree growth and development, particularly for water conduction and mechanical support. These different transgenic lines of poplars have been already evaluated in two different previous field trials, for agricultural performances and for evaluation of the technological properties of wood for pulp and paper making. This extension aims to produce enough wood from lignin modified poplars in order to evaluate its properties for bio-energy production. Both lignin/cellulose ratio and the accessibility to cellulose are critical for the production of bioethanol from ligno-cellulosic feedstock. Likewise, lignin content and properties (calorific properties, tar formation) may be very important for wood combustion and biogas production. In addition, the poplar trees will be grown as short rotation intensive culture on low-grade agricultural soils (marginal lands) using sustainable low-input conditions.
The second goal for this release is environmental. In the frame of national and international collaborative projects, environmental studies on this material will become possible: namely the effect of lignin modifications on wood degradation rate and on the biological diversity will be investigated.


2. Geographical location of the site:
Nursery of the Breeding Experimental Unit on the ground of the INRA-Orleans Centre located in Saint Cyr en Val, in France.

3. Size of the site (m2):
1363.5 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:
There have been two previous releases with the same GM poplar plants:
- 1) Initial notification number # B/FR/95.03.05 and extension notification number #B/FR/03.06.01, regarding CAD and COMT lignin modified poplars
- 2) Notification number # B/FR/99.02.15, regarding CCR and CCoAOMT lignin modified poplars.

During these two previous field trials, we did not observed any significant differences between GM and wild type poplars with regards to reproductive aspects. Lignin modified poplar flowering time and intensity did not appear affected by the genetic modifications.
However, lignin is involved in major biological functions for tree growth and development such as mechanical support, water conduction and pathogen defence: during more than 12 years, we performed field trial assessment of lignin modified trees; we consistently observed that important lignin modifications very rapidly translated in alteration in the function of conduction and/or support. Moreover, it also appeared that some lignin modified poplars that were shown to grow normally in the greenhouse (i.e. in optimal growth conditions), were unable to do so in the nursery (uncontrolled conditions where trees are submitted to important climatic variations, although these conditions remain less stringent than natural conditions) and for some transgenic lines were even unable to survive. In fact, it is not trivial to find the right balance between lignin modifications that can be of interest for a given utilization of wood and that is not too deleterious for tree growth and development. Altogether, this suggests that lignin modified poplars exhibit at the most an even fitness than their wild type counterpart.


Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
Note especially if the introduced traits could directly or indirectly confer an increased selective advantage in natural environments; also explain any significant expected environmental benefits

As outlined above, there is no expected selective advantage of the GM poplar and no identified effect on non target species.


Brief description of any measures taken for the management of risks:

Hybrid aspen is dioecious (every tree is either male or female). The INRA #717-1B4 clone is female. In consequence, there is no risk of dissemination through pollen. Moreover, as flower development occurs before vegetative bud burst and leaf development, it is very easy to identify and eliminate female catkins, before their full development. INRA technical staff is very familiar with such operation. Finally, the modified poplars will be grown as short rotation intensive culture during 5 years. As hybrid aspen is mature after 4 to 5 years, the first flowering will be present, at the most, the 2 last years of the trial and we know that the first years flowering is very weak with a low number of catkins: therefore flower collection and destruction will be easy.
Suckers are also regularly monitored and destroyed once a year using a contact herbicide.
At the end of the trial, the trees will be cut. New stems from the rootstock will grow until the next winter. We will then apply a glyphosate (systemic) treatment that will destroy both aerial parts and roots.
The experimental site is located in a controlled zone where only authorized staff or visitors can access.


Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
As pointed out at §C.1, the second goal for this field trial is to gain new data on the environmental impact of the release, namely the effect of lignin modifications on wood degradation rate and on the biodiversity.

Final report


European Commission administrative information

Consent given by the Member State Competent Authority:
Not known