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Notification report


General information

Notification Number
B/FR/12/12/01

Member State to which the notification was sent
France

Date of acknowledgement from the Member State Competent Authority
20/12/2012

Title of the Project
Field trial of genetically modified poplars for wood properties and bio energy production. Agronomical and environmental assessment.

Proposed period of release:
01/01/2013 to 31/12/2017

Name of the Institute(s) or Company(ies)
INRA - Institut National de la Recherche Agronomique, ;


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 tremula x populus tremuloides-INRA #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 regularly distributed 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; Leplé et al, 2007).
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 resulting 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. The 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…However, this application deals with short rotation coppice plantation of hybrid aspen trees that will never reach the flowering stage.

Experimental Release

1. Purpose of the release:
1) To complete the first analyses and results from the 2008 field trial. This application aimed to follow up on the previous application in 2007. Indeed, regular Short Rotation Coppice poplar plantation, involve at least 3 rotations with coppicing and subsequent regrowth of the initial plantation: therefore, there is no new GM material dissemination in the field. In addition, the different GM lines will express their real potential during the second and third rotation that yield more biomass than the first one. The analyses scheduled in the frame of this new application will make possible to confirm (or not) the results that will be currently obtained during the first rotation. However, this is not a simple replication of the previous experiment : indeed, during the first rotation, greenhouse-grown plants issued from in vitro plantlets acclimation were planted in the soil and have to get their root system sufficiently well installed to be able to efficiently adsorb water and mineral elements from the soil. At the beginning of the second rotation, the root system is well installed, hybrid aspens will be able to express their potential for growth and development, which may translate in increased biomass yield. Therefore in this application, we intend to assess agronomical and technological (particularly saccharification potential) performances of the different transgenic lines. This material will also be evaluated for stability of the modifications, particularly with regards to rejuvenation phenomenon induced by coppicing. The field trial is planted in Sologne on marginal land with a regular monitoring of the mineral elements until the end of the trial
2) To improve the ability of GM lignin-modified hybrid aspen to produce 2G ethanol using an optimized technological process, specially the pretreatment step. This optimization will certainly make possible to evaluate the wood produced by different GM lines using a mild pretreatment (*), in order to preserve as much as possible lignin chemical reactivity and the potential to use this fraction as high value added precursors in aromatic chemistry.
3) To compare natural variability to the variability generated by genetic engineering. The results that will be produced at the end of the GM trial will be compared to the results obtained by geneticist, while exploring natural genetic variability for traits important for biomass production: this comparison will makes possible to decide the most relevant approach:
i) Either GM variability is equivalent to the variability present in natural genetic resources: in this case, a biotechnological approach is not justified.
ii) Either GM variability exhibits phenotypes that are beyond the variability present in poplar natural population, with properties potentially more promising than properties present in natural population: in this case, this trial will be the demonstration on the possibilities opened by genetic engineering for improving saccharification potential and 2G bioethanol production in trees with regular growth and development.

(*) Regular process such as Kraft or bisulfite are not designed to protect lignin chemical integrity whereas a few others (Organosolv, CIMV) ease the recovery of lignins with a better chemical reactivity.


2. Geographical location of the site:
Nursery of the Breeding Experimental Unit (UE GBFOR) at 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 already been several releases with the same GM poplar plants:
1) Initial notification # B/FR/95.03.05 and extension notification #B/FR/03.06.01, regarding CAD and COMT lignin modified poplars
2) Notification # B/FR/99.02.15, regarding CCR and CCoAOMT lignin modified poplars.
3) Notification # B/FR/07.06.01, regarding CAD and COMT and CCR and CCoAOMT lignin modified poplars

During the 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 modification.
Lignin is involved in major biological functions for tree growth and development such as mechanical support, water conduction and pathogen defence. During more than 17 years, we performed field trial assessment of lignin modified trees and we consistently observed that an important decrease in lignin content very rapidly translated in alterations 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 some transgenic lines were even unable to survive. In fact, we'll have to find out the right balance between lignin modifications that can be both of interest for a given utilization of wood and 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 with coppicing every 2-3 years. As hybrid aspen is mature after 4 to 5 years, there will not any production of flowers during the course of this application.
Suckers are also regularly monitored and efficiently destroyed once a year using a contact herbicide treatment.
At the end of the trial, the trees will be cut. New stems from the rootstock will grow until the end of the next spring. 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.


Final report
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European Commission administrative information

Consent given by the Member State Competent Authority:
Not known