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General information Notification Number B/BE/21/V1 Member State to which the notification was sent Belgium Date of acknowledgement from the Member State Competent Authority 26/01/2021 Title of the Project Field evaluation of poplars with a modified wood composition Proposed period of release: 15/05/2021 to 01/03/2025 Name of the Institute(s) or Company(ies) VIB (Vlaams Interuniversitair Instituut voor Biotechnologie)
Is the same GMPt been notified elsewhere by the same notifier? 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 Name	Family Name	Genus	Species	Subspecies	Cultivar/breeding line
grey poplar	salicacee	populus	populus x canescens		717-1B4

Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: The genetically modified trees have an altered wood composition resulting from the downregulation of the Caffeoyl Shikimate Esterase (CSE) enzyme through RNAi. They possess less lignin and more cellulose. CSE is involved in the synthesis of monolignols which are the basic building blocks of lignin. Poplar has two homologous CSE genes: PtaCSE1 and PtaCSE2.The downregulation results in a residual messenger RNA production of between 15 to 50% of the amount of messenger RNA produced in wildtype trees. The amount of lignin that is produced is reduced between 19 and 25%, and the relative amount of cellulose has increased with 8 to 13%.The composition of the lignin is slightly altered as well. There is a shift towards more so-called ‘Hunits’ in the lignin polymer. The reduced amount of lignin together with its altered composition has a positive effect on the ease with which the lignin can be broken down to gain access to thevaluable sugar content in the cellulose and hemicellulose in the wood of the trees. Such types of alterations to the wood composition exist in nature too. Natural mutations have for instance been found in enzymes involved in lignin biosynthesis in the U.S. in loblolly pine (Pinus taeda) and inwild black poplar in Europe resulting in comparable effects on the wood composition.The modified trees also carry the NPT-II and cmR genes. The NPT-II gene is present as a selection marker gene allowing easy selection of transformed plants. The cmR gene which codes for a bacterial resistance to the antibiotic chloramphenicol is not expressed in the plants and therefore does not convey any new properties to the plant. Genetic modification 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 region which has been inserted, and which is flanked by the T-DNA borders from the Tiplasmid of Agrobacterium tumefaciens contains the following elements:Element/ Function / OriginLB / T-DNA left border / Agrobacterium tumefaciensTNOS / Transcription terminator / Agrobacterium tumefaciensKan (npt-II) / Neomycine phosphotransferase gene under control of thenopaline synthase (nos) promoter and nos terminator. Providesresistance to kanamycine, neomycine. / Tn5PNOS / Promotor / Agrobacterium tumefaciensT35S / Transcriptie terminator / Cauliflower mosaic virusAttB1* / Recombination site / Lysogenic Escherichia coliACSE / The first 120 bp of the PtaCSE2 coding sequence (antisenseorientation) /PoplarAttB2* / Recombination site / Lysogenic Escherichia coliintron / Selected from an Arabidopsis databank to achieve efficientsplicing / Arabidopsis thalianaCmR (cat)** /Provides resistance (in bacteria) against chloramphenicol. Notexpressed in the plant./ Tn9AttB2*/ Recombination site / Lysogenic Escherichia coliSCSE / The first 120 bp of the PtaCSE2 coding sequence (sense orientation) /PoplarAttB1*/ Recombination site / Lysogenic E.colip35S / Promotor / Cauliflower mosaic virusRB / T-DNA right border / Agrobacterium tumefaciens*the AttB1 and -2 recombination sites are synthetically altered versions of a recombination site originally isolated from Escherichia coli.**the CmR gene is removed during splicing and is not present in the final hairpin RNA molecule that results in downregulation of the CSE gene. 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. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination: Grey poplar (P. x canescens) can disseminate vegetatively through the production of suckers from superficial roots. Pollen and seed are disseminated by the wind, possibly over rather long distance. The seed is very small and devoid of albumen: for this reason the seed viability in thewild is rather short (between 2 and 4 weeks). In fact, seed regeneration is not often observed as ecological conditions necessary for 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 Purpose of the release: As already specified, the genetically modified poplars are modified in their lignin content. Lignin is very important for both tree growth and development, particularly for water conduction and mechanical support. Different transgenic lines of poplars with a modified lignin content havealready been evaluated in previous field trials in the UK, France, and Belgium, for agricultural performances and for evaluation of the technological properties of wood for pulp and paper making. This release has the purposes to test the performance of these new CSE-downregulatedpoplar lines under real life conditions and to produce wood to evaluate its properties to serve as a good biomass source for extracting sugars and other valuable compounds. Lignin composition, lignin/cellulose ratio and the accessibility to cellulose are critical for the extraction of sugars fromligno-cellulosic feedstock. The poplar trees will be grown as a short rotation intensive culture using sustainable low-input conditions. Geographical location of the site: The field trial will take place on grounds belonging to the ILVO research institute in the municipality of Wetteren. Size of the site (m2): The trial site is in total about 370 m2, which includes a surrounding non-GM buffer 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: The genetically modified plants have not been released before. Environmental Impact and Risk Management Summary of the potential environmental impact from the release of the GMPts: The environmental impact from the release is expected to be zero since it is expected that the GM poplars will not flower and any suckers from superficial roots will be destroyed. Spontaneous regrowing of trees from fallen branches is extremely unlikely, as it is known that P.x canescensand the clone 717-1-B4 does not easily shoot. Only under ideal conditions in the laboratory with the application of shooting powder, P. X canescens is able to shoot. This means that there will be no transfer of transgenes to native or cultivated poplars, and no spread of the GM poplars themselves. When poplar is grown in short rotation intensive culture the trunks and branches will not become older than three years, and therefore are not expected to flower. Grey poplar normally starts to flower between 5 – 8 years of age, only in some cases after 4 years. But anyhow, if monitoring would reveal any flowering, these flowers will be removed. For information:The clone used as a recipient is a female clone, unable to produce male flowers and therefore also unable to produce pollen.The modification of the trees is not expected to lead to environmentally relevant effects on nontarget species. In former trials only some effects were identified on the composition of bacteria living in the trees. The amount and species diversity of micro-organisms living in the rhizospherewas not affected. From scientific literature it can be deduced that lignin modified trees do not have an effect on the interaction with pathogens, that there is no or very limited effect on leafeating insects, and that for the decay of lignin-modified wood other factors like environmental conditions, the chosen poplar species and clone have more significant effects than the lignin modification.And as outlined above, there is no expected selective advantage of the GM poplar. It is more likely that the GM poplar will have a selective disadvantage.The toxicity or allergenicity of the trees has not been tested, but there are no reasons to suspect that an altered wood composition resulting from a lower lignin and higher cellulose content would have an effect on the toxicity or the allergenicity of the plant. Allergenicity of poplar is mostly associated with pollen, but as we are working with female trees that do not produce pollen this is not a concern.It is also known that trees with comparable alterations in the lignin content already exist in nature (in loblolly pine in the U.S. And in black poplar in Europe). If there would be any alteration of the way the modified trees interact with nature and in particular with non-target organisms, thisaltered interaction would be comparable with the interactions of those wild-type trees. Also, there are no indications from the loblolly pine and black poplar mutants that the modified wood would have any negative impact on the health of humans or animals. Brief description of any measures taken for the management of risks: Grey poplar (P. X canescens) is dioecious (every tree is either male or female). The 717-1B4 clone is female. There is therefore no risk of dissemination through pollen. Moreover, as flower development occurs before vegetative bud burst and leaf development, it is very easy to identifyand eliminate female catkins, before their full development. But as the modified poplars will be grown as short rotation intensive culture with a harvest of all trunks and branches after 3 years of growing, the GM poplars are not expected to flower. Suckers are also regularly monitored anddestroyed by either removing them or killing them with a contact herbicide. After a storm the site will be inspected for possible fallen branches and these will be removed. The site is designed in such a manner that fallen branches will not disperse by wind from the plot and will remain withinthe boundaries of a fence surrounding the trial.At the end of the trial, the rootstock will be mechanically removed and the soil will be worked with a rotary cultivator. The plot will be monitored for at least two years for suckers, which will be destroyed using a suitable contact herbicide. If necessary monitoring will be extended until therehas been one year without any suckers.The field trial plot will be surrounded by a 1.80 m high wire fence to prevent accidental trespassing and accidental removal or spread of GM material. Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release: There are no specific studies foreseen to gain new data on the environmental and human health impact from the release other than the study of the phenotype and growth characteristics of the trees. Final report - European Commission administrative Information Consent given by the Member State Competent Authority: Yes
06/02/2021