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General information Notification Number B/SE/23/21689 Member State to which the notification was sent Sweden Date of acknowledgement from the Member State Competent Authority 22/12/2023 Title of the Project Increased drought stress tolerance, Increased production of wood biomass by increased growth 3 - Prolongation, Changes in lignin with directed mutagenesis - Prolongation. Proposed period of release: 01/04/2024 to 31/12/2028 Name of the Institute(s) or Company(ies) SweTree Technologies AB
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
poplar	salicaceae	populus	populus deltoides		T89 and INRA 717-1B4

Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: In this application, we are seeking permission for a new trait gene (GOI) with the property "increased drought stress resistance" as well as renewal of permission for 3 trait genes (GOI) that affect the growth of the hybrid aspen T89 in previous field trials and/or in greenhouse trials.To gain knowledge about which expression level, timing and localisation that gives optimal effects, it is necessary to vary the expression by putting the gene under control of different promoters. Therefore the genes have been put under control of different promoters in individual constructs. In total 16 different constructs. In each of the constructs one of the GOI is under the control of one promoter.It may be important to mention that independently of selected promoter each trait from each trait gene (GOI) respectively is the same but the effect may vary. In this way we may find a more precise level for optimal effect. Four of these 16 GMO types have been chosen because they affect the drought stress resistance of the hybrid aspen and 12 of the GMO types have been chosen for this trial because they affect the growth of the hybrid aspen T89 in previous field trials and/or in greenhouse experiments.In the plantconstructs also the marker gene NPTII (Kan) was introduced toenable the selection of transformed plant cells. In this application, we are also seeking renewal of permission for gray poplar containing a mutation in each allele in the coding region of the gene encoding the enzyme cinnamoyl-CoA reductase 2 (CCR2). 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: We are applying for 16 hybrid aspen constructs each containing one of the 4 genes for the intended modification (GOI). These genes encode an inactive protease or transcription factor protein. All GOIs originate from Hybrid Aspen. The intended function of selected GOI is in 4 of the constructs to affect drought stress tolerance and in 12 of the constructs to affect growth.Genes and fragments included in the T-DNA, their origin and function, 1 or 2 depending on vector used: 1) Terminator t35S (Cauliflower mosaic virus), attB2 recombination site (Gateway Technology), Gene of interest, GOI (Hybrid aspen), attB1 recombination site (Gateway Technology), Ω leader (Tobacco mosaic virus), Promoter, p35S (Cauliflower mosaic virus), Promoter, pnos (Agrobacterium), Kanamycin marker gene, npt-II (E. coli), Terminator, tnos (Agrobacterium).2) AttB4 recombination site (Gateway Technology), promoter (Cauliflower mosaik virus or Eucalyptus), attB1 recombination site (Gateway Technology), Gene of interest GOI (Hybrid aspen or Arabidopsis), attB2 recombination site (Gateway Technology), Terminator T35S (Cauliflower mosaic virus), Promoter pnos (Agrobacterium Nopaline Plasmid), Kanamycin marker gene, npt-II (E. coli), Terminator, tnos (Agrobacterium) We are also applying for transgenic gray poplar that has undergone two Agrobacterium-mediated transformations with two different vectors and thus contains two T-DNAs.Vectors used in the transformations and properties: Brief description of the method used for the genetic modification: The transgenic trees were obtained using the well-known and established method of Agrobacterium-mediated genetic modification.In the gray poplars, the introduction of the transgene is to supply the plant with a CRISPR/Cas9 complex which in turn, with the help of the enzyme Cas9 and guide RNA (gRNA), makes a targeted gene edit at a pre-specified site in the plant's genome and thus inactivates or changes the function of a gene. This method is also well known and established.  If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination: Both hybrid aspen and gray poplar are wind-pollinated, and the seeds are wind-borne. The hybrid aspen clone is most likely a male clone and the gray poplar clone in the trial is a female clone. Furthermore, both species can propagate from suckers. Experimental Release Purpose of the release: The purpose is to study the growth and physiology as well as wood properties on trees to gain increased knowledge about drought stress resistance and/or growth, linked to the function and effect of the promoter-gene combinations we modified, with the aim of using this knowledge in the future to produce new tree species with increased production of wood biomass through increased growth or improved drought stress tolerance. These future trees can be either GMO trees or trees produced with marker-directed breeding or directed mutagenesis. The purpose of this trial is also to field test plants, with CRISPR/Cas9-generated mutations, which give a reduced level of lignin without affecting the growth of the plant, which have been shown in greenhouse trials. This is to provide a substantially increased fermentable sugar yield from wood biomass, which can lead to significant environmental benefits in the transition from a fossil-based to a bio-based economy.The purpose of prolongation of previous trial permits is to gain a deeper understanding of variation between lines and to confirm positive data generated at previous release sites. We want to confirm positive lines and test more lines of the same gene constructs. Geographical location of the site: The estate Alnarp 1:1, in municipality of Lomma. Alnarp land property, Swedish University of Agriculture Alnarp Size of the site (m2): Maximum 15000 m2 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 constructions for "Increased production of wood biomass by increased growth 3", for which we are applying for prolongation of the permit, have previously been in field under the following permissions: D.no. 4.6.18-11239/2020 and D.no. 4.6.18-11847/15.During the previous field trials, we have not seen any phenotypic effects other than the expected ones, i.e. changed growth. Lines from some of the trait gene constructs have shown increased growth, some lines grow at the same level and some lines grow slow, compared to wild-type control plants. Otherwise, the trees have behaved normally, like wild-type plants. We have not been able to see any phenotypic effects of the gene constructs that entailed any unexpected risk for human health or to the environment during the experimental period. We are also applying for the prolongation of the permit for the construction for: Changes in lignin with directed mutagenesis.This construction has previously been in the field trial in Våxtorp under experimental permission: Dnr. 4.6.18-22027/2021.During the previous field trial, we have not seen any unexpected phenotypic effects that indicate any increased risk for human health or the environment. Since the experiment was terminated earlier than planned, no analyzes of the lignin composition was done. When the experiment was terminated, the lines in the experiment were smaller compared to wild-type control plants. Otherwise, the plants were similar to wild-type plants. 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 signifant expected environmental benefitsNone of the modifications intend to change any reproductive organ of the plants. It is unlikely that the plants will form flower buds during the trial period. None of the modifications have shown signs of premature flowering in greenhouse trials or in previous releases in the field.The goal of some of the lines in the trial is that they show improved drought stress resistance and/or increased growth in the field.In terms of survivability, the trait "increased drought stress tolerance", an abiotic stress, can be considered to contribute to an increased ability to survive drier climates. During the trial period, however, a drier climate is not expected, but possibly drier periods, for example a hot summer. Our hope then is that one or a few of the lines can show such a positive effect compared to wild-type control plants in the same trial.Aspens with a greatly increased growth in the field could have a competitive advantage in the natural environment. Regarding the property "increased growth", no change in survival ability compared to wild type could be noted in previous field trials with these lines. On the other hand, we have seen that some of the constructions tested in previous trials have reduced survivability, i.a. they have had problems with in-wintering in the autumn or bud bursting that occurs too early in the spring, which means that they do not survive or are damaged and suffer frost damage. This is one of the reasons why some the constructs from previous permits are no longer part of the application for prolongation.Positive environmental effects of properties such as "increased drought stress resistance" and/or "increased growth" are that they can lead to increased knowledge about how we can increase the production of biomass. This increased biomass can be used for several different purposes, e.g. for wood, fiber, biochemical and energy purposes. You can also choose to produce the same amount of biomass on a smaller area. Or to make trees that survive in a changed climate. Brief description of any measures taken for the management of risks: Regular inspection to detect if any tree would form flower buds. It is possible to identify flower buds long before they start flowering. Should any of the trees in the trial, in contrary to expectations, form flower buds, all branches on that tree, with and without flower buds, will be cut off and destroyed. The rest of the tree is destroyed after the end of the season.The field site will be fenced and a surrounding safety zone will prevent root suckers from spreading from the experiment. After the end of the trial, all test plants will be terminated and the site will continue to be inspected annually for at least 3 years Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release: Not applicable Final report - European Commission administrative Information Consent given by the Member State Competent Authority: Yes
04/12/2024