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General information Notification Number B/FI/18/1MB Member State to which the notification was sent Finland Date of acknowledgement from the Member State Competent Authority 26/06/2018 Title of the Project Wood formation and stomatal regulation in genetically modified hybrid aspen Proposed period of release: 01/11/2018 to 01/11/2023 Name of the Institute(s) or Company(ies) University of Helsinki, Faculty of Biological and Environmental Sciences and Institute of Biotechnology
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
hybrid aspen salicaceae populus populus tremula x populus tremuloides - Clone 51
Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: The genetically modified hybrid aspens overexpress Ethylene Response Factor (ERF) genes or an Isopentenyl Transferase 7 (IPT7) gene enhancing cytokinin signaling under a constitutive LMX5 promoter, which directs the expression to wood forming tissues. Overexpression of these genes modifies wood properties and enhances the growth. In another set of genetically modified hybrid aspens the function of stomata has been modified by silencing Arabidopsis thaliana SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) and SLAC1 homolog 3 (SLAH3) orthologs of hybrid aspen under a guard-cell-specific, Arabidopsis thaliana, SLAC1 promoter. We also express the A. Thaliana SLAC1 gene in hybrid aspen since poplar AtSLAC1 orthologs might have a different function.

Hygromycin phosphotransferase (hpt) marker gene confers hygromycin B-resistance and/or neomycin phosphotransferase (nptII) marker gene kanamycin-resistance to the genetically modified hybrid aspens.
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: Four different vectors are used: 1) LMX5 vector (based on pPCV812 binary vector, which was modified by Gateway technique); 2) pK7GWIWG2(II) RNAi vector; 3) pGWB13 vector and pGWB40 vector. All the vectors have the LB (Left Border) and RB (Right Border) sequences from Agrobacterium tumefaciens to delimit the insert transfer into plant cells. All the vectors have also A. Tumefaciens 3’ untranslated region of a nopaline synthase (nos) gene and/or a TL-DNA gene 4 (only in vector 1) or Cauliflower mosaic virus (CaMV) 35S-terminator (only in vector 3) as a polyadenylation signal for expressed transgenes/antibiotic resistance genes. The antibiotic resistance genes are regulated by a nos promoter from A. Tumefaciens or by a CaMV 35S promoter (only in vector 3).

In addition, the vectors have the following individual constituents:

Vector 1) Three different ERF genes (ERF21, ERF30 and ERF33) from P. Trichocarpa and a IPT7 gene enhancing cytokinin response from A. Thaliana are overexpressed. The overexpressed genes are regulated by a constitutive LMX5 promoter from hybrid aspen. The hpt gene from A. Tumefaciens confers hygromycin B resistance. The vector has also ColE1 replicon and β-lactamase-gene for bacterial selection from Escherichia coli pBR322 plasmid that are non-functional in plants.

Vector 2) In this RNAi vector we silence the expression of AtSLAC1 ortholog from hybrid aspen (corresponding Potri.001G114300 gene model from P. Trichocarpa) and the expression of AtSLAH3 ortholog from hybrid aspen (corresponding joined Potri.015G026800 + Potri.015G026700 gene models from P. Trichocarpa). The silencing is regulated by a guard-cell-specific SLAC1 promoter from A. Thaliana. The vector confers kanamycin resistance by a nptII gene. The vector has also a chloramphenicol resistance gene from E. Coli that is non-functional in plants.

Vector 3) In this vector we express the A. Thaliana SLAC1 gene in hybrid aspen. The AtSLAC1 expression is regulated as in vector 2. To confirm SLAC1 expression in guard cells, the vector has either hemagglutinin (HA) tag from influenza virus. The vector confers both hygromycin B resistance (by hpt gene) and kanamycin resistance (by nptII gene).

Vector 4) This vector differs from vector 3 only by the protein tag used. Here the protein tag is enhanced yellow fluorescence protein tag from Aequorea Victoria instead of HA-tag.
Brief description of the method used for the genetic modification: Agrobacterium-mediated transformation. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination: Hybrid aspen is a dioecious wind-pollinated tree, and the seeds are dispersed by wind. However, hybrid aspen is mainly propagated by root suckers. Experimental Release Purpose of the release: The aim of this release is to conduct basic research on hybrid aspen and understand the role and function of plant hormones ethylene and cytokinin in wood formation. Our second major aim is to understand the function of stomatal regulation in hybrid aspen under natural growth conditions. The results obtained from this experiment could also provide important knowledge for traditional tree breeding. Geographical location of the site: Helsinki, University of Helsinki, field experimental site of Viikki greenhouses (60°13´ N, 25°1´ E) Size of the site (m2): 2896 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: According to our previous greenhouse experiments or ongoing field experiment (since 2014), none of our transgenic lines show any potential health impacts to environment and/or humans. Environmental Impact and Risk Management Summary of the potential environmental impact from the release of the GMPts: There might be a slight possibility for selective advantage in ERF-lines due to enhanced height growth and in lines with enhanced cytokinin signaling due to enhanced branching. Those factors may offer some selective advantage through improved light competition in natural environments. The potential horizontal transfer of the transgenes (especially the antibiotic resistance genes) from genetically modified hybrid aspens e.g. To bacteria represents a remote environmental risk since used transgenes do not offer selective advantage. Therefore, the risk associated with the horizontal gene transfer is very small. Overall, we do not expect any direct consequences for other organisms in the environment. Brief description of any measures taken for the management of risks: Hybrid aspen is dioecious tree species. For the field trial experiment (from 2014 to 2023), we use clone 51, which is female. Thus, there is no risk through pollen dissemination. In any case, we will monitor experimental trees for flower development at regular intervals throughout the experimental periods. In hybrid aspen flower buds burst in spring before leaves and therefore it is easy to identify and eliminate all the possible catkins.
Additionally, the field area will be monitored at regular intervals for the formation of root suckers that will be uprooted and eliminated. Field experiment will be terminated by cutting down all the trees and the remaining stumps will be uprooted. After that, the experimental site will be monitored for any vegetative hybrid aspen material for additional three years. The field is surrounded by a fence and only authorized persons may access the area. So far, we have monitored field experiment for 5 years; no flowering or formation of root suckers has been detected. There are no reports on unexpected behavior of transgenic trees.
Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release: We do not expect that this release will cause any negative influence on natural environments or human health. Final report - European Commission administrative Information Consent given by the Member State Competent Authority: Not known
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