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General information Notification Number B/ES/20/01 Member State to which the notification was sent Spain Date of acknowledgement from the Member State Competent Authority 30/08/2019 Title of the Project Field test of the development of tobacco cv K326 plants derived (by self-pollination) from lines L157-5, L192-6, L226-2 and L259-1, with mutations in the sequence of SPL transcription factors, generated by CRISPR / Cas9, 2020 campaign. Proposed period of release: 01/03/2020 to 31/10/2020 Name of the Institute(s) or Company(ies) Agencia Estatal Consejo Superior de Investigaciones Cientificas., Instituto de Biologia Molecular y Celular de Plantas
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
tobacco solanaceae nicotiana nicotiana tabacum tabacum K326
Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: The tobacco plants to be released carry mutations (small deletions and insertions) in the following endogenous genes that belong to the SPL family of transcription factors: Nitab4.5_0003348g0050.1, Nitab4.5_0003942g0050.1, Nitab4.5_0007487g0020.1, Nitab4.5_0002219g0060.1, Nitab4.5_0000638g0040.1, Nitab4.5_0001752g0040.1, Nitab4.5_0003572g0010.1, Nitab4.5_0000016g0300.1 ((Nitab4.5 Scaffolds Edwards 2017; These mutations have been generated by the CRISPR/Cas9 system. These plants do not contain any transgene. Genetic modification Type of genetic modification: Insertion;Other; Other

Genome editing by CRISPR/Cas9.
In case of insertion of genetic material, give the source and intended function of each constituent fragment of the region to be inserted: The tobacco lines of the proposed release carry mutations in genes of the SPL family belonging to groups VI and VIII. These genes play an important role in regulating the transition from the juvenile to the adult and vegetative to the reproductive phase1,2,3,4,5. During the early stages of development, elevated levels of miR156 repress the expression of the target SPL genes. As the plant develops, there is a decrease in the levels of miR156 with the concomitant increase in the levels of SPLs, which allows the transition to the adult phase and the reproductive phase, with the acquisition of the competence to flower. The mutations present in the tobacco plants consist of insertions of a nucleotide or deletions of between 1 and 24 nucleotides in the coding sequence of the gene that result in the production of truncated forms of the proteins and, potentially, in their loss of function. Based on the function described for these genes, this loss of function is expected to result in a longer vegetative phase with a delay in flowering time. Brief description of the method used for the genetic modification: Tobacco transformation. Generation of transgenic T0 lines. The tobacco lines of the proposed release have been obtained via CRISPR/Cas9 from tobacco cv K326 plants. Plants were transformed with the plasmid GB2138 by Agrobacterium tumefaciens mediated genetic transformation following the protocol of Horsch et al.6. Plasmid GB2138 contains the transcriptional units coding for DsRed and NptII proteins (as selection markers), the transcriptional unit of the endonuclease Cas9, and the transcriptional units for the expression of the gRNAs specific for the target genes (transcription factors belonging to the SPL family).Generation of T1 lines. T1 lines were obtained by self-pollination of T0 lines.Selection of T-DNA free T1 lines. The analysis of presence of T-DNA and vector backbone in the selected T1 lines was carried out by PCR amplification with five primer pairs that amplify a fragment of the 35S promoter, a fragment of the Tnos terminator, and three backbone fragments next to the right and left border of the T-DNA, respectively.Generation of T2 lines. The T2 lines of the proposed reléase were obtained by self-pollination of T1 lines devoid of T-DNA.Identification of mutations present in the lines of the proposed release. Characterisation of the mutations present in the T1 lines was carried out by PCR amplification and sequencing of the gRNAs target regions. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination: Not applicable. Experimental Release Purpose of the release: The purpose of the proposed release is the analysis of the development, under standard growing conditions, of the offspring of tobacco cv K326 T1 lines L157-5, L192-6, L226-2 y L259-1, all of which carry mutations in endogenous genes of the SPL family, and their comparison with the wild type line of K326 in the following:• Analysis of the effect of the introduced mutations on the duration of the vegetative phase and on biomass production.• Analysis of the potential of the modified plants for biorefinery. Geographical location of the site: CTAEX Experimental field, Villafranco del Guadiana, Badajoz. Size of the site (m2): 500 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: Not applicable. Environmental Impact and Risk Management Summary of the potential environmental impact from the release of the GMPts: The mutations present in the plants of the proposed release do not affect the survivability or confer any selective advantage to the plant. There is no risk to human health derive from the modified plants other than the risks associated to conventional tobacco plants.With respect to the potential of gene transfer to other plants, tobacco has no compatible wild species in Europe. There is a potential risk of cross-pollination of modified plants with commercial tobacco crops. The control measures described in the following section, however, eliminate the risk of transfer of genetic material.No negative environmental effects of cultivation, management and harvesting techniques are foreseen since they are not different from those used during commercial production. Brief description of any measures taken for the management of risks: The following measures will be taken:• Seeds will be transported to the release site in labelled closed containers• plants will be topped before the opening of the first flower buds preventing pollen and seed production. Growth of axillary buds will be inhibited by treatment with contact agents. Any axillary bud escaping chemical control will be manually eliminated. There will be, therefore, no production of pollen or seeds.• an isolation distance of at least 2000 meters will be maintained between the experimental plot and any other tobacco production area.• plant material left after harvest will be crushed with a disc harrow and buried in the ground, which will prevent vegetative propagation.• The release site will be regularly monitored by personnel from CTAEX to register any unexpected adverse environmental effect. Should this happen the competent authorities will be immediately notified.• the experimental plot will be monitored the year following the release allowing the detection and removal of potential volunteer plants. 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
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