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General information Notification Number B/ES/21/01 Member State to which the notification was sent Spain Date of acknowledgement from the Member State Competent Authority 01/09/2020 Title of the Project Characterization of tobacco cv K326 plants derived (by self-pollination) from lines FT3-4, FT15-5, SPL2-3, SPL10-5, SPL11-3, SPL15-1, SPL22-2, FT-SPL3-7, FT-SPL4-7, FT-SPL5-1, FT-SPL7-4 y FT-SPL9-8 (with mutations in the sequence of SPL and/or FT endogenous genes generated by CRISPR/Cas9 that give a non-flowering and/or juvenility phenotype); and lines MPO24-1-4-2, MPO24-1-7-1, BBL133 y BBL135 (with mutations in the sequence of MPO1 or BBLs endogenous genes generated by CRISPR/Cas9 that give phenotypes with different alkaloid composition). 2021 campaign. Proposed period of release: 01/03/2021 to 31/10/2021 Name of the Institute(s) or Company(ies) Instituto de BiologĂ­a Molecular y Celular de Plantas, Agencia Estatal Consejo Superior de Investigaciones CientĂ­ficas.
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 (deletions and insertions) in different combinations of endogenous genes of the SPL family (SPL and FT-SPL lines), endogenous FT5 genes (FT and FT-SPL lines), endogenous MPO1 genes (MPO lines) or endogenous BBL genes (BBL lines). The mutations have been generated using the CRISPR / Cas9 system. These plants do not contain any transgene. Genetic modification Type of genetic modification: Deletion; Other; Other

Genome editing by CRISPR/Cas9
In case of deletion of genetic material, give information on the function of the deleted sequences: FT lines carry mutations in the tobacco FT5 (flowering locus T) gene, a photoperiod-independent flowering activator1. The introduced mutations consist of insertions or deletions in the gene sequence that mostly result in the production of truncated proteins and potentially in their loss of function. In greenhouse conditions these lines have presented a non-flowering phenotype.

The SPL lines carry mutations in genes of the SPL family (SQUAMOSA promoter binding protein like) belonging to groups V, VI, VII, and VIII. These genes play an important role in regulating the transition from juvenile to adult and vegetative to reproductive phases2,3,4,5,6,7. During the early stages of development, elevated levels of miR156 repress the expression of target SPL genes. As the plant develops, there is a decrease in the levels of miR156 with the concomitant increase in the levels of the 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 or deletions in the gene sequence that mostly result in the production of truncated proteins and potentially in their loss of function. In greenhouse conditions these lines have shown a prolonged juvenile phase, increased number of lateral branches and, some of them, a delay in the flowering time.

FT-SPL lines combine mutations in SPL and FT5 genes (described in the previous paragraphs). These lines present a non-flowering phenotype. Some of these lines also present a prolonged juvenile phase and / or increased number of lateral branches.

MPO lines carry mutations in tobacco MPO1 (N-methylputrescin oxidase 1) genes, an enzyme that catalyzes the second step of the alkaloid biosynthesis pathway in tobacco. Introduced mutations consist of insertions or deletions in the gene sequence that result in the production of truncated proteins and potentially their loss of function. Under greenhouse conditions, these lines show a reduction in nicotine levels and an increase in anatabine levels.
BBL lines have mutations in BBL (Berberine Bridge Enzyme-Like) genes. BBLs are involved in the last stages of alkaloid biosynthesis in the tobacco plant8. Introduced mutations consist of insertions or deletions in the gene sequence that result in the production of truncated proteins and potentially their loss of function. Under greenhouse conditions these lines show a reduction in the levels of nicotine and anatabine.
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 plants of the commercial cultivar K326 (FT, MPO and BBL lines) or from the tobacco line cv K326 L157-5 (with mutations in seven SPL genes generated by CRISPR / Cas9; SPL and FT-SPL lines). For this, the tobacco plants were transformed by Agrobacterium tumefaciens (FT, SPL and FT-SPL lines) or Agrobacterium rhizogenes (MOP and BBL lines) with the corresponding transformation vectors, which contain the transcriptional units of the DsRed and / or NptII proteins (selection markers), the transcriptional unit of the Cas9 protein, and the transcriptional units for the expression of gRNAs complementary to the target genes.

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 in the selected T1 lines was carried out by PCR amplification with two pairs of specific primers that amplify a fragment of the 35S promoter and a fragment of the Tnos terminator (FT, SPL, FT-SPL and MPO lines) or a pair of specific primers that amplify a fragment of the Cas9 protein (BBL lines).Analysis of the presence of vector backbone fragments was performed by PCR with two pairs of specific primers that amplify two regions of the vector adjacent to the right and left borders of the T-DNA.

Generation of T2 lines. The T2 lines of the proposed release 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 and T2 lines 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, under standard growing conditions, of the performance of tobacco cv K326 lines genetically edited in genes involved in the transition from the juvenile to adult and vegetative to reproductive phase and/or regulation of flowering (SPL lines , FT, and FT-SPL), as well as genes of the alkaloid biosynthetic pathway (MPO and BBL lines), and their comparison with the wild type line of K326 in the following:
- SPL, FT, and FTSPL lines; analysis of the effect of the introduced mutations on the duration of the juvenile / vegetative phase and flowering and on biomass production.
- MPO and BBL lines; analysis of the alkaloid content; analysis of biomass production; evaluation at pilot scale of anatabine yield of the MPO24-1-7-1 line.
Geographical location of the site: CTAEX Experimental field, Villafranco del Guadiana, Badajoz. Size of the site (m2): 950 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 (in the event that flower buds appear before the time of harvest) 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. In plants with a non-flowering phenotype, this treatment is not expected to be necessary.
- An isolation distance of at 100 km will be maintained between the experimental plot and any other tobacco production area.
A second tobacco release trial is planned to be carried out at CTAEX facilities by the same notifier. This trial will apply the same risk control measures described in this section. In addition, a distance of 100 m will be maintained between the two trials. This distance ensures that, even in the hypothetical case of pollen production, there is no risk of cross pollination.
- 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.
- Plant material left after harvest will be crushed with a disc harrow and buried in the ground, which will prevent vegetative propagation. The experimental plot will be monitored the year following the release allowing the detection and removal of potential volunteer plants. To facilitate this task, the plot will be left fallow or cultivated with other sexually incompatible species.
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: Not known
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