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Notification report


General information

Notification Number
B/ES/21/02

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
Evaluation of an intragenic tobacco line (T33SQL49) enriched in squalene - 2021 campaign.

Proposed period of release:
01/03/2020 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., ;


3. Is the same GMPt release planned elsewhere in the Community?
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 NameFamily NameGenusSpeciesSubspeciesCultivar/breeding line
tobaccosolanaceaenicotiananicotiana tabacumtabacumK326

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
The intragenic tobacco line T33SQL49 is characterized by an increase in squalene content. This increase has been obtained through the overexpression of two of the enzymes responsible for the production of this compound, farnesyl pyrophosphate synthase and squalene synthase, targeted to the chloroplast. T33SQL49 is an intragenic line, all the sequences used in the genetic modification come from tobacco or from the sexually compatible species N. benthamiana.

Genetic modification

3. 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:
Line T33SQL49 is an intragenic line. In order to increase the squalene levels in tobacco, we have introduced the coding sequences of the N. benthamiana farnesyl pyrophosphate synthase (fpps) and squalene synthase (sqs), directed by the tobacco Ubi4 promoter and the tobacco Ubc terminator, and targeted to the chloroplast by the chloroplast transit peptide of the tobacco rubisco small subunit. Farnesyl pyrophosphate synthase and squalene synthase are two of the enzymes responsible for the synthesis of squalene in the cytoplasm where squalene is converted to sterols. The targeting of these proteins to chloroplast, which lacks the necessary enzymes to catalyze the conversion of squalene to sterols, allows their accumulation in this cell compartment.

To facilitate the selection of transformants in the transformation process, we have introduced the coding sequence of the phytoene desaturase (pds) of N. benthamiana, directed by the Ubi4 promoter and the tobacco Ubc terminator, with a point mutation that confers resistance to low concentrations of Norflurazon in tissue culture.


6. Brief description of the method used for the genetic modification:
Tobacco transformation. Generation of transgenic T0 lines. The tobacco T33SQL49 line has been generated by genetic transformation mediated by Agrobacterium tumefaciens (strain LBA4404). Leaf discs from 6-8 weeks old tobacco in vitro plants were incubated for 30 min with a culture of agrobacterium carrying the construct to be transformed. Discs were then transferred to co-culture medium (MS30B5 supplemented with the plant hormons BAP and NAA). After 4 days, discs were transferred to selection medium (MS30B5 supplemented with BAP and NAA, timenthine and the herbicide norflurazon) until the development of shoots. Shoots were transferred to elongation medium (selection medium without hormons) as they developed. Once rooted plantlets were transferred to soil and moved to the glasshouse.

Molecular analysis. The presence of the transgenes was confirmed by PCR amplification using primer pairs specific to the transit peptide and gene of interest. Plants that tested positive were further screened by Southern blotting to confirm insert number using a probe specific to a region of sequence spanning the transit peptide and sqs, and a single insert line was selected. GC-MS analysis confirmed the accumulation of squalene in the transgenic line.

Generation of T1 lines. Line T33SQL49 was obtained by self-pollination of a T0 line.


7. 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

1. Purpose of the release:
The objective of the present release is to evaluate the agronomic behavior of the intragenic line T33SQL49, enriched in squalene, under standard culture conditions and its potential as a source of this compound.

2. Geographical location of the site:
CTAEX Experimental field, Villafranco del Guadiana, Badajoz.

3. Size of the site (m2):
550 m2

4. 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 modifications present in the plants of the proposed release do not affect the survivability or confer any selective advantage to the plant. The tolerance to norflurazon has only been shown at very low concentrations (2M Norflurazon) in tissue culture. Norflurazon is an experimental herbicide with no agricultural use in the European Union. There is no risk to human health derived 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 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
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European Commission administrative information

Consent given by the Member State Competent Authority:
Not known