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Notification report


General information

Notification Number
B/IT/03/01

Member State to which the notification was sent
Italy

Date of acknowledgement from the Member State Competent Authority
10/12/2003

Title of the Project
"Italian Lycopersycon (ITA.LYCO): Biologia avanzata e innovazione di processo al servizio della qualità del pomodoro da industria italiana"

Proposed period of release:
31/03/2004 to 31/08/2004

Name of the Institute(s) or Company(ies)
Metapontum Agrobios s.c.a r.l., S.S. Jonica 106 Km. 448,2
75010 Metaponto (MT)
Italy;


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
tomatosolanaceaelycopersiconlycopersicon esculentumesculentumRed Setter codice varietà O 1270

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
From a genetic point of view, modifications brought to GMPt consist in the insertion within the genomic DNA of two transgenes:
- tLcy cDNA isolated from tomato and coding for the lycopene cyclase enzyme. In the expression cassette this coding sequence is under the control of 35SCaMV promoter and the NOS terminator. Lycopene cyclase catalyses the cyclization of lycopene in beta-carotene.
- NptII gene that codifies for the enzyme neomycin phosphotransferase II which catalyses the phosphorilation of the antibiotics neomycin and kanamycin.

As a consequence of the genetic modifications GMPt has got the following traits:
- Overexpression of lycopene cyclase in tomato plants. This modification allows the accumulation of beta-carotene instead of lycopene in the tomato fruits. In the transgenic plants the ripe fruit has a deep orange colour.
- Kanamycin resistance phenotype, determined as the ability of plants or tissues to grow on kanamycin containing selective media.


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:
Inserted DNA region corresponds to the Agrobacterium plasmid pJLcy T-DNA, which includes two physically linked expression cassettes:
- 2780 bp chimeric cassette, which confers the high beta-carotene phenotype, obtained by the transcriptional fusion of the 35S promoter (850 bp) of the Cauliflower Mosaic Virus (CaMV) which allows constitutive expression in transgenic tissues, tLcy gene from tomato (1560 bp) coding for lycopene cyclase enzyme, and NOS terminator (300 bp), from the nopaline synthase gene of Agrobacterium tumefaciens, which carries the termination signal for transcription.
- 1566 bp chimeric construct, which confers the kanamycin resistance phenotype, obtained by the insertion of the neomycin phosphotransferase Il gene (nptII of 792 bp) which codifies for the detoxifying enzyme, between the NOS promoter (474 bp) and the NOS terminator (300 bp) from the nopaline synthase gene of Agrobacterium tumefaciens, which allow respectively the start and the end of transcription in transgenic tissues. Up to date, nptII is the most selective marker gene used in plant transformation and kanamycin is the antibiotic more frequently used for the selection of nptII-transgenic plants (Brasileiro A:C:M and Aragao F:J:L:, 2001, J.Plant Biotechnology 3 (3): 113-221).


6. Brief description of the method used for the genetic modification:
Plasmid pJLcy containing the tlcy-b sequence was transferred to A. tumefaciens LBA4404 by electroporation. Tomato (Lycopersicon lycopersicum L.Karsten ex Farw cv Red Setter) cotyledons were transformed as described (Van Roekel, J.S.C., et al. 1993. Plant Cell Rep. 12, 644-647.), except that the time of co-cultivation was of 10 min and no feeder layer was used. Putative transformants were selected on Kanamycin containing (50 μg/ml-1) substrate. Selected plants were advanced in greenhouse and checked by Southern blot analysis. The resulting transgenic plants were self-fertilised (D’Ambrosio C. et al. 2004 Plant Science 166: 207-214). Four generations were necessary to get the homozygous HC3B6 line.

7. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination:
The GMPt of this release is not a forest tree.

Experimental Release

1. Purpose of the release:
The purpose of this release will be the evaluation of the biological risk derived by the cultivation of the GMP in open field in a strictly controlled experimental field. Concurrently, GMP will be evaluated for some agronomical characteristics in comparison to other two varieties: the REDSETTER, from which GMP was derided, as well as an elite commercial variety. Biological risk evaluation of the GMP release will be mainly concentrated on the possible dissemination of transgenic pollen to the sides of the experimental plot which will be covered with plants of REDSETTER variety as test plants. Fruits produced by test tomato plants will be collected in order to obtain seeds potentially derived from fertilization of ovules with transgenic pollen. Seeds will be planted in greenhouse and plants allowed to set fruit. Plants will be inspected to look for orange coloured fruits that demonstrates the pollen transfer.

2. Geographical location of the site:
The trial will be located at Metaponto (MT) in Southern Italy, in a field of Pantanello Farm, which belong to Basilicata Region Local Government (Regione Basilicata) 100 meters far the "Metapontum Agrobios" Research Centre (S.S. Jonica 106, Km 448.2, Metaponto (MT), ITALY.

3. Size of the site (m2):
6400 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:
No previous release were carried out with this GM-plant.

Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
No factor unique to this field test has been identified that would have an effect on any plant or animal species. In these plants, there were no intentional changes influencing their susceptibility to disease causing organisms. Moreover there are no reasons to believe that the GMPt peculiarity (high content of beta-carotene in ripe fruits) affects the dissemination and the palatability of fruits and seeds to insects. In fact, natural mutants having similar characteristics exist and none difference has been detected. Cross pollination with wild species is not possible because in this environment no wild type species compatible with tomato are present. Crosses of the transgenic pollen with tomato commercial varieties will be avoided since the plot will be surrounded by maize and tomato barriers and is fairly distant from other tomato fields. Moreover, previous field test conducted from the notifier (Not. B/IT/00/09) on a different GMPs demonstrated that there was no transfer of exogenous DNA from the transgenic plants to the tomato barrier plants (Grieco P.D. et al., 2001, Relazione finale su POM A15 "Scelte tecnologiche del sistema Agro-alimentare e qualità dei prodotti" A1: 1-31).

Brief description of any measures taken for the management of risks:
The field trial will take place on one small plot on agricultural land in the Basilicata (ITALY). The site will provide adequate physical security. Site monitoring of the field trial and agronomic management practices creating controlled environment are expected to provide the necessary degree of both biological and physical containment. Pollen dispersal will be avoided by maize and tomato barriers, which will surround the entire field. After sample collection and fruit harvesting, the remaining plants or parts will be removed, autoclaved and/or burned and finally disposed off. Possible plant residues left on the field will be harvested and burned on site. The test area will be marked to monitor re-emergence of volunteer tomato plants during the following seasons. The plots will not be planted the following season but will be checked several times to destroy any plant material.

Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
The GMP under discussion does not possess any form of resistance or tolerance to pathogens or insects, so it does not have any selective advantage respect to the control plant.

Final report
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European Commission administrative information

Consent given by the Member State Competent Authority:
Yes
01/04/2004 00:00:00
Remarks: