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Notification report


General information

Notification Number
B/ES/08/02

Member State to which the notification was sent
Spain

Date of acknowledgement from the Member State Competent Authority
28/01/2008

Title of the Project
Field trial with transgenic orange overexpressing a limonene synthase gene in sense and antisense with the aim of modifying fruit aroma and them their interaction with herbivore insects and predators of herbivores.

Proposed period of release:
30/03/2008 to 30/03/2018

Name of the Institute(s) or Company(ies)
Instituto Valenciano de Investigaciones Agrarias., Generalitat Valenciana.;


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
sweet orangerutaceaecitruscitrus sinensisNavelina

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
The endogenous content of limonene has been altered in the transgenic plants by overexpression of a limonene synthase gene from citrus in sense or antisense. As a consequence of the genetic modification, frut aroma has been modified in the transgenic sweet orange plants.
All sense and antisense transformed plants are also carrying a nptII transgene conferring resistance to kanamycin.


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:
Cassette NOSpro:: nptII:: NOSter: 1758 nucleotides. The nptII gene comes from Tn5 transposon of E. coli. NOS regulatory regions (promoter and terminator) come from the nopaline synthase (NOS) gene from Agrobacterium tumefaciens. Transgene expression confers resistance to kanamycin to the transgenic cells and tissues. This cassette is present in the T-DNA from pBI121, which was used as the binary vector for A. tumefaciens mediated transformation of citrange explants (GenBank accesión of pBI121: nº AF485783).
Cassette 35Spro 2X::limonene synthase sense or antisense:: NOSter: 852, 1821 and 265 nucleotides, respectively. Promoter and terminator regions come from the 35S gene from the cauliflower mosaic virus (CaMV) and confer high and constitutive expression of the transgene. The limonene synthase transgene comes satsuma mandarin (Citrus unshiu (Mak.) Marc.). Its overexpression in sense or antisense induces modification of the limonene content and emission in the flavedo from fruits of the transgenic plants. Consequently, sense plants show altered aroma compared to control plants.


6. Brief description of the method used for the genetic modification:
Co-cultivation of internodal stem segments from mature Navelina sweet orange with the disarmed strain EHA 105 of Agrobacterium tumefaciens, and regeneration of whole plants from the transgenic cells through organogenesis using proper tissue culture media and conditions.

Experimental Release

1. Purpose of the release:
Field trial of transgenic sweet orange over-expressing in sense or antisense a gene encoding a limonene synthase to study:
- modulation of fruit aroma in plants growing in field conditions. For this purpose, we propose to transfer to the field 5 antisense lines (1-1, 2-3, 3-1, 5-1 and 7-1), one sense line (8-1) plus one control Navelina line and one control Navelina negra line (8 propagation per transgenic line and 16 propagations from each of the two controls).
- phenological and morphological characteristics of the transgenic trees and fruit quality issues.
- possible new interaction of transgenic fruit with herbivore insects, as Ceratitis capitata, and predators of herbivores

We only have data from plants growing in the greenhouse that shows that limonene content and emission has been strongly decreased in fruit flavedo from antisense lines and it has not changed in sense lines. Southern and Northern blot analyses demonstrated transgene integrity and expression, respectively. Determination of limonene content in fruit flavedo was assayed through GC-MS.


2. Geographical location of the site:
The release site will be located at the experimental fields of the Instituto Valenciano de Investigaciones Agrarias in the STA of Villareal, Castellón, Spain.

3. Size of the site (m2):
About 7500 m2 in total for four field trials with transgenic citrus plants. This assay will cover 750 m2 approximately.

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:
These transgenic lines have not been released previously, but we have two other releases with transgenic citrus trees planted in Valencia region.

Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
Commercial citrus varieties are propagated vegetatively by grafting of well-known genotypes onto well-known rootstocks. In our Mediterranean conditions and considering the citrus genotypes used, it is not possible that transgenic plants could become weeds.
Citrus cultivars grown in the area are sexually compatible with the transgenic plants. There are not wild citrus species and relatives in Europe, so there are not possibilities of compatible interactions between transgenic and wild plants.
Under natural conditions, cross-pollination between transgenic lines and cultivated genotypes (citrus species or hybrids) is theoretically possible. Pollination in citrus is exclusively performed by insects, being bees the most successful pollinators. However, there are laws in Valencia region forbidding beehives where clementine is being planted. Moreover, it is permitted to treat with insecticides against bees during the citrus flowering season. This rule was adopted to avoid cross-pollination and consequently seed production in fruits of certain varieties. Presence of seeds in the fruit drastically reduces its price, preventing possible commercialisation. Most citrus species are parthenocarpic, meaning that they usually produce fruit without seeds. This is also the case of Navelina sweet orange as this genotype is female sterile. In any case, if cross-pollination occurs, transgenes will be only expressed in the seed, which is never consumed.
Citrus varieties are not reproduced by seeds. In the incidental case that transgenic seedlings could germinate in an orchard, they would be removed by farmers as it is usually done with any citrus seedling germinating in any orchard. Moreover, these seedlings would never flower before being removed because citrus seedlings need several years to start flowering.


Brief description of any measures taken for the management of risks:
The field is being prepared according to normal practices for citrus cultivation. Former trees were removed and all their rests (including roots) were eliminated, the soil has been treated and cleaned to avoid weeds and phytopathological problems and it has been laser-levelled. The standard system of citrus cultivation will be used, removing weeds.
We propose to plant in the field 5 antisense lines (1-1, 2-3, 3-1, 5-1 and 7-1), one sense line (8-1) plus one control Navelina line and one control Navelina negra line (8 propagation per transgenic line and 16 propagations from each of the two controls).
There will be about 80 trees in this field trial.
The experimental field will be surrounded by a border line of Clemenules clementine trees that will serve as a buffer to investigate transgenic pollen flow frequency.

All rests coming from the transgenic trees of the field (whole plants, rests from pruning, fruit, etc.) will be burned at the incinerators that are being constructed in the area.


Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
Pollen dispersal frequency is being assessed annually since 2001 in a transgenic field trial authorized in 1997 (notification nº B/ES/96/15).
No experiment is being designed to study possible impact of the transgenic fruit in human health.


Final report
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European Commission administrative information

Consent given by the Member State Competent Authority:
Not known