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Notification report


General information

Notification Number
B/ES/08/04

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 oranges over-expressing a PR P23 osmotin-like protein to investigate tolerance to Phytophthora when these new genotypes are used as rootstocks.

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 sinensisPineapple

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
Ectopic overexpression of the PR P23 osmotin-like protein confers to some transgenic lines higher tolerance to the oomycete pathogen Phytophthora citrophthora, as shown in challenges performed under greenhouse conditions with potted propagations of the sweet orange transformants.

All transformed plants are also carrying uidA and nptII transgenes conferring β-glucuronidase activity and resistance to kanamycin, respectively.


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 orange explants (GenBank accesión of pBI121: nº AF485783).
Cassette NOSpro:: uidA:: NOSter: 3005 nucleotides. The uidA gene comes from E. coli. NOS regulatory regions (promoter and terminator) come from the nopaline synthase (NOS) gene from Agrobacterium tumefaciens. Transgene expression confers β-glucuronidase activity 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 orange explants (GenBank accesión of pBI121: nº AF485783).
Cassette 35Spro 2X:: AlMV RNA4 leader:: PR P23:: NOSter: 847, 34, 967 and 277 nucleotides, respectively. Promoter and terminator regions come from the 35S gene from the cauliflower mosaic virus (CaMV) and NOS gene from Agrobacterium tumefaciens, respectively, and they confer high and constitutive expression of the transgene. The leader sequence from RNA 4 of Alfalfa mosaic virus (AlMV) favors transcript stability and stable translation. The PR P23 transgene comes from tomato (Solanum lycopersicum). Its overexpression confers higher tolerance to Phythophthora to the transgenic plants.


6. Brief description of the method used for the genetic modification:
Co-cultivation of internodal stem segments from Pineapple sweet orange seedlings 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 with transgenic oranges over-expressing a PR P23 osmotin-like protein to investigate:
- potential higher tolerance to Phytophthora-induced gummosis of the transgenic orange trees used as rootstocks. Non-transgenic clementine will be used as scion.

Southern and Western blot analyses demonstrate transgene integrity and expression.


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 700 m2 approximately.

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. 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.

In this particular case, only the rootstock will be transgenic. Then the possibility of transgene dispersal through the pollen will be insignificant.


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 15 cuttings from lines 2.31a and 9.1a, and 2.15 control. All of them will be grafted with buds from non-transformed Clemenules clementine.
There will be about 75 trees in the field.
In all trees only the rootstock will be transgenic.

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