Notification report

General information

Notification Number

Member State to which the notification was sent

Date of acknowledgement from the Member State Competent Authority

Title of the Project
Field trial with transgenic oranges over-expressing a flowering-time gene from Arabidopsis thaliana to investigate possible modification of growth and development.

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?

Has the same GMPt been notified elsewhere by the same notifier?

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 floral meristem identity gene APETALA 1 (AP1), from Arabidopsis thaliana in Pineapple sweet orange to induce early flowering in the transgenic plants and their progeny, thus generating rapid cycling trees. We want now to study whether over-expression of this transgene modifies growth and development of trrasgenic trees growing in the field.
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:

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 pROKII, which was used as the binary vector for A. tumefaciens mediated transformation of orange and citrange explants.

Cassette 35Spro::uidA::35SpA: 2800 nucleotides. The uidA gene comes from E. coli. 35S regulatory regions (promoter and terminator) come from the 35S gene from Cauliflower mosaic virus (CaMV). Transgene expression confers β-glucuronidase activity to the transgenic cells and tissues. This cassette is present in the T-DNA from pROKII, which was used as the binary vector for A. tumefaciens mediated transformation of orange and citrange explants.

Cassette 35Spro::AP1:: NOSter: 870, 1054 y 275 nucleotides, respectively.

Promoter and terminator regions come from the NOS gene from Agrobacterium tumefaciens, and they confer high and constitutive expression of the transgene. The AP1 transgene come from Arabidopsis thaliana. Its overexpression induces early flowering in transgenic citrus 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 flowering meristem identity genes from A. thaliana to investigate:
- Possible modification of tree growth due to profuse flowering.
- Possible modification of flower and fruit development.
- Fruit quality characteristics of fruits from AP1-sweet orange trees.

Southern and Northern 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 500 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 5 propagations from Pineapple sweet orange transgenic lines: AP1 166, AP1 393, AP1 481, AP1 532, AP1 554, AP1 555, AP1 569 and AP1 583, plus 5 propagations of Pineapple sweet orange control 1-7. There will be about 45 trees in the field.
All transgenic and control scions will be grafted onto vigorous citrange or C. macrophylla non-transgenic rootstocks.

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

European Commission administrative information

Consent given by the Member State Competent Authority:
Not known