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 Carrizo citrange overexpressing a GA20-oxidase gene in sense and antisense with the aim of modifying plant architecture, flowering and fruiting behaviour, and to investigate the performance of a non-transgenic variety grafted onto transgenic lines.

Proposed period of release:
30/03/2007 to 30/03/2017

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
carrizo citrangerutaceaecitronciruscitrus sinensis x poncirus trifoliata

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
The endogenous content of the gibberellin GA1 has been altered in the transgenic plants by overexpression of a GA 20-oxidase gene from citrus in sense or antisense. As a consequence of the genetic modification, plant architecture has been modified in the Carrizo citrange transgenic plants.
All sense, antisense and transformed control plants are also carrying a nptII transgene conferring resistance to kanamycin.

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 pBin19, which was used as the binary vector for A. tumefaciens mediated transformation of citrange explants (GenBank accesion of pBin19: nº U09365).
Cassette 35Spro 2X::CcGA 20-oxi1 sense or antisense:: 35Ster: 747, 1200 y 735 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 CcGA 20-oxi1 transgene comes from Carrizo citrange, more specifically from the Poncirus trifoliata parent. Its overexpression in sense or antisense induces an increase or decrease, respectively, of the accumulation of GA1 in growing shoots of the transgenic plants. Consequently, sense plants show an elongated growth pattern compared to control plants. Conversely, antisense plants are semi-dwarf and show shorter flushes than control transformed with the empty vector (pBin19).

6. Brief description of the method used for the genetic modification:
Co-cultivation of epicotyl segments from Carrizo citrange 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.

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:
Field trial of transgenic Carrizo citrange overexpressing in sense or antisense a gene encoding a GA 20-oxidase to study:
- modulation of tree architecture in plants growing in field conditions. For this purpose, we propose to transfer to the field 4 antisense lines (5A-8A, 5A-4, 5A 10 and 5A-33) and 3 sense lines (8S-8A, 8S-22 and 8S-23), plus one control line (CT-81A; empty vector) (1 sample tree per line).
- flowering and fruit set behaviour in the same trees.
- potential growth modulator effect of the transgenic trees used as rootstocks on a non-transgenic variety grafted onto them. For this purpose, we propose to transfer to the field between 8 and 13 cuttings from lines: 5A-8A, 5A-4, 5A 10 and 5A-33, 8S-8A, 8S-22 and 8S-23, CT-81A and Carrizo citrange non-transgenic control. All of them will be grafted with buds from non-transformed Clemenules clementine. We will investigate possible modulation of tree size, phenological characteristics of the transgenic trees and fruit production and quality.

2. Geographical location of the site:
The release site will be located at the experimental fields of the Instituto Valenciano de Investigaciones Agrarias, Moncada, Valencia, Spain.

3. Size of the site (m2):
About 1000 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:
Notification B/ES/96/15. The experiment has an extension of 1.638 m2. It has a total of 130 trees, including 16 transgenic plants of Pineapple sweet orange, 16 transgenic plants of Mexican lime and 16 transgenic plants of Carrizo citrange. In addition it has a similar number of non-transgenic plants of the same species and an external border of non-transgenic trees of Nules clementine. It has been introduced in the plants´ genome the marker genes nptII and uidA (GUS), that encode respectively for the neomycin phosphotransferase II and ß-glucuronidase enzymes.
We are investigating: Morphological and phenological characteristics of the transgenic trees, fruit quality, expression of the transgenes in leaves, flowers and fruits, stability of integration and expression of the transgenes, transmission of the transgenes to the progeny, and possibility of transgene dispersal through the pollen to non-transgenic monoembryonic citrus trees surrounding the transgenic area.
Since 2001, the number of transgenic seeds expressing the uidA gene has been yearly assessed in fruit produced by the border trees.
As border trees are very close to the transgenic trees (3 to 6 m), our results indicate that possibilities of transgene dispersal are very low in our conditions.

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. At the IVIA area, clementine orchards are very common, so there are not beehives in the surroundings (neither at IVIA experimental fields), preventing the possibilities of uncontrolled cross-pollination. 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, it has been laser-levelled, and a dropping watering system has been already installed. The standard system of citrus cultivation will be used, removing weeds and treating against pests.
We propose to plant in the field 4 antisense lines (5A-8A, 5A-4, 5A-10 and 5A-33) and 3 sense lines (8S-8A, 8S-22 and 8S-23), plus a control line (CT-81A; empty vector) (1 sample tree per line).
For the third objective of the trial, we propose to transfer to the field between 8 and 13 cuttings from lines: 5A-8A, 5A-4, 5A-10 and 5A-33, 8S-8A, 8S-22 and 8S-23, CT-81A and Carrizo citrange non-transgenic control. All of them will be grafted with buds from non-transformed Clemenules clementine.
There will be about 90 trees in the field.
In most trees only the rootstock will be transgenic, and the 6 transgenic Carrizo citrange non-grafted trees will be planted in the center of the field. In this way, we will favour that in case of presence of bees during the flowering season transgenic pollen will most likely be used to pollinate clementine flowers from the 80 surrounding trees of the experimental field.
All rests coming from the transgenic trees of the field (whole plants, rests from pruning, fruit, etc.) will be burned at the IVIA incinerator.

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