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

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
carrizo citrangerutaceaecitronciruscitrus sinensis x poncirus trifoliataCarrizo citrange

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 antisense. As a consequence of the genetic modification, plant architecture has been modified in the Carrizo citrange transgenic plants.

All transformed 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 accesión of pBin19: nº U09365).
Cassette 35Spro 2X::CcGA 20-oxi1 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 antisense induces a decrease of the accumulation of GA1 in growing shoots of the transgenic plants. Consequently, 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.

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:
- 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 20 cuttings from lines: 5A-8A and 5A-4, and CT-81A 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.
Southern and Northern blot analyses to demonstrate transgene integrity and expression as well as quantitative PCR analysis to determine the number of transgene integrations in each transgenic line.

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 575 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 20 cuttings from lines 5A-8A and 5A-4, and CT-81A control. All of them will be grafted with buds from non-transformed Clemenules clementine.
There will be about 60 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

European Commission administrative information

Consent given by the Member State Competent Authority:
Not known