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Notification report


General information

Notification Number
B/ES/03/37

Member State to which the notification was sent
Spain

Date of acknowledgement from the Member State Competent Authority
14/04/2003

Title of the Project
Evaluation of isolation distances and containment barriers to control the gene flow between transgenic and non- transgenic rice plants from the same cultivars. Introgression to the red rice weed.

Proposed period of release:
05/01/2004 to 30/10/2006

Name of the Institute(s) or Company(ies)
IRTA (Institut de Recerca i Tecnologia Agroalimentàries), Passeig de Gràcia 44. Barcelona.Spain;


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
ricepoaceaeoryzaoryza sativaSenia

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
Three transgenic lines containing the bar gene, conferring resistance to the herbicide ammonium glifosinate will be used. These lines are: line S-1B containing bar and gusA genes; line G9 bar-gfp, containing bar, gusA and hptII genes and line G9-bar, containing only the bar gene.

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:
All lines were obtained by using the Agrobacterium mediated transformation technique. The T-DNA contains:

Line S 1B : p35S:bar:t35S::p35S:gusA:tnos (This line has been used in previous field trials (B7ES/00/07 and B/ES/01/07)
Line G9-bar: pUbi:bar:tnos
Line G9-bar gus: (two T-DNAs inserted in the same loci): pUbi:bar:nos and pgos2:gfp: trbsc::p35S:hptII:t35S.

The source and function of each constituent fragments are:

p35S: function: promoter from ARN 35S from CaMV
Source: cauliflower mosaic virus.


pgos2: function: promoter from gos2 rice gene (de Pater et al., 1992).
Source: Oryza sativa


pUbi: function: ubiquitin constitutive promoter (Christensen et al., 1992)
Source: Zea mays

tNos: function: nopaline-syntase terminator.
Source: pTiT37 plasmid from Agrobacterium tumefaciens.

trbcS: function: terminator from rice rbcS gene (Matsuoka et al., 1988)
Source: Oryza sativa


t35S: function: terminator from CaMV ARN 35S
Source: cauliflower mosaic virus.

lacZ alpha: function: -galactosidase codifying sequence. It is not express in plants, only in bacteria.
Source: Escherichia coli

bar: function: phosfinotricine acetyl transferase codifying sequence.
Source: Stretomyces hygroscopicus

gusA: function: codifying sequence of -glucuronidase.
Source: Escherichia coli (Jefferson et al. 1987). Modified by CAMBIA by introducing an intron to avoid its expression in plant tissues (Ohta et al., 1990).

hptII Function: hygromycin phosfotransferase II codifying sequence.
Source E. Coli (Gritz y Davies, 1983).

GFPS65T: Function: codifying sequence from the mutant protein GFP: Ser 65Thr .
Source: gene gfp from Aequorea victoria (Heim et al, 1995).


6. Brief description of the method used for the genetic modification:
Rice Senia cv was modified by using the Agrobacterium mediated transformation technique according to Pons et al, 2000.

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:
There are two different field trials:
Field trial A: aimed at establishing the security distances between transgenic and non-transgenic plants and to study the efficiency of containment barriers on controlling the gene flow. Transgenic lines with different markers will be placed at different distances from non-transgenic plants in such a way that cross-pollination inside and outside the field will be monitored by analyzing the samples of seeds collected at the end of the culture in different places and taking into account the wind directions. Moreover, a “natural barrier” will be placed between transgenic and non-transgenic plants in one half of the field. This field trial will be carried out on 2005.

Field trial B: aimed at assess the introgression of transgenes into red rice, the only one weed compatible with cultivated rice in Europe. This field trial will be carried out during 3 years (2004-2006). Each year transgenic plants with a different molecular marker will be used in combination with four different agricultural practices, the most commonly used by rice growers to control the red rice weed. Moreover, the first year a known number of red rice plants, equivalent to a high infestation level will be planted among transgenic plants. This strategy will allow, at the end of the field trial, to evaluate the contribution of each year to the final introgression produced and will give a valuable overview of the effect of the different agricultural methods on red rice control among the three years of culture. The plots will be divided in three sections in such a way that the first year only line S-bar will be planted. On second year, line S-bar-gus will be planted in 2/3 parts of the plot whereas the third year, only th1/3 of the plot will be planted with the S-bar-gfp line. Non-transgenic plants from the same variety will surround all these plots. At the end of the growing season samples of red rice seeds from each sub-parcel and from surrounding non-transgenic plants will be analyzed to determine the hybridization and the introgression rate.

As described in C.4, field trials carried out in the same place (B/ES/99/16, B7ES/00/07 and B/ES/01/07) clearly demonstrated that gene flow from transgenic to non-transgenic plants takes place to some extent but strongly decreases as distance increases from 1 to 10m and there is a clear influence of the dominant wind. Nevertheless, results obtained with these field trials -that had a circular design and little size- needs to be confirmed in a commercial-size field, in order to be able to establish some general regulations to be applied in rice growing areas.

On the other hand, hybridization between transgenic and red rice takes place (see C.4) but there are not enough data to establish de degree of introgression that could take place when a proper agricultural technique focused to the control of this weed is applied.

Genes transferred to transgenic plants used in this release do not represent any selective advantage in comparison with non-transgenic plants, except in the use of ammonium glufosinate herbicide. Nevertheless, this herbicide is not currently used in rice crop. Safety of phosphinothricin acetyltransferase is well known. In relation to the marker genes, gusA gene encodes for -glucuronidase protein, widely present in nature and the gfp protein is widely used in medical tests.

Studies carried out in greenhouse conditions have shown that introduced genes do not change the dissemination ability of transgenic plants and that its agricultural behavior is similar to that of non-transgenic plants.


2. Geographical location of the site:
IRTA Experimental Station. (Amposta) Tarragona, Spain

3. Size of the site (m2):
Field trial A: determination of security distances and efficiency of containment barriers: 1720 m2 (880 m2 with transgenic plants).
Field trial B: introgression to red rice: 2700 m2 (1920 m2 with transgenic plants)


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:
A circular field trials designs (B/ES/00/07 and B/ES/01/07) were carried out to assess the frequency of pollen-mediated gene flow from a transgenic rice line S 1B, harbouring the gusA gene and the bar gene encoding respectively ß-glucuronidase and phosphinothricin acetyl transferase as markers, to conventional rice in the Spanish japonica cultivars Senia. Frequencies of gene flow based on detection of herbicide resistant, GUS positive seedlings among seed progenies of recipient plants and averaged over all the wind directions were 0.086 ± 0.007. However, a clear asymmetric distribution was observed with pollination frequency favoured in plants placed under the local dominant winds. Southern analyses confirmed the hemizygous status and the origin of the transgenes in progenies of surviving, GUS positive plants. Examination of the influence on gene flow frequency of the distance from the transgenic source to recipient plots of conventional rice planted at 1, 2, 5 and 10 m distance revealed a clear decrease with increasing distance which was less dramatic under the dominant wind direction. The precise determination of the local wind conditions at flowering period and pollination day time appear of primary importance for setting up suitable isolation distances.

The same field trials designs were used to evaluate the gene flow to red rice placed at a different distances from the transgenic plants and how the wind could influence the gene flow to red rice plants growing in the borders. Frequencies of gene flow averaged over all the wind directions were 0.036 ± 0.006 %. However, as in the case of conventional rice, a clear asymmetric distribution was observed with pollination frequency favored in plants placed under the local dominant winds. Nevertheless within a commercial transgenic rice field the influence of the wind appears a less determinant factor because red rice plants usually will grow isolated or in patches surrounded by transgenic plants and consequently can be pollinated by all of them. On the other hand, the wind influence on cross-pollination has to be taken into account for the plants growing in the borders. This is a very essential question to consider because the real introgression of the genes will be minimized inside the field by the usual control practices tending to destroy the red rice but the wild plants in the borders can act as reservoirs of the transgenic characters. Moreover, although the gene flow values are relatively low, the shattering and dormancy of the red rice seeds, which ensure their persistence in the field, lead into an undesirable effect of durability of the transferred genes. In consequence, whether one wants to avoid gene flow to the red rice, crop management has to be changed. In this sense, further studies are needed to evaluate how different agricultural practices may control the effective introgression of transgenes into the red rice. These studies are needed to design new methodological tools for assessing systemic effects within the diversity of environmental systems in which GMOs may be cultivated.


Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
Introduced genes could not confer an increased selective advantage in natural environments to transgenic plants because the herbicide ammonium glufosinate is not commonly use in rice fields.

One of the most potential environmental impacts of the release of the GMPts is the risk of transgene spread throughout cross-pollination. As has been described in C.4, we have quantified the gene flow by using circular designs. The objective of the field trials proposed here is to confirm if it is possible to minimize the gene flow by increasing security distances or by establishing containment barriers. On the other hand, it is very important to establish at what degree the introgression of transgenes to the red rice takes place in field conditions and to know if it can be controlled by the agricultural practices commonly used in controlling this weed.


Brief description of any measures taken for the management of risks:
To prevent out-crossing with neighboring rice fields the trial will be located 30m from any conventional rice field. It has to be taken into account that the security distance recommended by plant breeders is of 10 m.

Field trial will be keep free of weeds with the exception of red rice introduced in field trial B. Weekly controls of agronomic traits will be performed.

At the end of culture samples of seed will be harvested manually. The rest of seed will be burned. The vegetal parts will be burned on the plot or shallow incorporated in the soil. The plot will be monitoring for re-growth and eventual volunteer plants will be eradicated or analysed depending on the field trial. In case of an emergency the plants can be destroyed mechanically or by applying herbicides.


Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
As described in C.4 and D, these field trials will contribute to better knowledge on gene flow in rice and to establish proper regulations to be applied in case transgenic rice plants could be introduced in Europe.

Final report
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European Commission administrative information

Consent given by the Member State Competent Authority:
Yes
01/12/2003 00:00:00
Remarks:
The Competent Authority for the consent of these field trials is Cataluña.