Member State to which the notification was sent
Date of acknowledgement from the Member State Competent Authority
Title of the Project
Validation of a concept of long-term resistance to the rhizomania virus (BNYVV): Field evaluation of sugar beet hybrids genetically modified to be resistant to BNYVV (experimental programme: 2003-2006)
Proposed period of release:
01/03/2003 to 30/09/2006
Name of the Institute(s) or Company(ies)
ADVANTA France, Zone Industrielle
Route de Lavardac
3. Is the same GMPt release planned elsewhere in the Community?
Has the same GMPt been notified elsewhere by the same notifier?
If yes, notification number(s):
Genetically modified plant
Complete name of the recipient or parental plant(s)
vulgaris var. saccharifera
2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
The sugar beet was modified with two sequences intended to be expressed in the transformed plant:
- The P15-4 gene
The widespread virus disease of the sugar beet plant called rhizomania is caused by a benyvirus, the beet necrotic yellow vein virus (BNYVV), which is transmitted to the root of the beet by a soilborne fungus Polymyxa betae.
The genome of the BNYVV consists of five plus-sense RNAs, two of which (RNAs 1 and 2) encode functions essential for infection of all plants, while the other three (RNAs 3, 4 and 5) are implicated in vector-mediated infection of sugar beet roots. Cell-to-cell movement of BNYVV is governed by a set of three viral genes on RNA2, so called 'triple gene block' (TGB), which encodes the viral protein known as P42, P13, and P15.
The sugar beet has been modified by the insertion of a nucleotide sequence derived from the P15 gene of BNYVV. The P15 gene encodes protein that is involved in the formation of a specific complex necessary to the cell-to-cell movement of the virus.
The original P15 gene was modified by substitution of two amino acids, prior to sugar beet transformation. The resulting modified P15 sequence, called P15-4, is not functional in the virus.
The suggested action mechanism is that the expression of the modified P15 sequence in sugar beet transformants interferes with the mechanisms of virus translocation within the sugar beet. The modified gene encodes a product that competes with the wild P15 protein of the virus, for the formation of a complex with the other proteins of the TGB or with other sites of the complex.
The expression of the modified P15 sequences in sugar beet confers a high level of resistance to BNYVV. In particular the transformation events described in the present application, MOX 63 and MOA 20, are resistant to the virus.
The modified P15 sequence is linked to the promoter of the ubiquitin gene of Arabidopsis thaliana (Ubi) and the NOS terminator.
Ubiquitin is a highly conserved protein that is found in all eucaryotes. The ubiquitin pathway is required for the degradation of cellular proteins and nitrogen recycling. In plants, ubiquitin is involved in the regulation of stress responses and in the processus of senescence.
The Ubi promoter constitutively expresses the genes it is linked to.
- The pat gene
Synthetic pat gene encodes phosphinotricine acetyltransferase that confers tolerance to the herbicide glufosinate-ammonium by catalysing a reaction that inactivates it.
In this project, the pat gene was used as a selection marker during the genetic transformation and tissue culture phases.
The pat gene is flanked by the CaMV 35S promoter and terminator sequences.
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:
Two transformation events are described in this application. They are named MOX 63 and MOA 20.
The plasmid pS48 was used to produce the transformation event MOX 63. The plasmid pS51 was used to produce the transformation event MOA 20.
Both pS48 and pS51 plasmids contain two genes with regulatory sequences, which are expressed in the plant.
- The P15-4 gene
The modified P15 gene derives from the P15 gene of BNYVV. The P15 gene encodes a protein that is involved in the formation of a specific complex necessary for the cell-to-cell movement of the virus.
The original P15 gene of BNYVV was modified by the substitution of two amino acids prior to sugar beet transformation. The resulting modified P15 sequence, P15-4, is not functional in the virus.
It is suggested that the expression of the modified P15 sequence in sugar beet transformants interferes with the mechanisms of virus translocation within the plant. The modified genes encode a product that competes with the wild protein of the virus for the formation of a complex with the other protein of the TGB or with other sites of the complex.
The expression of the modified P15 sequences in sugar beet confers a high level of resistance to BNYVV.
The modified P15 sequence is linked to the promoter of the ubiquitin gene of Arabidopsis thaliana (Ubi) and the NOS terminator from Agrobacterium tumefaciens.
- The pat gene
The synthetic pat gene conferring tolerance to the herbicide glufosinate-ammonium is linked to the 35S promoter and the 35S polyadenylation signal from CaMV.
The pat gene was used as marker gene during the phases of transformation and regeneration.
The plasmid pS48 derives from pUC18. pS48 contains the bacterial gene, amp, which codes for the (beta-lactamase and confers resistance to ampicillin.
The amp gene was used to select the E. coli containing the plasmid while preparing sufficient DNA for protoplast transformation experiments. The expression of the amp gene in E. coli complements non-resistant E. coli, and the transformed cells can be selected on ampicillin containing medium. The amp gene is under control of bacterial regulatory sequences and is not expressed in the transgenic plants.
The plasmid also contains the origin of replication of the plasmid in E. coli (ORI ColE1)
The plasmid pS51 derives from the pIGPD7 plasmid. pS51 contains the yeast HIS3 gene, isolated from Saccharomyces cerevisiae, which was used to select E.coli cells containing the construct while preparing sufficient DNA for protoplast transformation experiments.
The HIS3 gene encodes the sequence for the enzyme imidazolglycerol phosphate dehydratase of the histidine pathway. The expression of that gene in E. coli complements E. coli HisB- mutants and transformed cells can be selected on minimal growth media. This gene is under control of yeast regulatory sequences and is not expressed in plant if present in the transgenic plant. Absence of expression in the transformants has been showed experimentally.
Plasmid pS51 also contains the origin of replication of the plasmid in E. coli (ORI: ColE1)
6. Brief description of the method used for the genetic modification:
The primary transformants MOX 63, MOA 20 were obtained by applying PEG mediated DNA transformation to stomatal guard cell protoplasts from which a plant can be regenerated.
1. Purpose of the release:
The purpose of the experimental release programme is to validate a concept of long-term resistance to the rhizomania virus (BNYVV), based on the interaction of the transgene products in the plant cell with the processus of virus movement in the infected plant.
The release programme is part of a research project aimed at the diversification of sugar beet rhizomania resistance sources. The major objective of a transgenic resistance is to confer the immunity of the beet in all viral infection conditions.
The experimental transformants described in this application encode a sequence coding for a modified BNYVV P15 protein (P15-4). The first data obtained from bioassays conducted under controlled conditions indicate that the expression of the P 15-4 sequence in transgenic sugar beet confer a high level of resistance to BNYVV. In particular, the primary transformants MOX 63 and MOA 20 are resistant. These data were confirmed in the field experiments conducted in France, in 2001 and 2002, in soil naturally infected with BNYVV (release permit B/FR/01/02/02).
The field experiments that are proposed in this application are necessary to demonstrate that
(1) the expected mechanism leads to the rapid blocking of the virus multiplication and
diffusion through the beet root, in natural infection conditions;
(2) the expression of the tested sequence confers resistance to different BNYVV sources.
(3) the constitutive expression of the modified P15 sequence confers immunity to the
beet, all over the season;
(4) the expression of the tested sequence restores normal root yield.
2. Geographical location of the site:
There will be two trials in 2003, which will be conducted in BNYVV infected soil.
One trial site will be chosen in an area, where the most agressive strain of the BNYVV virus is found (pathotype P). The other trial site will be selected in an area where the most common strain of BNYVV is present (pathotype B).
3. Size of the site (m2):
The size of each trial will not exceed 2500m2.
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:
The transformation events were produced to validate a new concept of resistance to BNYVV, based on the interaction of the transgene products in the plant cell with the processus of virus movement in the infected plant.
The transgenic material described in this application is strictly experimental. The sugar beet produced in the trials are not intended to feed or to food. At the end of the trials, the beets will be destroyed and incorporated into the soil.
The first data obtained from bio-assays conducted under confined culture conditions indicate that the primary transformants MOX 63 and MOA 20 express a high level of rhizomania resistance. These data were confirmed in the first field trial conducted in France, in 2001 and 2002, in soil naturally infected with BNYVV (release permit B/FR/01 /02/02).
The first data collected from greenhouse or in the field did not show significant phenotypic differences between the plants derived from the transformation events described in this application and non-transgenic plants grown under similar conditions.
Two generations of seeds were produced from each transformation event. The observations made during these crosses indicate that the characteristics of reproduction of the transgenic plants are not significantly different from those of the non-transgenic lines grown under similar conditions.
Sugar beet is a biennial plant that requires a period of vemalisation to flower. Like the original non-transgenic line used in transformation, both the transformation events MOX 63 and MOA 20 need a period of vernalisation to bolt and to flower.
In normal culture conditions, the sugar beet root crops do not flower. As expected, all the plants derived from MOX 63 and MOA 20 remained vegetative during the field releases conducted in 2001 and 2002 (release permit B/FR/O1/02/02).
The time needed after the vernalisation period to flower and to produce mature seeds is similar to that of non-transgenic lines grown under the same conditions. The amount of seeds harvested per plant is in the range observed for non-transgenic lines grown in similar conditions.
The data collected in greenhouse and in the field from lines and hybrids derived from transformation events MOA 20 and MOX 63 do not show any behavior significantly different from that of non-transgenic lines or hybrids grown under similar conditions.
c. Survival potential
The criteria's generally used to characterise sugar beet seeds were applied to the seeds derived from the transformation event MOX 63 and MOA 20. The obtained data were not significantly different from those of non-transgenic seeds produced under similar conditions.
In the field releases of 2001 and 2002, the beets were mechanically harvested at the end of the experiment. According to the protocol generally used by Advanta, the leaves and the pieces of roots were left in the trial site and they were incorporated into the soil by rotavation. The release sites were regularly monitored after harvest. No sugar beet regrowth was observed in the sites of the release.
Environmental Impact and Risk Management
Summary of the potential environmental impact from the release of the GMPts:
Sugar beet is not recorded as an invasive species, nor is sugar beet or any species of the genus Beta a weedy species in any other environment nor in any other crop, except in the sugar beet crop. The data obtained so far from lines and hybrids derived from the transformation events MOX 63 and MOA 20 do not indicate that the genetic modification changes the behavior of the sugar beet in the environment, except in the presence of BNYVV.
In normal culture conditions, the sugar beet root crops do not flower. The monitoring visits conducted regularly in the trial will ensure that any bolting beet occurring in the site will be identified and will be destroyed immediately, prior to flowering. The probability of diffusion of the transgene through pollen or hybridization with other non-genetically modified sugar beet plants or volunteer beet is considered to be negligible.
The release sites are in an agricultural ecosystem. The agronomic practices will be those traditionally used in a sugar beet yield trial. No particular effect of the genetically modified plants is expected on the environment that would differ from that of a conventional sugar beet crop.
The strategy explored in this project is to confer BNYVV resistance to the plant by blocking the cell-to-cell movement of the virus. To achieve this goal, a gene of the `triple gene block' necessary to the movement mechanism of the virus, the P15 gene, was isolated and mutated so that its expression in the plant blocks virus multiplication and diffusion mechanism into the plant.
The P 15 gene was modified so that the product it encodes is different from the original viral protein and is consequently inactive in the virus. It is not expected that the genetic modification have an ecological incidence on the BNYVV virus.
The environmental impact of the interactions between the genetically modified sugar beet plants and non-target organisms is not expected to be different from that arising from a trial of non-genetically modified sugar beet.
The transgenic material described in this application is strictly experimental. The data produced in the field releases will contribute to evaluate the opportunity of developing this new technology.
The global risk to the environment associated with the proposed trials is considered to be negligible.
Brief description of any measures taken for the management of risks:
A detailed protocol will be produced prior to sowing, and will be communicated to the team in charge of the trial. The protocol will describe all the operations to conduct in the release site, including observations, notations and sampling and in particular the specific measures taken in a release of genetically modified sugar beets. The technicians in charge of the trial will use the trial logbook to record all the operations carried on in the trial. The logbook will be validated by the responsible in charge of the trial.
The trials will be surrounded by a 5m border of naked soil that will isolate the transgenic beets from the other crops.
Before sowing, the trial seeds will be packed in closed bags. After sowing, the drill machine will be cleaned. Any remaining seed will be put in a closed bag and will be sent back to the laboratory to be destroyed.
The beets in the trials will not be allowed to flower. Regular visits of the trial by experienced staff will ensure the early detection of any bolting plant. Procedures will then followed to destroy the bolting plant, long before flowering.
At the end of the experiment, the beets will be mechanically' harvested. The leaves will be cut off, the roots will be harvested, washed and weighted. They will be sliced and pulp samples will be taken and frozen in closed plastic vials. All these operation will be done in the trial area.
After harvest of the yield trials, plant debris and wastewater will be spread on the trial site and incorporated into the soil by rotavation.
The site will not be used for sugar beet cultivation for the following two years, during which all volunteer beets that may appear will be destroyed. The only authorised crops will be those using herbicides lethal to the beets (for example, cereals).
The trial site will be visited during the two years following the release by experienced technicians of Advanta.
Treatment of the site, other than already described, will be based on previous experience of sugar beet cultivation.
European Commission administrative information
Consent given by the Member State Competent Authority: