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Notification report


General information

Notification Number
B/CZ/09/05

Member State to which the notification was sent
Czech Republic

Date of acknowledgement from the Member State Competent Authority
28/12/2009

Title of the Project
Biotechnology Tools for Improving Disease Resistance and Seed Quality in Legumes; Functional Genomics and Proteomics in Plant Breeding

Proposed period of release:
01/04/2010 to 01/10/2019

Name of the Institute(s) or Company(ies)
Agritec, Research, Breeding & Services, Ltd., ;


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
peafabaceaepisumpisum sativum

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
Six types of modified peas a) – f) will be tested in field trials:

a) for resistance to fungal pathogenes or insect pests mediated by the insertion of a modified gene for serine protease inhibitor (gmSPI-2) fused to uidA (GUS) reporter gene, with a selectable bar gene, no previous modification

b) for resistance to fungal pathogenes or insect pests mediated by the insertion of a fused gmSPI-2 gene for serine protease inhibitor with green fluorescent protein (GFP) reporter gene, with a selectable bar gene, no previous modification

c) showing enhanced accumulation of seed proteins due to the insertion of plant Dof gene, with a selectable nptII marker gene, no previous modification

d) with earlier development of seed embryo caused by the insertion of plant L1L gene, with a selectable nptII marker gene, no previous modification

e) for resistance to Pea Enation Mosaic Virus (PEMV) mediated by the insertion of a sequence coding for viral coat protein

f) for resistance to Pea Seed-borne Mosaic Virus (PSbMV) mediated by the insertion of a sequence coding for viral coat protein


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:
a) Expression of a new (fused) serine protease inhibitor gmSPI-2gene of Wax Moth (Galleria mellonella) could under certain conditions reduce the attack of pea plants by fungal pathogenes or insect pests. The gene sequence of this protease inhibitor used for plant transformation has been codon-optimized to allow its expression in plants.
The CaMV 35S promoter from the Cauliflower mosaic virus controls expression of a fused bacterial (Escherichia coli) reporter uidA gene (β-glucuronidase) with gmSPI-2 of Wax Moth (Galleria mellonella) gene sequence. The CaMV 35S promoter enhancer Triple X is included to increase the transcription of gmSPI-2 gene. The octopine synthase (OCS) terminator of Agrobacterium tumefaciens terminates transcription of gmSPI-2 gene. There are two fragments of lacZ operon of E. coli present between the fused target gene was cloned in. The nopaline synthase (NOS) promoter of Agrobacterium tumefaciens drives the expression of a selectable bar gene. The bar gene (phosphinothricin acetyltransferase) of the soilborne bacterium Streptomyces hygroscopicus mediates resistance against the herbicide Bialaphos (glufosinate ammonium). Its intended function is to enable the in vitro selection of transformed pea plants.
Left and right border sequences from Agrobacterium tumefaciens.

b) Expression of a serine protease inhibitor gmSPI-2 gene (another fused variant) of Wax Moth (Galleria mellonella) could under certain conditions reduce the attack of pea plants caused by fungal pathogenes or insect pests.
All components of the insert are same as in a), except that the gmSPI-2 gene is covalently bond to a sequence of a second reporter – the green flourescent protein (gfp) gene of jelly fish Aequorea victoria

c) Plant Dof (DNA-binding with one finger) gene coding for a domain protein which is one of specific plant transcription factors with highly conservative DNA-binging domain containing mainly simple C(2)-C(2) zinc finger. It is supposed that Dof could enhance the accumulation of seed proteins and thus increase the yield of pea transgenic lines.
The CaMV 35S promoter from the Cauliflower mosaic virus controls expression of plant (Arabidopsis thaliana L.) Dof gene. A. tumefaciens octopine synthase (OCS) terminator terminates the transcription of the Dof gene. The nopaline synthase (NOS) promoter of A. tumefaciens is used to control expression of the selectable nptII gene. The nptII gene of E. coli coding for an enzyme neomycin phosphotransferase serves as a selectable marker during transformation.
Left and right border sequences from Agrobacterium tumefaciens

d) Plant gene L1L (LEC1-LIKE) belongs into a group of genes influencing plant embryo development, especially during its early stage. It is a transcription factor coding for Hap3 subunit of CCAAT box-binding factor. The gene is expressed in embryogenic cells only. Enhanced L1L gene expression in plants could stimulate earlier seed embryo development and thus the earlines (sooner development) of modified pea lines and cultivars.
All components of the insert are the same as in c), except that the L1L gene is present instead of Dof.
Left and right border sequences from Agrobacterium tumefaciens

e) Expression of PEMV coat protein sequence (cpPEMV) in transgenic plants interferes with the mechanism of virus multiplication what causes delay in a disease progress.
The CaMV 35S promoter from the Cauliflower mosaic virus controls expression of a bacterial (E. coli) reporter uidA gene (β-glucuronidase). The CaMV 35S promoter drives the expression of PEMV coat protein sequence, its transciption is terminated by polyA terminator of CaMV. There are two fragments of lacZ operon of E. coli present between which the target- and reporter genes were cloned. The nopaline synthase (NOS) promoter of A. tumefaciens drives the expression of bacterial (S. hygroscopicus) selectable bar gene. The bar gene (phosphinothricin acetyltransferase) mediates resistance to the herbicide Bialaphos (glufosinate ammonium). Its intended function is to enable the in vitro selection of transformed pea plants. Left and right border sequences from Agrobacterium tumefaciens.

f) Expression of PSbMV coat protein sequence (cpPSbMV) in transgenic plants interferes with the mechanism of virus multiplication what causes delay in a disease progress.
The CaMV 35S promoter controls the expression of a bacterial (E. coli) reporter uidA gene (β-glucuronidase). The CaMV 35S promoter drives the expression of PSbMV coat protein sequence linked motifs in a sense- and antisense orientation, transciption its is terminated by polyA terminator of CaMV. There are two fragments of lacZ operon of E. coli present between which the target- and reporter genes were cloned. The nopaline synthase (NOS) promoter of A. tumefaciens drives the expression of bacterial (S. hygroscopicus) selectable bar gene. The bar gene (phosphinothricin acetyltransferase) mediates resistance against the herbicide Bialaphos (glufosinate ammonium). Its intended function is to enable the in vitro selection of transformed pea plants. Left and right border sequences from Agrobacterium tumefaciens.


6. Brief description of the method used for the genetic modification:
co-cultivation with Agrobacterium tumefaciens

7. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination:
not applicabe

Experimental Release

1. Purpose of the release:
All transgenic lines – selection of breeding material, next breeding, evaluation of qualitative and quantitative parameters, assessment of safety to the environment
gmSPI-2 transformants – tests of resistance to biotic and abiotic stress, resistance to fungal diseases and insect pests
Dof and L1L transformants – field performance, yield characteristics
cpPEMV and cpPSbMV transformants – virus resistance, field performance, yield characteristics


2. Geographical location of the site:
Vikyrovice, region Olomouc (Nothern Moravia, district of Sumperk)

3. Size of the site (m2):
total max. 1000 m2 , 100–300 m2 actual plot size for planting of transgenic pea with conventional pea controls

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:
not released yet

Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
As to the present state of knowledge we do not expect any increased selective advantage of modified peas in natural environment. Peas are strictly self pollinating, no other crossable species exist on the territory of the Czech Republic, seed loss during the trials is prevented by several measures (listed below). If any seed is consumed by animals, no impact on the health of these organisms is expected, because all introduced target genes are of natural origin, do not code for any known toxins or alergens. Safety of reporter genes (uidA and gfp) to animals was confirmed repeatedly in previous literature studies. Also potential risks of selectable markers (nptII and bar) were assessed earlier (EFSA) and found as negligible. Therefore because of the risk assessment results and in combination with the planned measures (as listed at point E) no impact on the surrounding environment of the experimental plot is expected.
In case of gmSPI-2, gmSPI-2::GFP modifications the effect is targeted to insect pests of peas, especially the following species Sitona lineatus, Bruchus pisorum and Acyrtosiphon pisum. Serine protease inhibitors produced by GM plants may inhibit the activity of proteases in digestive organs of these pests what could in some inhibitor concentration cause their increased mortality due to the starvation. The effect of gmSPI-2 and gmSPI-2::GFP genes against pathogenic fungi is based on an inhibition of fungal proteases active during the pathogene throughout. Modifications with L1L and Dof transgens are not aimed to have any influence on other organisms. Based on a wide prevalence of L1L and Dof genes among higher plants the existence of target organisms is not expected.


Brief description of any measures taken for the management of risks:
Planting, sampling and harvesting will be done either by a special small scale machinery or by hand (depending on individual plot size).
The plot will be situated to inner parts of the whole experimental area only. Next plot will be isolated by a separating distance (at least 100 m of conventional peas), 3 m wide band of soil free of any plants around the whole plot, borders of pea variants will be surrounded by cereals.
The whole plot will be covered by a net which prevents seed dispersal by birds.
Harvested plants will be additionally covered by a second net to prevent seed loss.
Harvested seed material will be transported under containment (closed boxes).
Observation of the site in regular intervals (at least 1 × in 2 weeks careful evaluations) is planned, checks in shorter period of time (up to daily during two the growing season).
After the termination of the field trial, such plant species will be planted at the site which allow easy detection of any volunteer pea plants upcoming in the following year.


Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
not applicable

Final report
-

European Commission administrative information

Consent given by the Member State Competent Authority:
Yes
30/04/2010 00:00:00
Remarks: