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Notification report


General information

Notification Number
B/ES/16/03


This notification has been withdrawn by the notifier
07/12/2016


Member State to which the notification was sent
Spain

Date of acknowledgement from the Member State Competent Authority
01/02/2016

Title of the Project
Evaluation of a transgenic corn Bt line (Cry1Ac, Cry1C and Vip3) and its hybrid Carolight (CaroBt).

Proposed period of release:
15/05/2016 to 31/12/2016

Name of the Institute(s) or Company(ies)
University of Lleida, ;


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
maizepoaceaezeazea maysmays

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
The transgenic line Carolight that accumulates 169-times the normal amount of β-carotene, 6 times the normal amount of ascorbate and twice the normal amount of folic acid do not is protected against the corn borers. Corn attack by these plagues causes damage in corn stem and cob so we generated a transgenic plant (L-Bt-4) containing 3 genes with insecticidal activity (Cry1Ac and Cry1C Vip3) derived from the bacterium Bacillus thuringiensis. These genes are described in detail in subsequent sections. This L-Bt-4 line was crossed with the Carolight maize line. Thus we have achieved nutritionally improved maize and resistance to insect CaroBt.

The objective of this notification is describe how it will be the first release of the inbreed line L-Bt-4 of genetically modified corn (20 plants) and the hybrid of its cross with the transgenic line Carolight (hybrid CaroBt, 20 plants). The evaluation of L-Bt-4 with respect to their original line Carolight will be done in the following aspects:
- agronomical characterization.
- effects on pests and natural fauna.


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:
The marker gene bar and 3 genes/cDNAs encoding 3 insecticide proteins:
Gen Cry1Ac
Cry1Ac cDNA (1.8 Kb) was cloned according from Bacillus thruringiensis and inserted in PAL76 (BamHI /EcoRI) plasmid containing the constitutive promoter of corn Ubicuitine-1 and the first and terminal intron of nopalina sintase.

Cry1C
Cry1C gene (2.856 bp) encoding for a 108-kDa protein was cloned from Bacillus thruringiensis and inserted in PAL76 plasmid (BamHI/HindIII) containing the constitutive promoter of corn Ubicuitine-1 and the first and terminal intron of nopalina sintase.

Vip3
Vip3Ba1 gene was cloned from Bacillus thuringiensis strain ucr8. The whole sequence of vip3Ba1 gen is 2,406 bp and encode the VipBa1 protein of 801 amino acids (AY823271). The cDNA was inserted in AL76 plasmid (BamHI/HindIII) under the control of the constitutive promoter of of corn Ubicuitine-1 and the first and terminal intron of nopalina sintase.


6. Brief description of the method used for the genetic modification:
Corn Transformation. Corn plants (Zea mays L., cv. M37W) were grown in the greenhouse and growth room at 28/20 °C day/night temperature with a 10-h photoperiod and 60–90% relative humidity for the first 50 days, followed by maintenance at 21/18 °C day/night temperature with a 16-h photoperiod thereafter. Immature zygotic embryos were excised at 10–14 days after pollination and cultured on N6 medium. After a further 5 days, the embryos were transferred to N6 medium containing high osmoticum (0.2Mmannitol, 0.2M sorbitol) for 5–6 h before bombardment, then bombarded with 10 mg of coated gold particles, as previously described. Bombarded callus was selected on phosphinothricin-supplemented medium, and transgenic plantlets were regenerated and hardened off in soil. A population of 75 transgenic plants was regenerated and screened by genomic PCR to identify primary transformants containing all 3 input transgenes.

Molecular analysis. The genomic PCR was carried out by using sets of primers for each gene, generating overlapping products. Transgene expression was verified by mRNA blot analysis, and one transgenic line (L-Bt-4) was identified expressing 4 genes.

Independent transgenic events were identified and characterized by PCR (see below) and then self-pollinated to produce T1 seeds. Homozygous T2, T3 and subsequent T4 generations were derived through selfing.

A homozygous T3 L-Bt-4 plant was pollinated with multivitamin Carolight pollen and the hybrid offspring will be evaluated. The hybrid CaroBt accumulates 169-times the normal amount of β-carotene, 6 times the normal amount of ascorbate and twice the normal amount of folic acid and is protected against the corn borers when is compared with the original line M37W.


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:
The objective of this notification is to describe how will be the first release of the inbreed line L-Bt-4 of genetically modified corn (20 plants) and the hybrid of its cross with the transgenic line Carolight (hybrid CaroBt, 20 plants). The evaluation of L-Bt-4 with respect to their original line Carolight will be done in the following aspects:
- agronomical characterization.
- effects on the pests and natural fauna.


2. Geographical location of the site:
We plan only Lleida municipality.

3. Size of the site (m2):
20 m2 surrounded by more than 6 rows of conventional corn.

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:
Commercial Bt corn in Europe contains the event MON 810 (Cry1Ab, YieldGard) and it has been authorized by EFSA for food and feed (http://ec.europa.eu/food/dyna/gm_register/index_en.cfm). For this reason no toxic effect are expected of Cry1Ac y Cry1C proteins.
Syngenta seeds Inc has developed transgenic corn varieties (MIR 162, BT11xMIR162. y BT11xMIR162xMIR604) containing Vip3A protein and they are n commercials en USA, Australia, Taiwan and Brazil (http://www.agbios.com/dbase.php) For this reason it isn’t waited any toxic effect of Vip3A protein.


Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
- Within the consecutives multiplication generations no morphological, physiological or agronomic differences were detected between transformed plants and their conventional counterparts except for the presence of the three insecticide proteins.
- The genetic modification introduced in the corn plant consists on the incorporation of different genes involved in the synthesis of the three insecticide proteins, in order to protect the plant against corn borers. Any negative effect on human health is predicted.
- The genetic modification does not target any organism but therefore no toxic effects on other organisms within the ecosystem are predicted according to the previous experience in other events analyzed.
- They are no sexually compatible species (neither cultivated nor wild-type) in Europe, since the corn plant has its origins in Central America.
- As cultivated specie, corn varieties have been bred to germinate in the consecutive crop cycle when the appropriate environmental conditions are present. Their survival out of the crop is very unlikely since they are highly adapted to crop conditions. Moreover, in the region where the trial field would be placed, those seeds able to germinate after the harvest period die due to autumn and winter colds. Therefore, any selective advantage of the transformed plants over wild-type plants in natural environments is predicted.
- There are no predicted effects on the biogeochemical cycles since the newly synthesized vitamins are mineralized like most of other organic compounds.


Brief description of any measures taken for the management of risks:
In the case of this Bt resistant transgenic corn the risk is equivalent to the Bt corn authorized in Europe. Despite of this, however, the following safety measures will be implemented:
- The field will be placed in an urban zone where corn is not cultivated.
- The field for transgenic corn multiplication will be surrounded by at least 6 rows of an authorized conventional variety in order to decrease the amount of released pollen outside the growing area.
- No wild relative are present within the total area of land used for the experiment, therefore there is no risk of crosses with wild relatives.
- The grain harvested from the corn in the edges of the field will be destroyed and buried.
- The grain harvested from the transgenic corn L1 will be manipulated, transported and packaged by qualified staff.
- The equipments used in the process (planter and thrasher) will be appropriately cleaned in the trial field. Corn grain will be harvested manually. We have provided a procedure for cleaning the equipment together with the documents of the B/ES/14/04 release.
- Transgenic and conventional corn plants will be crushed and buried in the soil.
- Alter the corn harvest, we will inspect visually the field and collect by hand any cop left to prevent the possible growth of seed remnants. In addition, we will control manually (by rogue or with herbicide) any volunteer that could appear in autumn or at the end of winter.
- The trial will be periodically checked in order to register any information about adverse effects towards the environment and / or food safety, which will be notified to the corresponding authority.
- If the winter weather was mild, and volunteers would appear, they would be eliminated manually or mechanically.
- In the end of the trial a report will be sent to the corresponding authority.


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:
No
07/12/2016 00:00:00
Remarks:
Withdrawn by the notifier.