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Notification report


General information

Notification Number
B/SE/11/11749

Member State to which the notification was sent
Sweden

Date of acknowledgement from the Member State Competent Authority
03/11/2011

Title of the Project
Developing industrial quality oils in crambe

Proposed period of release:
01/05/2011 to 31/12/2016

Name of the Institute(s) or Company(ies)
Swedish University of Agricultural Sciences, Department of Plant Breeding and Biotechnology;


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
CrambeBrassicaceaeCrambeCrambe abyssinicaGalactica

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
Three transgenic lines will be included in the field trial: XLZeru, XLZwax2, XLZwax3.
1. Three oil modifying genetic traits have been introduced in line XLZeru. BnFAE that elongates 18:1 fatty acids to 22:1 (erucic acid), LdLPAAT that insert 22:1 on the second position (sn2) on the triacylglycerol molecule and CaFAD2-RNAi that shut down the desaturation of 18:1 to 18:2. The result of the introduction of these three traits is a significant increase in the erucic acid content of the crambe seed oil. Analyses of the seed oil from 3 generations show a steady increase in the average erucic acid content of the seed oil with each generation, indicating the line is getting more and more genetically stable. In the 3rd generation, the average erucic acid content is about 70% compared with 60% in non-transformed crambe. Since the line is not yet homozygous, we expect to have even higher erucic acid content in the subsequent generations. For the field trial 2012, we will use the seeds from the 4th generation. We expect the line to have reached homozygozy in the 5th generation i.e. the seeds that will be harvested from the field trial by the end of 2012. The erucic acid content in the seed oil should then have established at levels well above 70% and these seeds will be used for later field trials in the subsequent years starting from 2013.
The XLZeru line also contains the nptII gene that is used as a marker, and which has previously been proved not to affect the plant growth and development.
2. Two oil modifying genetic traits have been introduced in line XLZwax2, ScFAR that reduces fatty acids to fatty alcohols and ScWS that catalyses formation of wax esters from fatty acids and fatty alcohols. Introduction of these two traits results in production of wax esters (manly C44 made up of 22:1 fatty acids and 22:1 fatty alcohols) that is accumulating in the crambe seed oil. Analyses show that the average wax ester content in the seed oil increases with each generation and about 20% of the seed oil in 3rd generation seeds are made up of wax esters. This is due to that the line is not yet homozygous for the introduced traits. For the field trial 2012, we will use the seeds from the 4th generation and the seeds that will be harvested from the field trial by the end of 2012 will be used in 2013 years field trials. We estimate that the seed material harvested from 2013 years field trial (6th generation) will be homozygous for the introduced genetic traits. This seed material will be used in 2014 years field trial.
The XLZwax2 line also contains the nptII gene that is used as a marker and the DsRed gene that is used as a reporter. Neither of the two introduced genetic traits, nptII and DsRed, effects plant growth or development.
3. Three oil modifying genetic traits have been introduced in line XLZwax3, ScFAR that reduces fatty acids to fatty alcohols, ScFAE that elongates 18:1 fatty acids to 22:1 and 24:1, and ScWS that catalyses formation of wax esters from fatty acids and fatty alcohols. Introduction of these three traits results in production of wax esters (manly C44 and small amounts C48 made up of 22:1/24:1 fatty acids and 22:1/24:1 fatty alcohols) that is accumulating in the crambe seed oil. Analyses show that the average wax ester content in the seed oil increases with each generation and about 25% of the seed oil in 3rd generation seeds are made up of wax esters. This is due to that the line is not yet homozygous for the introduced traits. For the field trial 2012, we will use the seeds from the 4th generation and the seeds that will be harvested from the field trial by the end of 2012 will be used in 2013 years field trials. We estimate that the seed material harvested from 2013 years field trial (6th generation) will be homozygous for the introduced genetic traits. This seed material will be used in 2014 years field trial.
The XLZwax3 line also contains the nptII gene that is used as a marker and a non-functional DsRed gene. During the making of this vector construct the nptII gene was inserted within the DsRed coding sequence, interrupting it and making it non-functional.


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 construct with the following sequences have been introduced in line XLZeru.

RB; 25 bp; Agrobacterium tumefaciens; is needed for T-DNA transfer to start
Nos promoter; 184 bp; Agrobacterium tumefaciens; promoter that controls expression of NPTII
NPTII; 821 bp; Escherichia coli; confer resistance to kanamycin that is used for selecting transformants
Nos terminator; 706 bp; Agrobacterium tumefaciens; adenylation sequence to NPTII
BnFAE terminator; 90 bp; Brassica napus; adenylation sequence to BnFAE
BnFAE; 1520 bp; Brassica napus; elongates 18:1 fatty acids to 22:1 fatty acids
Napin promoter; 2146 bp; Brassica napus; promoter that controls expression of BnFAE
Napin terminator; 222 bp; Brassica napus; adenylation sequence to LdLPAAT
LdLPAAT; 845 bp; Limnanthes douglasii; insert erucic at the 2nd position on the triacylglycerol molecule to produce trierucin.
Napin promoter; 2047 bp; Brassica napus; promoter that controls expression of LdLPAAT
Intron sequences; 777/ 183 bp; Flaveria trinerva/ Ricinus communis; allow for downregulation of expression of FAD2
Napin promoter; 2134 bp; Brassica napus; promoter that controls expression of FAD2-RNAi
CaFAD2, two fragments; 354 bp x 2 =708 bp; Crambe abyssinica; downregulate expression of FAD2 through the RNAi mechanism and resulting in increased production of erucic acid
Ocs terminator; 737 bp; Agrobacterium tumefaciens; adenylation sequence to FAD2
LB; 25 bp; Agrobacterium tumefaciens; is needed to stop T-DNA transfer


A construct with the following sequences have been introduced in line XLZwax2.

RB; 25 bp; Agrobacterium tumefaciens; is needed for T-DNA transfer to start
Nos-promoter; 183 bp; Agrobacterium tumefaciens; promoter that controls expression of NPTII
NPTII; 821bp; Escherichia coli; confer resistance to kanamycin that is used for selecting transformants
Nos terminator; 705 bp; Agrobacterium tumefaciens; adenylation sequence to NPTII
Glycinin-1 promoter; 701 bp; Glycine max; promoter that controls expression of ScWS
ScWS; 1062 bp; Simmondsia chinensis; form wax esters from fatty acids and fatty alcohols
3’UTR of the Glycinin-1 gene; 457 bp; Glycine max; adenylation sequence to ScWS
Glycinin-1 promoter; 687 bp; Glycine max; promoter that controls expression of ScFAR
ScFAR; 1484 bp; Simmondsia chinensis; reducing fatty acids to fatty alcohols
3’UTR of the Glycinin-1 gene; 457 bp; Glycine max; adenylation sequence to ScFAR
CMV-promoter; 527 bp; Cassava mosaic virus; promoter that controls expression of DsRed
DsRed; 684 bp; Discosoma sp.; fluorescence protein that is used as a reporter
Nos-terminator; 705 bp; Agrobacterium tumefaciens; adenylation sequence to DsRed
LB; 25 bp; Agrobacterium tumefaciens; is needed to stop T-DNA transfer


A construct with the following sequences have been introduced in line XLZwax3.

RB; 25 bp; Agrobacterium tumefaciens; is needed for T-DNA transfer to start
Nos-promoter; 183 bp; Agrobacterium tumefaciens; promoter that controls expression of NPTII
NPTII; 821 bp; Escherichia coli; confer resistance to kanamycin that is used for selecting transformants
Nos terminator; 705 bp; Agrobacterium tumefaciens; adenylation sequence to NPTII
Glycinin-1 promoter; 701bp; Glycine max; promoter that controls expression of ScWS
ScWS; 1062 bp; Simmondsia chinensis; form wax esters from fatty acids and fatty alcohols 3’UTR of the Glycinin-1 gene; 457 bp; Glycine max; adenylation sequence to ScWS
CMV-promoter; 527 bp; Cassava mosaic virus; promoter that controls expression of DsRed
DsRed; 684 bp; Discosoma sp.; fluorescence protein that is used as a reporter, here non-functioning due to interruption in its sequence from NPTII
Nos-terminator; 252 bp; Escherichia coli; adenylation sequence to DsRed
Glycinin-1 promoter; 687 bp; Simmondsia chinensis; promoter that controls expression of ScFAR
ScFAR; 1484 bp; Simmondsia chinensis; reducing fatty acids to fatty alcohols
3’UTR of the Glycinin-1 gene; 457 bp; Simmondsia chinensis; adenylation sequence to ScFAR
Glycinin-1 promoter; 687 bp; Simmondsia chinensis; promoter that controls expression of ScFAR
ScFAE; 1571 bp; Simmondsia chinensis; elongates 18:1 fatty acids to 22:1 and 24:1fatty acids
3’UTR of the Glycinin-1 gene; 457 bp; Simmondsia chinensis; adenylation sequence to ScFAE
LB; 25 bp; Agrobacterium tumefaciens; is needed to stop T-DNA transfer


6. Brief description of the method used for the genetic modification:
Agrobaterium-mediated gene transfer was used for the genetic modification.

Experimental Release

1. Purpose of the release:
The aim of the field trial is to investigate performance of the GM crambe lines under field conditions regarding the oil composition, especially the erucic acid content and wax ester production, together with seed setting and yield. We also intend to observe the resistance of the GM lines to biotic and abiotic stresses, such as insect and disease resistance. In addition, we will also evaluate birds behavior regarding the GM lines with wax esters. The release will also allow us to multiply the seed material from the three lines to be used in future field trials as well as use the seed material for application tests.

2. Geographical location of the site:
During 2012 the release will be at Helgegården in Skepparslöv outside Kristianstad. In the period 2013-2016 release can also be done in Lomma and Kävlinge municipallities.

3. Size of the site (m2):
The total area of release in one year during 2012-2016 will not exceed 30000 m2.
In 2012 the total area is 4 X 120 m2 + 10 meter barrier crop (barley) = 2940 m2


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:
Genetic modified crambe has not been released before in Sweden.

Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
The introduced genetic traits in the three GM-crambe lines bring about changes in the composition of the seed oil. In the XLZeru line, the level of the existing erucic acid has been increased from 60% to 70% in comparison with non-GM crambe. The XLZwax2 and XLZwax3 lines, have seed oil compositions that apart from triacylglycerols, also contain wax esters, a new oil component for crambe seed. Wax esters are composed of one alcohol and one fatty acid with chain lengths of 22-24 carbons. Wax esters are found naturally in high content in the seeds of the desert plant jojoba (Simmondsia chinensis) from America and they also exist as natural components in e.g. the cuticle of plants.

The increased level in erucic acid in the XLZeru line should not give any selective advantages in a natural envirionment since erucic acid already exists at up till 60% in the seeds of non-GM-crambe and non-GM high erucic acid rapeseed.

The XLZwax2 and XLZwax3 lines contain up to 25% wax esters in the seed oil and we don’t expect this change will lead to selective advantages in a natural environment.


Brief description of any measures taken for the management of risks:
The trial fields with the GM-crambe lines will be sourrounded by a 10 meters wide border of a spring planted barrier crop (barley). The border is wide enough so that mashineries are able to drive, turn and to clean out the harvesting equipment within the zone of barrier crop.
After harvesting the seeds from the GM-crambe lines, the trial field will be burned to destroy any remaining crop or crop rest (including barrier crop). Additional straw will be added onto the field to facilitate the burning.
Measures are made as so to limit the buildup of a seed bank after the harvest by first burning the field followed by a light harrowing that will stimulate GM-crambe seeds dropped to the ground to germinate. Over the following year the field will lay in fallow and during this period it will be harrowed interveningly over the year to facilitate seeds to grow and seedlings to be controlled. The field will be monitored over four years after the trial and any emerging crambe plants will be destroyed. If crambe plants still are observered during the fourth year monitoring will be estended
It can be added that growing crambe plants can not survive a normal winter in the South of Sweden.
Beside the dates for recording the crops development, the crambe field will be monitored at least twice a month.
The GM-crambe trial field will not be used for crambe over the following four year.
No presence of bee-keeping in the neighbourhood has been verified


Final report


European Commission administrative information

Consent given by the Member State Competent Authority:
Yes
04/04/2012 00:00:00
Remarks: