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Notification report


General information

Notification Number
B/GB/19/R08/01

Member State to which the notification was sent
United Kingdom

Date of acknowledgement from the Member State Competent Authority
06/02/2019

Title of the Project
Synthesis and accumulation of seed storage compounds in Camelina sativa

Proposed period of release:
01/05/2019 to 31/10/2023

Name of the Institute(s) or Company(ies)
Rothamsted Research, West Common, Harpenden
Hertfordshire,
AL5 2JQ
UK;


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
Gold-of-pleasure, false flaxBrassicaceaeCamelinasativaCeline

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
Construct ID Phenotype # of genes Marker
1 EPA-D6 Omega-3 LC PUFAs in seed oil Four1 or more DsRed
2 DHA-D6 Omega-3 LC PUFAs in seed oil Six1 or more DsRed
3 EPA-D9 Omega-3 LC PUFAs in seed oil Four1 or more DsRed
4 DHA-D9 Omega-3 LC PUFAs in seed oil Six1 or more DsRed
5 C18 NMI-PUFA C18 non-methylene-interrupted polyunsaturated in seed oil One1 or more DsRed
6 C20 NMI-PUFA C20 non-methylene-interrupted polyunsaturated in seed oil Two1 or more DsRed
7 CRISPR-Cas9 – fad2 Disruption of the FAD2 desaturase – high oleic acid phenotype Zero None
8 CRISPR-Cas9 – fae1 Disruption of the FAE1 elongase – increased C18 fatty acids in seed oil Zero None
9 HO High levels of oleic acid in seeds Three BAR
10 LPAT Increased seed oil Two DsRed
11 FAT Increased seed oil One DsRed
12 SAD Reduced sinapoyl-esters in seeds One DsRed

1 = Minimum number of genes required to generate phenotype are indicated


Genetic modification

3. Type of genetic modification:
Insertion; Deletion;

In case of insertion of genetic material, give the source and intended function of each constituent fragment of the region to be inserted:
Element Donor Organism Description, size, and Intended Function
RB Agrobacterium tumefaciens T-DNA Right border (24 bp)
LB Agrobacterium tumefaciens T-DNA Left border (23 bp)
Seed-specific promoters (Group P)
pUSP Vicia faba Unknown Seed Protein Seed-specific promoter (684 bp)
pNP Brassica napus Napin seed specific promoter (664 bp)
pCNL Linum usitatissimum 2S seed storage protein (Conlinin) promoter (1064 bp)
pGLY Glycine max 11S Seed storage protein (Glycinin) promoter (702 bp)
pOLEO Arabidopsis thaliana Oleosin seed storage protein promoter (579 bp)

Regulatory elements (Group T)
OCSt Agrobacterium tumefaciens octopine synthase gene terminator sequence (192 bp)
Glyt Glycine max 11S storage protein (Glycinin) terminator (451 bp)
HSPt Arabidopsis thaliana Heat shock protein 18.2 (HSP) gene terminator sequence (245 bp)
Phast Phaseolus vulgaris Alpha-phaseolin gene terminator sequence (183 bp)
Fad2t Camelina sativa Microsomal oleate desaturases (FAD2) gene terminator sequence (182 bp)
PDK-i3 Flaveria trinervia Pyruvate orthophosphate dikinase intron 3 (i3) (1597 bp)

Regulatory elements used to direct Cas9 expression (Group X)
Cas9 Synthetic RNA-guided DNA endonuclease from Streptococcus pyogenes (4104 bp)
pEC1.2 Arabidopsis thaliana Egg-cell specific promoter to direct restricted expression of Cas9 (1000 bp)
pPcUbi4‐2 Petroselinum crispum Ubiquitin4–2 promoter to direct expression of Cas9 (971 bp)
pU3-26 Arabidopsis thaliana Promoter to express gRNAs for Cas9 targeting (400 bp)
pU6-26 Arabidopsis thaliana Promoter to express gRNAs for Cas9 targeting (400 bp)
U6-26t Synthetic Terminator sequence behind gRNAs for Cas9 targeting (120 bp)
Pea3At Pisum sativa Terminator sequence behind Cas9 (470 bp)
Synthetic Target-specific templates for guide RNAs (<24bp)

Biosynthetic genes

&#61508;6-desaturases (Group A)
OtD6 Synthetic Encodes a fatty acid &#61508;6-desaturase from Ostreococcus tauri (1665 bp)
AaD6 Synthetic Encodes a fatty acid &#61508;6-desaturase from Aureococcus anophagefferens (1350 bp)
MsqD6 Synthetic Encodes a fatty acid &#916;6-desaturase from Mantoniella squamata (1350 bp)
O809D6 Synthetic Encodes a fatty acid &#61508;6-desaturase from Ostreococcus RCC809 (1662 bp)
PvD6 Synthetic Encodes a fatty acid &#61508;6-desaturase from Primula vialii (1360 bp)
PfD6 Synthetic Encodes a fatty acid &#61508;6-desaturase from Primula farinosa (1360 bp
ThalpD6 Synthetic Encodes a fatty acid &#61508;6-desaturase from Thalassiosira pseudonana (1455 bp)
AnelD6 Synthetic Encodes a fatty acid &#61508;6-desaturase from Anemone leveillei (1341 bp)
MnnD6 Synthetic Encodes a fatty acid &#61508;6-desaturase from Mamiellales spp. (1456bp)

&#61508;6-elongases (Group B)
AaELO6 Synthetic Encodes a fatty acid &#61508;6-elongase from Aureococcus anophagefferens (900 bp)
BpELO6 Synthetic Encodes a fatty acid &#61508;6-elongase from Bathycoccus prasinos (900 bp)
FcELO6 Synthetic Encodes a fatty acid &#61508;6-elongase from Fragilariopsis cylindrus (825 bp)
MsqELO6 Synthetic Encodes a fatty acid &#916;6-elongase from Mantoniella squamata (900 bp)
MnnELO6 Synthetic Encodes a fatty acid &#61508;6-elongase from Mamiellales spp. (903 bp)
PpELO6 Synthetic Encodes a fatty acid &#61508;6-elongase from Physcomitrella patens (873 bp)
MoalELO6 Synthetic Encodes a fatty acid &#61508;6-elongase from Mortierella alpine (954 bp)
ThalpELO6 Synthetic Encodes a fatty acid &#61508;6-elongase from Thalassiosira pseudonana (819 bp)

&#61508;5-desaturases (C20) (Group C)
EmiD5 Synthetic Encodes a fatty acid &#916;5-desaturase from Emiliania huxleyi (1368 bp)

AaD5 Synthetic Encodes a fatty acid &#61508;5-desaturase from microalga Aureococcus anophagefferens (1380 bp)
MsqD5 Synthetic Encodes a fatty acid &#916;5-desaturase from Mantoniella squamata (1380 bp)
O809D5 Synthetic Encodes a fatty acid &#61508;5-desaturase from picoalgae Ostreococcus RCC809 (1380 bp)
ThraD5 Encodes a fatty acid &#61508;5-desaturase from Thraustochytrium spp. (1317 bp)
MnnD5 Synthetic Encodes a fatty acid &#61508;5-desaturase from Mamiellales spp. (1450 bp)
ThalpD5 Synthetic Encodes a fatty acid &#61508;5-desaturase from Thalassiosira pseudonana (1449 bp)
PhaeoD5 Synthetic Encodes a fatty acid &#61508;5-desaturase from Phaeodactylum tricornutum (1410 bp)

&#61508;5-elongases (Group D)
OtElo5 Synthetic Encodes a fatty acid &#61508;5-elongase from Ostreococcus tauri (903 bp)
BpELO5 Synthetic Encodes a fatty acid &#61508;5-elongase from Bathycoccus prasinos (900 bp)
AaELO5 Synthetic Encodes a fatty acid &#61508;5-elongase from Aureococcus anophagefferens (900 bp)
MsqELO5 Synthetic Encodes a fatty acid &#916;5-elongase from Mantoniella squamata (900 bp)
O809E5 Synthetic Encodes a fatty acid &#916;5-elongase from Ostreococcus RCC809 (900 bp)

ThalpELO5 Synthetic Encodes a fatty acid &#61508;5-elongase from Thalassiosira pseudonana (879 bp)

MnnELO5 Synthetic Encodes a fatty acid &#61508;5-elongase from Mamiellales spp. (900 bp)
PhaeoELO5 Synthetic Encodes a fatty acid &#61508;5-elongase from Phaeodactylum tricornutum (909 bp)

&#61508;4-desaturases (Group E)
BpD4 Synthetic Encodes a fatty acid &#61508;4-desaturase from Bathycoccus prasinos (1500 bp)
TpD4 Synthetic Encodes a fatty acid &#61508;4-desaturase from Thalassiosira pseudonana (1650 bp)
O809D4 Synthetic Encodes a fatty acid &#61508;4-desaturase Ostreococcus RCC809 (1480 bp)
EmiD4 Synthetic Encodes a fatty acid &#916;4-desaturase Emiliania huxleyi (1467 bp)

OtD4 Synthetic Encodes a fatty acid &#61508;4-desaturase from Ostreococcus tauri (1611 bp)

OlD4 Synthetic Encodes a fatty acid &#61508;4-desaturase from Ostreococcus lucimarinus (1398 bp)

w3-desaturases (Group F)
PveW3 Synthetic Encodes a fatty acid w3-desaturase from Pythium spp (1092 bp)
PolW3 Synthetic Encodes a fatty acid w3-desaturase from Pythium spp (1080 bp)
ParW3 Synthetic Encodes a fatty acid w3-desaturase from Pythium spp (1074 bp)
PiW3 Synthetic Encodes a fatty acid w3-desaturase from Phytophora infestans (1086 bp)
AdvaW3 Synthetic Encodes a fatty acid w3-desaturase from Adineta vaga (1122 bp)
PlduW3 Synthetic Encodes a fatty acid w3-desaturase from Platynereis dumerilii (1167 bp)
PlvuW3 Synthetic Encodes a fatty acid w3-desaturase from Patella vulgate (1263 bp)
AcmiW3 Synthetic Encodes a fatty acid w3-desaturase from Caenorhabditis elegans (1179 bp)

&#61508;12/&#61508;15-desaturases (Group I)
BoofD12 Synthetic Encodes a fatty acid &#61508;12-desaturase FAD2 activity from Borago officinalis (1149 bp)
TpD12 Synthetic Encodes a fatty acid &#61508;12-desaturase from Thalassiosira pseudonana (1182bp)
PsD12 Synthetic Encodes a fatty acid &#61508;12-desaturase activity from Phytophora sojae (1143 bp)
CsD12 Synthetic Encodes a fatty acid &#61508;12-desaturase FAD2 activity from Camelina sativa (1149 bp)
CsD15 Synthetic Encodes a fatty acid &#61508;15-desaturase FAD3 activity from Camelina sativa (1158 bp)

&#61508;9-elongases (Group J)
EhuxELO9 Synthetic Encodes a fatty acid &#61508;9-elongase from Emiliania huxleyi (300 bp)
IsocrELO9 Synthetic Encodes a fatty acid &#61508;9-elongase from Isochrysis galbana (300 bp)
PmarFAE9 Synthetic Encodes a fatty acid &#61508;9-elongase from Perkinsus marinus (1320 bp)

&#61508;8-desaturases (Group K)
PsD8 Synthetic Encodes a fatty acid &#916;8-desaturase from Pavlova salina (1440 bp)

EhD8 Synthetic Encodes a fatty acid &#916;8-desaturase from Emiliania huxleyi (1440 bp)

AcD8 Synthetic Encodes a fatty acid &#916;8-desaturase from Acanthamoeba castellanii (1440 bp)


&#61508;5-desaturases (C18) (Group L)
AqvuD5 Synthetic Encodes a fatty acid &#61508;5-desaturase from Aquilegia vulgaris (990 bp)
AnleD5-Al10 Synthetic Encodes a fatty acid &#61508;5-desaturase from Anemone leveillei (AL10) (990 bp)
AnleD5-Al21 Synthetic Encodes a fatty acid &#61508;5-desaturase from Anemone leveillei (AL21) (990 bp)
ChlamyD5 Synthetic Encodes a fatty acid &#61508;5-desaturase from Chlamydomonas reinhardtii (1800 bp)

Acyltransferases (Group M)
DGAT1 Synthetic Encodes acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the triacylglycerol synthesis pathway – isoform 1 (1563 bp)
DGAT2 Synthetic Encodes acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the triacylglycerol synthesis pathway – isoform 2 (1330 bp)
PDAT Synthetic Encodes the phospholipid:diacylglycerol acyltransferase involved in the biosynthesis of triacylglycerol (2016 bp)

GPAT Synthetic Encodes the glycerol-3-phosphate acyltransferase involved in the first committed step on the Kennedy pathway for the biosynthesis of glycerolipids. (1131 bp)
LPAAT Synthetic Encodes the acyl-CoA sn-2 acyltransferase involved in the second step on the Kennedy pathway for the biosynthesis of glycerolipids. (1071 bp)
LPCAT Synthetic Encodes the acyltransferase responsible for the conversion 1-acyl-sn-glycero-3-phosphocholine into phosphatidylcholine via the Lands cycle. (1389 bp)
LPAT1 Brassica napus Encodes a modified lysophosphatidic acid acyltransferase 1 that localises in the endoplasmic reticulum (768 bp)

Other transgenes (Group N)
AtWRI1 Arabidopsis thaliana Encodes the WRINKLED1 (WRI1) AP2/ERWEBP transcription factor involved in the control of storage compound biosynthesis – Arabidopsis homolog (1317 bp)
CsWRI1 Camelina sativa Encodes the WRINKLED1 (WRI1) AP2/ERWEBP transcription factor involved in the control of storage compound biosynthesis –Camelina homolog (1317 bp)
AtFATb Arabidopsis thaliana Encodes a plastidial palmitoyl-acyl carrier protein thioesterase (1239 bp)
CsFAD2-AS Camelina sativa Arabidopsis fatty acid desaturase 2 (FAD2) DNA fragment for antisense expression (299 bp)
CsFAD3-AS Camelina sativa Arabidopsis fatty acid desaturase 3 (FAD3) DNA fragment for antisense expression (323 bp)
CsFAE1-AS Camelina sativa Camelina fatty acid elongase 1 (FAE1) DNA fragment for antisense expression (251 bp)
LPAT2 amiRNA Synthetic Arabidopsis artificial microRNA mi319a adapted to target Camelina lysophosphatidic acid acyltransferase 2 (404 bp)
SAD Synthetic Encodes an engineered sinapic acid decarboxylase derived from Bacillus pumilis strain UI-670 (481 bp)

Marker genes (Group Z)
DsRed Synthetic Encodes a red florescent protein from Discosoma spp. (684 bp)
EGFP Synthetic Encodes a green fluorescent protein from Aequorea victoria (759 bp)
bar Synthetic Encodes phosphinothricin acetyl transferase, providing to resistance to Class H herbicides such as Basta ™, in which bialaphos is the active ingredient. (548 bp)
pZ Cassava vein mosaic virus CsVMV promoter sequence (528 bp)

pNOS Agrobacterium tumefaciens Nopaline synthase gene promoter (288 bp)
Zt Agrobacterium tumefaciens Nopaline synthase gene terminator sequence (256 bp)


In case of deletion of genetic material, give information on the function of the deleted sequences:
The deleted sequences (as a consequence of CRISPR-Cas9 gene-editing) are either in the homeologues of the C. sativa FAD2 gene (&#61508;12-desaturase) or the FAE1 gene (fatty acid elongase1). FAD2 is partially responsible for the synthesis of linoleic acid from oleic acid; FAE1 is responsible for the seed-specific synthesis of long chain (C20+) saturated and monosaturated fatty acids.

6. Brief description of the method used for the genetic modification:
Plasmid DNA was inserted into the C. sativa genome using Agrobacterium mediated floral transformation system and transgenic plants were identified on the basis of visual identification (DsRed expression) or bialaphos resistance.

7. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination:
n/a

Experimental Release

1. Purpose of the release:
The purpose of this experimental trial is to determine the performance of these different GM Camelina iterations in the field, with respect to oil composition and oil quantity for different lipids (omega-3 LC-PUFAs, NMI-PUFAs) as well as other aspects of seed composition. It is also to assess any additional phenotypic and agronomic variations.

2. Geographical location of the site:
It will be sited on the experimental farm at Rothamsted Research, Harpenden, at grid reference TL120130, and also at the experimental farm at Rothamsted Research, Brooms Barn, at grid reference TL756654.

3. Size of the site (m2):
The total area of GM plants per year will maximally be 4,675m2

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 lines listed in this application have not previously been released.

Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
Of the twelve different GM C. sativa lines listed above in (2), ten are indistinguishable from the non-GM equivalent except for the modified composition of their seeds, in particular by the presence of the health-beneficial omega-3 long chain polyunsaturated fatty acids EPA and DHA, NMI-PUFAs or variation in the endogenous components of the seed. This modified composition is found only in the seeds of the GM C. sativa and is absent from all other vegetative tissues (e.g. leaves, roots, stems). In the other two cases, although devoid of transgenes, the plants have undergone CRISPR-Cas9 gene-editing resulting in the disruption of particular genes (FAD2, FAE1), with the resulting chemotype manifest in all cells. However, this is a simple variation in endogenous fatty acid composition.

In all cases, the gene donor organisms are not known to be pathogenic or allergenic to humans, and none of the genes under investigation or the selectable or visual marker genes, are expected to result in the synthesis of products that are harmful to humans, other organisms or the environment. Any unknown hazards arising from the expression and ingestion of foreign proteins will not occur since the C. sativa plants will not be consumed by humans.

The probability of C. sativa seeds escaping from the trial site or the transfer of inserted characteristics to sexually-compatible species outside the trial area is estimated as very low. C. sativa seeds are moderate in size and not normally dispersed by wind. Management measures including the use of gas guns and hawk kites will be employed to mitigate the risk of seed removal by birds. Management procedures to minimise the spread of seeds or pollen will further reduce the probability of these events occurring. There will be no compatible species grown for 750 meters from the boundary of the site and no sexually-compatible wild relatives of C. sativa exist in the vicinity of the Rothamsted farm.

The risk of non-sexual, horizontal gene transfer to other species is extremely low. In the event of horizontal gene transfer to bacteria, neither the trait genes nor the marker genes would be expected to confer a selective advantage in the field environment under consideration. The area proposed to be planted with GMOs is small and temporary (lasting between 4 and 5 months).


Brief description of any measures taken for the management of risks:
No C. sativa or related species will be grown within 750m from the trial. The trial plots will be surrounded by a pollen barrier of non-GM camelina.


The release site will be visited by trained laboratory personnel who are working on the project at no less than weekly intervals (and at some periods, daily) during the growing season of each year of the trial. Any unexpected occurrences that could potentially result in adverse environmental effects or the possibility of adverse effects on human health will be notified to the Defra immediately. Should the need arise to terminate the release at any point the emergency plans detailed below will be followed.

At the end of each season, the plot will remain in stubble and monitored for volunteers during the remainder of the year and the following season. Any volunteers identified will be destroyed by herbicide treatment (e.g. glyphosate) or removed by hand and destroyed.

Following completion of the two-year trial the release site will remain fallow for a further season to enable easy identification of volunteers. The site will be inspected regularly, and any volunteers identified will be immediately destroyed either by application of a systematic broad leaf herbicide.

In the unlikely event that the integrity of the site is seriously compromised, the trial will be terminated and all plants, (including GM and control C. sativa plots, and cereal separator) will be destroyed using a suitable herbicide or harvesting as deemed appropriate. All harvested material will be removed from the site and disposed of by incineration or deep burial at a local authority-approved landfill site using an approved contractor. Transportation of waste materials will be in secure containers. The phone numbers of all key staff will be available to site security and farm.


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
01/05/2019 00:00:00
Remarks: