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WP3: Assessment of biological and physical containment strategies
Joachim Schiemann
Institute for Biosafety in Plant Biotechnology
Project meeting, EU-funded project PRICEBrussels, November 15-16, 2012
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Objectives
Assessing the potential of biological and physical containment strategies
Specific objectives
Validation of a hypothetical coexistence regulation under field conditionstaking into account flowering delays in combination with separation distances and buffer zones
Development of molecular detection tools for air-borne dispersal of GM material (pollen as model)
Validation and quantification of cytoplasmic male sterility (CMS) in maize
Determination of effective isolation methods and distances for GM-CMS-maize
WP 3: Objectives
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Participants
JRC-IHCP (Joint Research Centre, Institute for Health and Consumer Protection)
molecular method development for the detection of genetically modified pollen in bio-aerosol, developed methods for pollen entrapment in combination with molecular screening analysis of pollen DNA, development of “MPT Decision Support System” and evaluation in the field
JKI -SB (Julius Kühn-Institut, Institute for Biosafety in Plant Biotechnology)
Leading WP3, validation of CMS maize as a biological containment method, studies on CMS maize, studies on isolation methods and distances, recommendation for good agricultural practice to improve the coexistence between GM plants based on CMS and non-GM plant growing farmers
WP 3: Participants
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Participants
CULSP (Czech University of Life Sciences Prague)
Conduction of field trials: validation of CMS maize as a biological containment method
FMB/IRTA/UdG (Mas Badia Foundation / Institute for Food and Agricultural Research and Technology / University of Girona, Institute for Agro-Food Technology)
Assessment of flowering delays in combination with separation distances and buffer zones on coexistence, identification of a suitable agronomic region, study on coexistence in real agronomic fields, recommendation for good agricultural practice to guarantee the coexistence
Conduction of field trials: validation of CMS maize as a biological containment method
WP 3: Participants
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Tasks
Task 3.1: Assessment of flowering delays in combination with separation distances and buffer zones on coexistence (Leader: Joan Serra, FMB)
Task 3.2: Molecular methods development for the detection of GM pollen in bio-aerosol
(Leader: Marco Mazzarra, JRC-IHCP)
Task 3.3: Validation of CMS maize as a biological containment method
(Leader: Joachim Schiemann, JKI)
WP 3: Tasks
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Milestones – set for M18
MS 7: Summary report on the state of the art of physical containment methods
(FMB)
MS 8: Summary report on the state of the art of air-borne maize pollen dispersal
(JRC)
MS 9: Summary report on the state of the art of biological containment methods
(JKI)
WP 3: Milestones
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Deliverables
D 3.16: Completion of the 1st year trial, evaluation of the effectiveness of the hypothetical coexistence regulation mainly based on flowering asynchronicity, distances and buffer zones FMB - M 18
D 3.17: Completion of the 2nd year trial, evaluation of the effectiveness of the strategy of coexistence based on flowering asynchronicity FMB - M 30
D 3.18: Report on the efficacy of a coexistence regulation mainly based on asynchronous flowering FMB - M 32
D 3.19: Development of multi-target GMO detection strategies for low genome copy number samples JRC - M 12
D 3.20: Validation of “fit to purpose” multi-target GMO detection strategy for entrapped pollen GMO detection: laboratory pollen entrapment samples JRC - M 24
WP 3: Deliverables
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Deliverables
D 3.21: Validation of “fit to purpose” multi-target GMO detection strategy for entrapped pollen GMO detection: field pollen entrapment samples JRC - M
24
D 3.22: Completion of field trials and field assessments on CMS maize with regard to validation of sterility JKI - M 18
D 3.23: Completion of field trials and field assessments on cross pollination with regard to outcrossing potential, isolation distances and segregation measures JKI – M 30
D 3.24: Report on the efficacy of CMS maize for coexistence JKI – M 32
WP 3: Deliverables
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Task 3.1: Assessment of flowering delays in combination with separation distances and buffer zones on coexistence
(Leader: Joan Serra, FMB)
Subtasks
T3.1.1: Identification of a suitable agronomic region (FMB)
T3.1.2: Studies on coexistence in real agronomic fields (UdG)
T3.1.3: Recommendation for good agricultural practice to guarantee coexistence (FMB)
WP 3: Task 3.1
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Identification of a suitable agronomic region
NE Catalonia
Baix Empordà
Location 1: Ullà
Location 2: La Tallada d’Empordà
Location 3: Torroella de Montgrí
Google Maps
WP 3: Task 3.1
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Sowing date: April the 7th
Conventional hybrid: PR32T83Sowing area: 0,4 ha
Sowing date: April the 3th
GM hybrid: PR33Y72Buffer hybrid: PR32T16Sowing area: 0,7 ha
Sowing date: April the 9th
GM hybrid: DKC6667YGBuffer hybrid: P1921Sowing area: 2,5 ha
Sowing date: April the 7th
Conventional hybrid: PR32T83Sowing area: 0,5 ha
Location 1: Ullà
Identification of a suitable agronomic region
WP 3: Task 3.1
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Start date of assessments 22/06/2012
GM male flowering (DKC6667YG)
Conventional male flowering (PR32T83)
Buffer male flowering (P1921)
Conventional female flowering (PR32T83)
Days
% o
f flo
wer
ing
plan
ts
Location 1: Ullà
Studies on coexistence in real agronomic fields Task 3.1
Days
% o
f flo
wer
ing
plan
tsBuffer male flowering (PR32T16)GM male flowering (PR33Y72)
Conventional male flowering (PR32T83) Conventional female flowering (PR32T83)
GM male flowering (PR33Y72)
Conventional male flowering (PR32T83)
Buffer male flowering (PR32T16)
Conventional female flowering (PR32T83)
% o
f flo
wer
ing
plan
ts
Days
Conventional maize (PR32T83) Male flowering date (50 % plants stage 3) July 1Feminine flowering date (50 % plants stage 3) July 2GM maize (PR33Y72)Male flowering date (50 % plants stage 3) July 12Buffer conventional maize (PR32T16) Male flowering date (50 % plants stage 3) July 9 (?)
July 21 (?)
Conventional maize (PR32T83) Male flowering date (50 % plants stage 3) July 2Feminine flowering date (50 % plants stage 3) July 2GM maize (PR33Y72)Male flowering date (50 % plants stage 3) July 12Buffer conventional maize (PR32T16) Male flowering date (50 % plants stage 3) July 9 (?) July 16 (?)
Conventional maize (PR32T83) Male flowering date (50 % plants stage 3) July 1 Feminine flowering date (50 % plants stage 3) July 3GM maize (DKC6667YG) Male flowering date (50 % plants stage 3) July 8Buffer conventional maize (P1921) Male flowering date (50 % plants stage 3) July 10
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Buffer zone 1 (PR32T16)
Buffer zone 2 (PR32T16)
Buffer zone 3 (PR32T16)
Location 1: Ullà
Studies on coexistence in real agronomic fields
Male sterility on PR32T16 (Buffer) (?): 70-85 %
% o
f mal
e flo
wer
ing
plan
ts
Days
Buffer 1 (PR32T16) Buffer 2 (PR32T16) Buffer 3 (PR32T16)
Start date of assessments 22/06/2012
WP 3: Task 3.1
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GI (Global index) = ∑ Estimated cross pollination index
ECP (Estimated cross pollination index) = (10 – (Ft – Fc)) / (distance in decameters + 1)2
Where Ft and Fc are the flowering dates from transgenic and conventional fields
% GM = 0,068 x GI
Global Index tool (Messeguer et al., 2006)
Studies on coexistence in real agronomic fields
WP 3: Task 3.1
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GM 0,00% (?)
GM 0,09% (?) GM maize
Conventional maize
GM maize
Conventional maize
Studies on coexistence in real agronomic fields
WP 3: Task 3.1
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Location 1: Ullà Sampling methodology
Studies on coexistence in real agronomic fields
WP 3: Task 3.1
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Task 3.2: Molecular methods development for the detection of GM pollen in bio-aerosol
(Leader: Marco Mazzarra, JRC-IHCP)
Subtasks
Development of tools for the application of pollen entrapment combined with molecular screening analysis of isolated pollen DNA
Development of a “MPT (molecular pollen trap) decision support system” and evaluation under field conditions in (2012)2013 and 2014
WP 3: Task 3.2
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WP 3: Task 3.2
GMO analysis of bio-aerosol samplesGMO analysis of bio-aerosol samples Outdoor exposure of sampling devices
→ Technical pollen sampler PMF/Sigma2 (TIEM) → Hirst type Sampler: VPPS 2010 (Lanzoni)
Extraction of pollen grains from the entrapping surface (tape, filters) DNA purification from pollen grains Molecular analysis (Real-Time PCR methods)
Weight the maize pollen (1-10 mg)On-tape adhesion
Filter adhesion (PMF)
Microscopic count
+ H2O, glass beads, CTAB buffer
DNA extraction and purificationReal Time PCR
DNA extraction and purification
5-10 μl
PMF/Sigma-2Hirst type
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Pollen traps Location: Ullà
WP 3: Task 3.2
Photos:
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Task 3.3: Validation of CMS maize as a biological containment method
(Leader: Joachim Schiemann, JKI)
Subtasks
T3.3.1: Studies on CMS maize (JKI)
T3.3.2: Studies on isolation methods and distances (UdG)
T3.3.3: Recommendation for a good agricultural practice to improve the coexistence between GM plants based on CMS and non-GM plant growing farmers (JKI)
WP 3: Task 3.3
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CMS maizeCytplasmic male sterility (CMS) offers the potential
for efficient biological containment to facilitate coexistence in maize
WP 3: Task 3.3
Plus-Hybrid SystemThe Plus-Hybrid system offers the potential of a
biological confinement and a high and stable yield.
CMS effect: no pollen = more recources available
Xenia effect: allo-pollen influence on kernel weight
CMSeffect
Plus-Hybrideffect Xenia
effect
Hybrid AFertile
Hybrid ASterile
Hybrid AFertile
Hybrid AFertile
Hybrid AFertile
Hybrid BFertile
Hybrid ASterile
Hybrid BFertile
RelativYield
(Weingartner et al., 2002)
Sterile tassel Fertile tassel
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WP 3: Task 3.3
Field trial design – Germany and Czech
1. Rep. 2. Rep. 3. Rep.
5m
5m
5m
5m
5m
5m
5m
5m
5m
5m
5 mMixture Row Mixture Mixture Row Row Mixture Row Mixture
10% 15% 20% 15% 10% 20% 20% 20% 10%15 m
Hemp 5 m 45 m Row Mixture Row Row Mixture Mixture Mixture Row Row
10% 15% 20% 15% 10% 20% 15% 10% 15%15 m
5 m
15 m 15 m 15 m 15 m 15 m 15 m 15 m 15 m 15 m185 m
CMS maize hybrid TorresConventional maize White maize WM 17007LocationsCzech, Germany, Spain
Measurements
Flowering date Dates of female and male flowering Plant height Number of plants at harvesting and ears/plant Number of grains per ear Harvesting date Harvest humidity 1000 kernel weight Grain yield
Best proportion and arrangement of a pollen donor in a CMS-maize plot?
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Yield of the Plus-Hybrid trial in 2012
WP 3: Task 3.3
M10 / R 10 10 % WM + 90 % CMS maize M: mixture, WM mixed with CMS maizeM 15 / R 15 15 % WM + 85 % CMS maize R: row, WM designed in rowsM 20 / R 20 20 % WM + 80 % CMS maize
Plot size 225 m”
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Field trial design – Spain
WP 3: Task 3.3
MIXTURE 10 %MIXTURE 15 %MIXTURE 20 %ROW 10 %ROW 15 %ROW 20 %
SORGHUM
Yield in Mas Badia, 2012
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Mean Kernel Set after Self-pollination - test for pollen fertility
WP 3: Task 3.3
MKS = mean kernel setnuber of developed kernels per cob in relation to a fully pollinated cob
1
2
3
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WP 3: Task 3.3Cross-pollination
Reliability test of the Plus-Hybrid system as a confinement tool to facilitate coexistence
Cross-pollination of „mix 20%“ into white maize
mixture white maize20% 10 m
main wind direction
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VARIETIES Male sterility
FAO maturity
Company
PR32T16 No (?) 700 PIONEER HI-BRED
PR33W82 No (?) 600 PIONEER HI-BRED
DKC6666 No 700 MONSANTO
NK GIGANTIC STE Yes 700 SYNGENTA
NK FAMOSO No 600 SYNGENTA
NK ARMA STE Yes 700 SYNGENTA
SY NEPAL STE Yes 600 SYNGENTA
SY VERDEMAX STE Yes 700 SYNGENTA
SY NEPAL No 600 SYNGENTA
Cytoplasmic male sterile hybrids tested in Spain, FAO 600 – 700
Hybrids more adapted to Spanish climate?
WP 3: Task 3.3
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WP 3: Task 3.3 - Impressions
Harvesting in PragueMaize and Hemp in Quedlinburg
Trail in Mas Badia
Sowing in Prague
P-H-trial Quedlinburg
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Cross-pollination trial in Mas BadiaWork group meeting in Mas Badia
Work group meeting in Mas Badia
WP 3: Task 3.3 - Impressions
TorresVariety trial in Mas Badia
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Conclusions
WP 3: First Conclusions
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Those who want the world to continue as it is, do not want the world to continue
Thank you for your attention
Photo:Anke Schiemann