WP3: Assessment of biological and physical
containment strategies
Joachim Schiemann
Project meeting, EU-funded project PRICE
Brussels, November 15-16, 2012
Institute for Biosafety in Plant Biotechnology
www.jki.bund.de
WP 3: Objectives
 Objectives
Assessing the potential of biological and physical containment strategies
 Specific objectives
Validation of a hypothetical coexistence regulation under field conditions
taking 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-CMSmaize
www.jki.bund.de
WP 3: Participants
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
www.jki.bund.de
WP 3: Participants
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
www.jki.bund.de
WP 3: Tasks
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)
www.jki.bund.de
WP 3: Milestones
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)
www.jki.bund.de
WP 3: Deliverables
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
D 3.20:
JRC - M 12
Validation of “fit to purpose” multi-target GMO detection strategy
for entrapped pollen GMO detection: laboratory pollen
entrapment samples
JRC - M 24
www.jki.bund.de
WP 3: Deliverables
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
www.jki.bund.de
WP 3: Task 3.1
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)
www.jki.bund.de
WP 3: Task 3.1
Identification of a suitable agronomic region
Location 1: Ullà
Google Maps
Location 2: La Tallada
Location 3: Torroella de
d’Empordà
Montgrí
NE
Catalonia
Baix Empordà
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WP 3: Task 3.1
Identification of a suitable agronomic region
Location 1: Ullà
Sowing date: April the 7th
Conventional hybrid: PR32T83
Sowing area: 0,4 ha
Sowing date: April the 3th
GM hybrid: PR33Y72
Buffer hybrid: PR32T16
Sowing area: 0,7 ha
Sowing date: April the 7th
Conventional hybrid: PR32T83
Sowing area: 0,5 ha
Sowing date: April the 9th
GM hybrid: DKC6667YG
Buffer hybrid: P1921
Sowing area: 2,5 ha
www.jki.bund.de
Studies on coexistence in real agronomic fields
Location 1: Ullà
Task 3.1
GM male flowering (PR33Y72)
Buffer male flowering (PR32T16)
Conventional male flowering (PR32T83)
Conventional female flowering (PR32T83)
% of flowering plants
Conventional maize (PR32T83)
Male flowering date (50 % plants stage 3) July 2
Feminine flowering date (50 % plants stage 3) July 2
GM maize (PR33Y72)
Male flowering date (50 % plants stage 3) July 12
Buffer conventional maize (PR32T16)
Male flowering date (50 % plants stage 3) July 9 (?)
July 16 (?)
Days
Buffer male flowering (P1921)
Conventional male flowering (PR32T83)
Conventional female flowering (PR32T83)
GM male flowering (PR33Y72)
Buffer male flowering (PR32T16)
Conventional male flowering (PR32T83)
Conventional female flowering (PR32T83)
Conventional maize (PR32T83)
Male flowering date (50 % plants stage 3)
July 1
Feminine flowering date (50 % plants stage 3) July 2
GM maize (PR33Y72)
Male flowering date (50 % plants stage 3)
July 12
Buffer conventional maize (PR32T16)
Male flowering date (50 % plants stage 3) July 9 (?)
% of flowering plants
% of flowering plants
GM male flowering (DKC6667YG)
July 21 (?)
Days
Conventional maize (PR32T83)
Male flowering date (50 % plants stage 3)
Feminine flowering date (50 % plants stage 3)
GM maize (DKC6667YG)
Male flowering date (50 % plants stage 3)
Buffer conventional maize (P1921)
Male flowering date (50 % plants stage 3)
10
July 1
July 3
Days
Start date of assessments
22/06/2012
July 8
July
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WP 3: Task 3.1
Studies on coexistence in real agronomic fields
Location 1: Ullà
Buffer zone 1 (PR32T16)
Buffer zone 2 (PR32T16)
Buffer zone 3 (PR32T16)
Buffer 2 (PR32T16)
Buffer 3 (PR32T16)
% of male flowering plants
Buffer 1 (PR32T16)
Male sterility on PR32T16 (Buffer) (?):
70-85 %
Days
Start date of assessments 22/06/2012
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WP 3: Task 3.1
Studies on coexistence in real agronomic fields
Global Index
tool (Messeguer et al., 2006)
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
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WP 3: Task 3.1
Studies on coexistence in real agronomic fields
GM maize
Conventional
maize
GM maize
Conventional
maize
GM 0,00%
(?)
GM 0,09%
(?)
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WP 3: Task 3.1
Studies on coexistence in real agronomic fields
Location 1: Ullà
Sampling methodology
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WP 3: Task 3.2
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
www.jki.bund.de
WP 3: Task 3.2
GMO 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)
Hirst type
PMF/Sigma-2
Weight the maize pollen
(1-10 mg)
On-tape adhesion
Filter adhesion (PMF)
Microscopic count
DNA extraction and purification
+ H2O, glass beads, CTAB buffer
DNA extraction and purification
5-10 μl
Real Time PCR
www.jki.bund.de
WP 3: Task 3.2
Photos:
Pollen traps
Location: Ullà
www.jki.bund.de
WP 3: Task 3.3
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)
www.jki.bund.de
WP 3: Task 3.3
CMS maize
Plus-Hybrid System
Cytplasmic male sterility (CMS) offers the
potential for efficient biological containment to
facilitate coexistence in maize
The 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
Relativ
Yield
Sterile tassel
CMS
effect
Xenia
effect
Plus-Hybrid
effect
Fertile tassel
Hybrid A Hybrid A
Fertile Fertile
Hybrid A Hybrid A
Sterile Fertile
Hybrid A Hybrid B
Fertile Fertile
Hybrid A Hybrid B
Sterile Fertile
(Weingartner et al., 2002)
www.jki.bund.de
WP 3: Task 3.3
Field trial design – Germany and Czech
Best proportion and arrangement of a pollen donor in a CMS-maize plot?
Mixture
Row
10%
Mixture
15%
Mixture
20%
Row
15%
Row
10%
Mixture
20%
Row
20%
5m
5m
5m
5m
5m
3. Rep.
5m
5m
5m
2. Rep.
5m
5m
1. Rep.
5m
Mixture
20%
10%
15 m
Hemp
5m
Row
Mixture
10%
Row
15%
Row
20%
Mixture
15%
Mixture
10%
Mixture
20%
Row
15%
45 m
Row
10%
15%
15 m
5m
15 m
15 m
15 m
15 m
15 m
15 m
15 m
15 m
15 m
185 m
Measurements
CMS maize hybrid
Torres
Conventional maize
White maize WM 17007
Locations
Czech, Germany, Spain
 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
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WP 3: Task 3.3
Yield of the Plus-Hybrid trial in 2012
M10 / R 10
M 15 / R 15
M 20 / R 20
10 % WM + 90 % CMS maize M: mixture, WM mixed with CMS maize
15 % WM + 85 % CMS maize R: row, WM designed in rows
20 % WM + 80 % CMS maize
Plot size 225 m”
Prague
250
250
200
200
yield [kg/plot]
yield [kg/plot]
Quedlinburg
150
100
50
150
100
50
0
0
M10
M15
M20
R10
R15
R20
M10
M15
M20
R10
R15
R20
www.jki.bund.de
WP 3: Task 3.3
Field trial design – Spain
Yield in Mas Badia, 2012
MIXTURE 10 %
MIXTURE 15 %
MIXTURE 20 %
ROW 10 %
ROW 15 %
ROW 20 %
SORGHUM
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WP 3: Task 3.3
Mean Kernel Set after Self-pollination - test for pollen fertility
MKS = mean kernel set
nuber of developed kernels
per cob in relation to a fully
pollinated cob
Quedlinburg
30
25
2
MKS [%]
20
15
10
5
0
1
3
2
3
4
5 6 7 8 9
number of ears
10 11 12 13
Prague
30
25
MKS [%]
1
20
15
10
5
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
number of ears
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WP 3: Task 3.3
Cross-pollination
Reliability test of the Plus-Hybrid system as a confinement tool to facilitate
coexistence
Cross-pollination of „mix 20%“ into white maize
mixture
20%
white maize
0.60
10 m
main wind direction
cross-pollination [%]
0.50
0.40
Quedlinburg
0.30
Prague
Sickte
0.20
0.10
0.00
-0.10
-1
1
3
5
7
9
11
13
distance within the white maize plot [m]
15
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WP 3: Task 3.3
Cytoplasmic male sterile hybrids tested in Spain, FAO 600 – 700
Hybrids more adapted to Spanish climate?
VARIETIES
PR32T16
PR33W82
DKC6666
NK GIGANTIC STE
NK FAMOSO
NK ARMA STE
SY NEPAL STE
SY VERDEMAX STE
SY NEPAL
Male
sterility
FAO
maturity
No (?)
No (?)
No
Yes
No
Yes
Yes
Yes
No
700
600
700
700
600
700
600
700
600
Company
PIONEER HI-BRED
PIONEER HI-BRED
MONSANTO
SYNGENTA
SYNGENTA
SYNGENTA
SYNGENTA
SYNGENTA
SYNGENTA
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WP 3: Task 3.3 - Impressions
P-H-trial Quedlinburg
Sowing in Prague
Trail in Mas Badia
Maize and Hemp in Quedlinburg
Harvesting in Prague
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WP 3: Task 3.3 - Impressions
Work group meeting in Mas Badia
Cross-pollination trial in Mas Badia
Variety trial in Mas Badia
Work group meeting in Mas Badia
Torres
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WP 3: First Conclusions
Conclusions
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Thank you for your attention
Photo:
Anke Schiemann
Those who want the world to continue as it is, do not want the world to continue
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