WP3: Assessment of biological and physical containment strategies

1. 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

2. WP 3: Objectives • 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

3. 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

4. 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

5. 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)

6. 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)

7. 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 zonesFMB - 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

8. 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

9. 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)

10. WP 3: Task 3.1 Identification of a suitable agronomic region Location 1: Ullà Google Maps NE Catalonia Baix Empordà Location 2: La Tallada d’Empordà Location 3: Torroella de Montgrí

11. 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

12. Studies on coexistence in real agronomic fields Task 3.1 Location 1: Ullà GM male flowering (PR33Y72) Buffer male flowering (PR32T16) Conventional female flowering (PR32T83) Conventional male flowering (PR32T83) 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 (?) % of flowering plants Days Buffer male flowering (PR32T16) GM male flowering (PR33Y72) Buffer male flowering (P1921) GM male flowering (DKC6667YG) Conventional male flowering (PR32T83) Conventional female flowering (PR32T83) 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 (?) July 21 (?) % of flowering plants % of flowering plants Conventional maize (PR32T83) Male flowering date (50 % plants stage 3) July 1 Feminine flowering date (50 % plants stage 3) July 3 GM maize (DKC6667YG) Male flowering date (50 % plants stage 3) July 8 Buffer conventional maize (P1921) Male flowering date (50 % plants stage 3) July 10 Start date of assessments 22/06/2012 Days Days

13. 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 3 (PR32T16) Buffer 2 (PR32T16) Buffer 1 (PR32T16) % of male flowering plants Male sterility on PR32T16 (Buffer) (?): 70-85 % Days Start date of assessments 22/06/2012

14. 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

15. WP 3: Task 3.1 Studies on coexistence in real agronomic fields GM maize GM 0,00% (?) Conventional maize Conventional maize GM 0,09% (?) GM maize

16. WP 3: Task 3.1 Studies on coexistence in real agronomic fields Location 1: Ullà Sampling methodology

17. 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

18. On-tape adhesion Filter adhesion (PMF) Microscopic count DNA extraction and purification + H2O, glass beads, CTAB buffer 5-10 μl DNA extraction and purification Real Time PCR 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) PMF/Sigma-2 Hirst type Weight the maize pollen (1-10 mg)

19. WP 3: Task 3.2 Photos: Pollen traps Location: Ullà

20. 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)

21. Relativ Yield Plus-Hybrid effect CMS effect Xenia effect Hybrid A Fertile Hybrid A Fertile Hybrid A Sterile Hybrid A Fertile Hybrid A Fertile Hybrid B Fertile Hybrid A Sterile Hybrid B Fertile WP 3: Task 3.3 CMS maize Cytplasmic male sterility (CMS) offers the potential for efficient biological containment to facilitate coexistence in maize Plus-Hybrid System 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 Fertile tassel Sterile tassel (Weingartner et al., 2002)

22. WP 3: Task 3.3 Field trial design – Germany and Czech Best proportion and arrangement of a pollen donor in a CMS-maize plot? • 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 CMS maize hybrid Torres Conventional maize White maize WM 17007 Locations Czech, Germany, Spain

23. WP 3: Task 3.3 Yield of the Plus-Hybrid trial in 2012 M10 / R 10 10 % WM + 90 % CMS maize M: mixture, WM mixed with CMS maize M 15 / R 15 15 % WM + 85 % CMS maize R: row, WM designed in rows M 20 / R 20 20 % WM + 80 % CMS maize Plot size 225 m”

24. 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

25. 2 1 3 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

26. 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

27. WP 3: Task 3.3 Cytoplasmic male sterile hybrids tested in Spain, FAO 600 – 700 Hybrids more adapted to Spanish climate?

28. WP 3: Task 3.3 - Impressions P-H-trial Quedlinburg Sowing in Prague Trail in Mas Badia Harvesting in Prague Maize and Hemp in Quedlinburg

29. WP 3: Task 3.3 - Impressions Cross-pollination trial in Mas Badia Variety trial in Mas Badia Work group meeting in Mas Badia Work group meeting in Mas Badia Torres

30. WP 3: First Conclusions Conclusions

31. 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