Certification Report

Transcription

1 Certification Report (Revised 27 March 2009) The certification of Conventional, Roundup Ready, Bollgard, and Bollgard II Cottonseed Reference Materials AOCS 0804-A AOCS 0804-B AOCS 0804-C AOCS 0804-D G. Clapper and R. Cantrill AOCS, Champaign, IL, USA

2 Legal Notice Neither AOCS nor any person acting on behalf of AOCS is responsible for the use, which might be made of the following information. AOCS Mission Statement: To be a global forum to promote the exchange of ideas, information and experience, to enhance personal excellence, and to provide high standards of quality among those with a professional interest in the science and technology of fats, oils, surfactants, and related materials. More information regarding AOCS is available at AOCS, 2004

3 Table of Contents Page Abstract 4 Acknowledgements 5 Glossary 6 Introduction 8 Materials and Methods 8 Results Sample Homogeneity 10 Prepared Sample Verification 14 References 17

4 Abstract This report describes the preparation and certification of the cottonseed CRMs AOCS 0804-A, AOCS 0804-B, AOCS 0804-C, AOCS 0804-D, AOCS 0804-E, and AOCS 0804-F produced by AOCS Technical Services in These CRMs have been prepared according to ISO Guides and are intended to serve as a control group for third party testing of cottonseed or grain for transformation events. The purity of the conventional and genetically modified cottonseed was verified using DNA- and proteinbased detection methods. AOCS 0804-A, AOCS 0804-B, AOCS 0804-C, AOCS D, AOCS 0804-E, and AOCS 0804-F are available in 27-mL glass headspace vials. The conventional cottonseed (line 'Stoneville 474 '), Roundup Ready (line DP5690RR ) cottonseed, Bollgard (line DP448B ) cottonseed, Bollgard /Roundup Ready (line SG215BG/RR) cottonseed, and Bollgard II /Roundup Ready (line DP468BGII/RR ) cottonseed were clean seed quality provided by Monsanto Company, St. Louis, MO, USA. The Bollgard II (line Bollgard II ) material was bulk-grain quality provided by Monsanto Company, St. Louis, MO. The materials were prepared by grinding the bulk sources of seed/grain according to standard cottonseed processing protocols and were then packaged in a Nitrogen environment.

5 Acknowledgements The authors would like to express sincere appreciation and gratitude to several individuals and their companies for support and guidance throughout this project. Firstly to Markus Lipp, Monsanto Company, for offering AOCS the opportunity to manufacture these products and Kirk Remund for answering a plethora of statistical questions. Steve Gregory, Texas A&M University, provided expertise for grinding/processing the cottonseed into a uniform blend encompassing the entire matrix. Frank Spiegelhalter and Greg Ditta, GeneScan USA, were integral players regarding all PCR analyses, providing information on how to run the analyses and interpret the results. Brett Roberts, Agdia, explained the complexities of immuno-assays and assisted with interpretation of results.

6 Glossary 35S AOCS Bt-Cry1Ac Bt-Cry2Ab Promoter derived from the cauliflower mosaic virus American Oil Chemists' Society Sequence characteristic of various Bollgard crops Sequence characteristic of various Bollgard II crops Conventional Variety Crop variety with no history of genetic engineering and are produced through plant-breeding techniques that rely on selecting and mating parent plants possessing promising traits and repeatedly selecting for superior performance among their offspring CTP2/EPSPS CP4 Sequence characteristic of various Roundup Ready crops DNA Deoxyribonucleic Acid is the linear, double-helix macromolecule that makes up the genetic material of most organisms Detection Limit Lowest level at which target DNA can exist in a sample and be reliably tested by PCR methods. It is typically expressed as a percentage: the ratio of the number of transgenically derived genomes to the number of crop genomes times 100 percent EC GMO Genome European Commission Organism that has had genetic sequences modified using molecular-level techniques The full set of genes and associated DNA characteristics of an organism

7 ISO International Organisation for Standardisation ISTA International Seed Testing Association PCR Polymerase Chain Reaction: technique used to determine whether a sample of plant tissue contains a particular DNA sequence. PCR relies on primer sets that zero in on a particular target DNA sequence and a special DNA-copying enzyme (DNA polymerase) that makes enough copies of the target sequence for identification and measurement. Primer set Short pieces of DNA added to PCR mixtures to identify the pieces of target DNA that will be copied. Primer sets are synthesized to match sequences at the beginning and end of the target DNA, defining the exact segment of DNA to be duplicated by the DNA-copying enzyme. Promoter/Terminator Regulatory sequences of DNA that start and stop the processes by which cells manufacture proteins Qualitative PCR PCR methods that determine the presence or absence of a specific target DNA sequence at a particular level of detection. Quantitation Limit Lowest level at which the amount of target DNA sequence in a sample can be reproducible. It is typically expressed as the ration of the number of transgenic genomes to the number of crop genomes times 100 percent. Quantitative PCR PCR methods that estimate the relative amount of target DNA sequence in a mixture of DNA molecules

8 Introduction Plant biotechnology is an extension of traditional plant breeding. It allows plant breeders to develop crops with specific beneficial traits including insect, disease, and herbicide resistance; processing advantages; and nutritional enhancement. A useful tool for identifying these new traits is Certified Reference Material created from seed or grain with the new trait as well as CRM created from the conventionally bred matrix. The European Commission has mandated that a method for detecting a new biotech event and Certified Reference Material must be available before the EC will authorize acceptance of a new genetically modified trait. Several nations outside Europe also require grain and ingredients to have a threshold level ranging from 0.90 to 5% of authorized biotech events before accepting a shipment. With this in mind, AOCS 0804-A, AOCS 0804-B, AOCS 0804-C, AOCS 0804-D, AOCS 0804-E, and AOCS 0804-F were manufactured according to ISO Guides and in accordance with EC No 1829/2003. The CRMs are available through AOCS. Materials and Methods Monsanto Company (St. Louis, MO) delivered 75 kg (3x25 kg) conventional cottonseed (line Stoneville 474 ) and 25 kg each of Roundup Ready (line DP5690RR ) cottonseed, Bollgard (line DP448B ) cottonseed, Bollgard II (line Bollgard II ) cottonseed, Bollgard /Roundup Ready (line SG215BG/RR) cottonseed, and Bollgard II /Roundup Ready (line DP468BGII/RR ) cottonseed to AOCS. The materials were all clean seed quality, with the exception of Bollgard II (line Bollgard II ) which was bulk-grain quality. The International Seed Testing Association's (ISTA) Seed Science and Technology Rules state a minimum of 5 primary samples (small portion taken from one point in the lot) be taken from batches up to 500 kg. Gossypium spp., cottonseed, should have a maximum lot size of 25,000 kg, a submitted sample of 1 kg, and a working sample of 350 grams for detection of other species.

9 AOCS received the bulk materials from Monsanto Company and made arrangements for Texas A&M University to process the materials at two separate times (conventional materials first and the biotech materials two weeks later). Before the materials were shipped to Texas A&M University, samples were taken from randomly selected areas and depths in each container to form 6, 5kg composite samples. Five working samples of 100g each were prepared from each composite and sent to GeneScan USA, New Orleans, LA (ISO Accredited) for qualitative PCR analysis, followed with quantitative PCR if qualitative results for conventional seed indicated that CTP2/EPSPS CP4, Bt-Cry1Ac, and/or Bt-Cry-2Ab were present in the samples. This testing was for purity as well as homogeneity purposes. Five hundred seeds were randomly selected from each of the biotech composite samples and analyzed with Agdia s Bollgard II and Roundup Ready three trait (2A/1Ac/RR) ImmunoStrips to verify seed-lot purity. The Stoneville 474 seed (non-modified) was packaged in 27-mL headspace vials and sealed in a nitrogen environment. AOCS used the Random Number Generator function of Microsoft Excel 2000 to select samples for verification of purity, homogeneity, and to rule out contamination during packaging. Sample numbers AOCS 0804-A: 534, 1162, 1193, 1254, 1676, 2622, 2739, 2740, 3999, and 4354 were sent to GeneScan USA (New Orleans, LA) for qualitative PCR analysis, followed with quantitative PCR if qualitative results indicated that CTP2/EPSPS CP4, Bt-Cry1Ac, and/or Bt-Cry-2Ab were present in the sample. After the non-modified seed packaging was completed, the genetically modified cotton lines were packaged in 27-mL headspace vials and sealed in a nitrogen environment. AOCS used the Random Number Generator function of Microsoft Excel 2000 to select samples for verification of purity, homogeneity, and to rule out contamination during packaging. Sample numbers AOCS 0804-B: 14, 101, 139, 382, 407, 596, 863, 885, 899, 958; AOCS 0804-C: 65, 130, 454, 611, 731, 760, 778, 796, 870, 964; AOCS D: 66, 84, 234, 249, 349, 440, 501, 751, 855, 984; AOCS 0804-E: 239, 240, 259, 266, 590, 607, 618, 685, 790, 853; and AOCS 0804-F: 25, 45, 66, 67, 78, 84, 539, 579, 957,

10 981 were sent to GeneScan USA (New Orleans, LA) for qualitative PCR analysis to screen for CTP2/EPSPS CP4, Bt-Cry1Ac, and/or Bt-Cry-2Ab present in the samples. Stability of these CRMs has been listed as 1year from the introduction date. The seed used to manufacture these materials was clean seed quality with germination guaranteed for 1 year in the original packaging. The materials have been ground and are stored frozen under Nitrogen gas in a sealed, glass vial. These materials are expected to be stable for longer than the estimated expiration date. This information will be reevaluated at time of expiration. If the samples are still representative of the certified value, the certificates will be extended. Results and Discussion Sample Homogeneity The following tables are the purity data for the homogeneity samples. The non-modified cottonseed (line Stoneville 474 ) is presented in Table 1. Eight samples were negative for indicators of genetic modification; one sample was found to contain 0.4% quantifiable 35S in cotton; and one sample was found to contain 0.2% quantifiable RR in cotton. Results for the genetically modified cottonseeds are presented in Tables 3, 5, and 7. Tables 2, 4, and 6 include the data generated from Agdia s Bollgard II and Roundup Ready three trait (2A/1Ac/RR) ImmunoStrips. Five hundred seeds were tested from each sample to verify seed-lot purity. Samples were prepared as either identity preserved conventional or identity preserved genetically modified cottonseed. Sample heterogeneity can be ignored due to the absence of mixing conventional and genetically modified cottonseed into prepared blends.

11 Table 1. Results from GeneScan for the homogeneity of non-modified cottonseed (line Stoneville 474 ). biotech DNA Sample CTP2/EPSPS Bt-11 total cottonseed DNA CP4 Bollgard % % Table 2. Results from administering Agdia s Bollgard II and Roundup Ready three trait (2A/1Ac/RR) ImmunoStrips on 500 Roundup Ready cottonseeds. Cottonseeds Tested Results Conventional 0 Roundup Ready 497 Bollgard 0 Bollgard II 0 Bollgard /RR 3 Bollgard II /RR % of the seeds in this line exhibit the Roundup Ready trait (497/500 seeds with 95% confidence). All 500 seeds were genetically modified (500/500 seeds indicates 99.40% purity with 95% confidence).

12 Table 3. Results from GeneScan for the homogeneity of Roundup Ready modified cottonseed (line DP5690RR ). Sample CTP2/EPSPS CP4 Bt-11 Bollgard Table 4. Results from administering Agdia s Bollgard II and Roundup Ready three trait (2A/1Ac/RR) ImmunoStrips on 500 Bollgard cottonseeds. Cottonseeds Tested Results Conventional 7 Roundup Ready 0 Bollgard 492 Bollgard II 0 Bollgard /RR 1 Bollgard II /RR % of the seeds in this line exhibit the Bollgard trait (492/500 seeds with 95% confidence). All 493 seeds were genetically modified (493/500 seeds indicates 97.39% purity with 95% confidence).

13 Table 5. Results from GeneScan for the homogeneity of Bollgard cottonseed (line DP448B ). modified Sample CTP2/EPSPS CP4 Bt- Bollgard Table 6. Results from administering Agdia s Bollgard II and Roundup Ready three trait (2A/1Ac/RR) ImmunoStrips on 500 Bollgard II cottonseeds. Cottonseeds Tested Results Conventional 3 Roundup Ready 1 Bollgard 4 Bollgard II 408 Bt-Cry-2Ab only 81 Bollgard /RR 0 Bollgard II /RR % of the seeds in this line exhibit the Bollgard II trait (408/499 seeds with 95% confidence). 497 seeds were genetically modified (497/499 seeds indicates 98.45% purity with 95% confidence). Table 7. Results from GeneScan for the homogeneity of Bollgard II modified cottonseed (line Bollgard II ). Sample CTP2/EPSPS CP4 Bt-Bollgard

14 Prepared Sample Verification Once the ground cottonseed was packaged, 10 samples of each variety were identified by the Microsoft Excel Random Number Generator and sent to GeneScan USA (New Orleans, LA) for qualitative PCR analysis. The Bollgard and Bollgard II samples were also subjected to quantitative PCR if the qualitative result was positive for Roundup Ready. The 35S quantitation will detect 35S DNA in Bollgard, Bollgard II, and in Roundup Ready cotton, whereas, the Roundup Ready cotton quantitation detects the Roundup Ready cotton DNA in cotton DNA specifically. Therefore, it is not possible with this test to ascertain the level of Bollgard or Bollgard II presence in these Roundup Ready cotton samples. Table 8 verifies that no contamination was introduced during the packaging phase of AOCS 0804-A. Tables 9-11 display the results for Roundup Ready (line DP5690RR ) cottonseed, Bollgard (line DP448B ) cottonseed, Bollgard II (line Bollgard II ) cottonseed, Bollgard /Roundup Ready (line SG215BG/RR) cottonseed, and Bollgard II /Roundup Ready (line DP468BGII/RR ) cottonseed respectively. Table 13 indicates that both Bollgard and Roundup Ready traits were found in the samples. This information concurs with the results from Table 2; the seed lot does contain some seed with the stacked trait. The current testing strategies allow for determining the level of Roundup Ready trait presence in Bollgard and Bollgard II, but not the reverse. Tables 14 and 15 also concur the information presented in Tables 4 and 6, though the level of Roundup Ready trait was quantified.

15 Table 8. Results for the verification of non-modified cottonseed (line Stoneville 474 ) as tested by GeneScan USA. AOCS 0804-A CTP2/EPSPS CP4 Bt-Bollgard biotech DNA total cottonseed DNA Table 9. Results for the verification of Roundup Ready modified cottonseed (line DP5690RR ) as tested by GeneScan USA. AOCS 0804-B CTP2/EPSPS CP4 Bt-Bollgard *The 35S quantitation will detect 35S DNA in Bollgard, Bollgard II, and in Roundup Ready cotton, whereas, the Roundup Ready cotton quantitation detects the Roundup Ready cotton DNA in Cotton DNA specifically. Therefore, it is not possible with this test to ascertain the level of Bollgard or Bollgard II presence in these Roundup Ready cotton samples.

16 Table 10. Results for the verification of Bollgard DP448B ) as tested by GeneScan USA. modified cottonseed (line BioTech DNA Total Cottonseed DNA AOCS 0804-C CTP2/EPSPS CP4, % Bt-Bollgard < < < Table 11. Results for the verification of Bollgard II Bollgard II ) as tested by GeneScan USA. modified cottonseed (line biotech DNA Total Cottonseed DNA AOCS 0804-D CTP2/EPSPS CP4, % Bt-Bollgard > > > > > > > 2.0 +

17 References Agdia County Road 6 Elkhart, IN Telephone: Tollfree: AGDIA Fax: GeneScan USA 2315 N Causeway Blvd, Suite 200 Metairie, LA Telephone: Toll Free: Fax: ISO Guide 30:1992 (E/F), Terms and definitions used in connection with reference materials ISO Guide 31:2000 (E), Reference Materials-Contents of certificates and labels ISO Guide 32:1997 (E) Calibration in analytical chemistry and use of certified reference materials ISO Guide 33:2000 (E) Uses of certified reference materials ISO Guide 34:2000 (E) General requirements for the competence of reference material producers ISO Guide 35:1989 (E) Certification of reference materials-general and statistical principles International Seed Testing Association, International Rules of Seed Testing: Seed Science and Technology Rules, Volume 21, Supplement, Rules, 1993 Union of Concerned Scientists' Gone to Seed Report