Certified Reference Materials - AOCS

ISO 17034:2016

A2LA Certificate 3438.01

Certified Reference Materials

AOCS 0804-A

AOCS 0804-B

AOCS 0804-C

AOCS 0804-D

First Batch

Denise Williams Scott Bloomer

Technical Service Manager Technical Director

Street Address:

AOCS, 2710 S. Boulder Drive

Urbana, IL 61802- 6996 USA

Phone: +1-217-359-2344; Fax: +1-217-351-8091;

E-Mail: [email protected]; Web: www.aocs.org

Report of Certification for 0804-A, B. C, D

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©AOCS, 2004 Updated 2019

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 AOCS advances the science and technology of oils, fats, surfactants and related materials, enriching the lives of people everywhere. More information regarding AOCS is available at http://www.aocs.org


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Table of Contents

Page

Abstract 4

Acknowledgements 5

Glossary 6

Introduction 8

Materials and Methods 8

Stability 10

Results 10

Sample Homogeneity 10

Prepared Sample Verification 11

References 22


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Abstract

This report describes the preparation and certification of the cottonseed CRMs AOCS

0804-A, AOCS 0804-B, AOCS 0804-C and AOCS 0804-D produced by AOCS Technical

Services in 2004. These CRMs have been prepared according to ISO Guides 30-35 and

are intended to serve as a control material for third party testing of cottonseed for

transformation events. The purity of the conventional and genetically modified cottonseed

was verified using DNA- and protein- based detection methods. AOCS 0804-A, AOCS

0804-B, AOCS 0804-C and AOCS 0804- D 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® (lin ‘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. The powder sample shall be stored dry in a sealed

container at ambient or cooler conditions in the dark.


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Acknowledgements

The authors would like to express sincere appreciation and gratitude to several individuals

and their companies for support and guidance throughout this project. Thanks to Markus

Lipp, Monsanto Company, for offering AOCS the opportunity to manufacture these

products; to Kirk Remund for answering a plethora of statistical questions; to Steve

Gregory, Texas A&M University, provided expertise for grinding/processing the

cottonseed into a uniform blend encompassing the entire matrix; to Frank Spiegelhalter

and Greg Ditta, GeneScan USA, who were integral players regarding all PCR analyses,

providing information on how to run the analyses and interpret the results; and to Brett

Roberts, Agdia, for explaining the complexities of immuno-assays and assisted with

interpretation of results.


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Glossary

AOCS American Oil Chemists' Society Bt-Cry1Ac Sequence characteristic of various Bollgard® crops Bt-Cry2Ab Sequence characteristic of various Bollgard II® crops Conventional Crop Crop variety with no history of transgenic technology and is

produced through traditional 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 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 be detected in a

sample.

EC European Commission Genome The full set of genes and associated DNA characteristic of

an organism

ISO International Organisation for Standardisation


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ISTA International Seed Testing Association GMO Organism that has had genetic sequences modified using

molecular-level techniques

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 to be copied. Primer sets are

synthesized to match sequences at the beginning and the

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 protein

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.

Quantitative PCR PCR methods that estimate the relative amount of target

DNA sequence in a mixture of DNA molecules

35S Promoter derived from cauliflower mosaic virus


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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 and AOCS 0804-D were

manufactured according to ISO Guides 30-35 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


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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.

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 material 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 17025 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


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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 0804-

D: 66, 84, 234, 249, 349, 440, 501, 751, 855, 984

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

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.


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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.

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

1 - - -

2 - - -

3 - - -

4 - - -

5 - + 0.4%

6 - - -

7 - - -

8 + - 0.2 %

9 - - -

10 - - -


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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® 0

98.46% 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 indicate

99.40% purity with 95% confidence).


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Table 3. Results from GeneScan for the homogeneity of Roundup Ready®

modified cottonseed (line ‘DP5690RR’).

Sample

CTP2/EPSPS

CP4 Bt-11 Bollgard

11 + +

12 + +

13 + +

14 + +

15 + +


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three trait (2A/1Ac/RR) ImmunoStrips™ on 500 Bollgard® cottonseeds.


Conventional 7

Roundup Ready® 0

Bollgard® 492

Bollgard II® 0

Bollgard®/RR® 1


97.13% of the seeds in this line exhibit the Bollgard® trait (492/500 seeds with 95%

confidence). All 493 seeds were genetically modified (493/500 seed indicates 97.39%

purity with 95% confidence).


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Table 5. Results from GeneScan for the homogeneity of Bollgard® modified

cottonseed (line ‘DP448B’).

Sample CTP2/EPSPS CP4 Bt-

Bollgard

16 + +

17 + +

18 + +

19 + +

20 + +


three trait (2A/1Ac/RR) ImmunoStrips™ on 500 Bollgard II® cottonseeds.


Conventional 3

Roundup Ready® 1

Bollgard® 4


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Bollgard II® 408

Bt-Cry-2Ab only 81

Bollgard®/RR® 0


78.68% 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 indicate 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

21 + +

22 + +

23 + +

24 + +

25 + +


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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.


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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

534 - - -

1162 - - -

1193 - - -

1254 - - -

1676 - - -

2622 - - -

2739 - - -

2740 - - -

3999 - - -

4354 - - -


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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

14 - -

101 + +

139 + +

382 + +

407 + +

596 + +

863 + +

885 + +

899 + +

958 + +

*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.


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Table 10. Results for the verification of Bollgard® modified cottonseed (line

‘DP448B’) as tested by GeneScan USA.

BioTech DNA

Total Cottonseed DNA

AOCS 0804-C CTP2/EPSPS CP4, % Bt-Bollgard

65 0.9 +

130 1.0 +

454 0.2 +

611 < 0.1 +

731 0.2 +

760 0.2 +

778 0.2 +

796 < 0.1 +

870 0.2 +

964 < 0.1 +


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Table 11. Results for the verification of Bollgard II® modified cottonseed (line

‘Bollgard II’) as tested by GeneScan USA.

biotech DNA

Total Cottonseed DNA

AOCS 0804-D CTP2/EPSPS CP4, % Bt-Bollgard

66 0.6 +

84 0.8 +

234 1.1 +

249 > 2.0 +

349 > 2.0 +

440 > 2.0 +

501 > 2.0 +

751 > 2.0 +

855 > 2.0 +

984 > 2.0 +


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References

Agdia

30380 County Road 6

Elkhart, IN 46514

Telephone: +1 574 264 2014

Tollfree: +1 800 62 AGDI Fax: +1 574 264 2153 http://www.agdia.com

GeneScan USA

2315 N Causeway Blvd, Suite 200

Metairie, LA 70001

Telephone: +1 504 297 4330

Toll Free: +1 866 535 273 Fax: +1 504 297 4335 http://www.gmotesting.com

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


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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 http://www.ucsusa.org/documents/seedreport_fullreport.pdf

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