Certified Reference Materials · for CTP2/EPSPS CP4, Bt-Cry1Ac, and/or Bt-Cry-2Ab present in the...
Transcript of Certified Reference Materials · for CTP2/EPSPS CP4, Bt-Cry1Ac, and/or Bt-Cry-2Ab present in the...
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
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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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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® 0
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 + +
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
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Bollgard II® 408
Bt-Cry-2Ab only 81
Bollgard®/RR® 0
Bollgard II®/RR® 2
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