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Biotechnology Consultation
Note to the File
BNF No. 000094
Date:07/07/2005
Subject: Biotechnology Notification File (BNF) BNF 000094: VIP3A Cotton Transformation Event COT102
Keywords: Cotton, Gossypium hirsutum L., Coker 312 variety, transformation event COT102, lepidopteran insect resistance, VIP3A protein, vip3A(a) gene, Bacillus thuringiensis strain AB88, hygromycin B phosphotransferase (APH4 protein), aph4 gene, Escherichia coli
In a submission dated October 27, 2003, Syngenta Seeds, Inc. (Syngenta) submitted to FDA a summary of the safety and nutritional assessment they have conducted on a new line of genetically engineered insect-resistant cotton, transformation event COT102 (hereafter referred to as COT102). The company provided additional information in submissions dated February 13, 2004, February 17, 2004, March 29, 2004, and July 26, 2004. Syngenta has concluded that COT102 is as safe and nutritious as conventional cotton varieties currently being marketed.
The intended effect of this genetic modification of cotton is to confer broad-spectrum lepidopteran insect resistance. To accomplish this objective, Syngenta introduced the vip3A(a) gene from B. thuringiensis, strain AB88 into the cotton variety Coker 312. The vip3A(a) gene encodes the pesticidal protein VIP3A. Syngenta states that plants expressing the VIP3A protein have resistance to several lepidopteran species including, but not limited to, Helicoverpa zea (cotton bollworm), Heliothis virescens (tobacco budworm), Pectinophora gossypiella (pink bollworm), Spodoptera frugiperda (fall armyworm), Spodoptera exigua (beet armyworm), Pseudoplusia includens (soybean looper), Trichoplusia ni (cabbage looper), and Bucculatrix thurberiella (cotton leaf perforator). Syngenta also introduced into the Coker 312 genome the aph4 gene from Escherichia coli, that encodes hygromycin B phosphotransferase protein (hereafter referred to as APH4). The APH4 protein serves as a selectable marker in the identification of the plant cells transformed with the vip3A(a) gene.
The Environmental Protection Agency (EPA) regulates plant-incorporated protectants under the Federal Food, Drug, and Cosmetic Act and the Federal Insecticide, Fungicide, and Rodenticide Act. Under EPA regulations, the VIP3A protein in COT102 is considered a pesticidal substance and the APH4 protein is considered an inert ingredient. Therefore, the safety assessment of these proteins falls under the regulatory purview of EPA.
Syngenta used Coker 312 as the parental variety for transformation. Coker 312 is a United States Protected Variety (PVP 7200100) currently owned by the SeedCo Corporation of Lubbock, TX. The Coker Pedigreed Seed company initially released Coker 312 in 1974. Syngenta states that they chose the Coker 312 variety because of its amenability to tissue culture and molecular transformation techniques. Syngenta also states that although Coker 312 is a viable commercial variety, currently it is not widely planted.
Syngenta constructed the binary Agrobacterium tumefaciens-based transformation plasmid vector, pCOT-1, using the following two expression cassettes within the left and right borders of the T-DNA region (the region designed to be transferred and integrated into the plant genome):
Transformation of cotton hypocotyl tissue was carried out by incubation of the plant tissue with cells of A. tumefaciens strain GV3101. This strain carried two separate plasmid vectors, the pCOT-1 vector and a disarmed helper plasmid vector, pMP90. The helper plasmid provided the virulence genes necessary for transfer of the pCOT-1 T-DNA. As pMP90 contained neither tumor inducing genes nor T-DNA borders, its DNA would not be expected to be transferred to the cotton genome. Following incubation with the A. tumefaciens cells, the hypocotyl tissue was plated onto synthetic medium containing the selection agent, hygromycin B. Transformed cotton cells expressing APH4 survived the selection and plants were subsequently regenerated.
In addition to the above-mentioned genetic elements, both vectors contain sequences outside of the T-DNA region that are necessary for selection and maintenance of the vectors in the appropriate bacterial hosts. Syngenta provides both published references and GenBank accession numbers for the expressed sequences and their regulatory elements in the notification.
Syngenta summarized the results of the genomic DNA blot (Southern) analyses used to assess the insertion event. Based on these analyses, Syngenta states that COT102 contains one insert with a single copy of the vip3A(a) and aph4 expression cassettes, without any transfer of vector sequences outside of the T-DNA region. Syngenta also summarized the cloning, amplification, and sequencing of the insertion event. Syngenta states that no open reading frames were found in the genomic sequences flanking the insertion site and that the sequence of the transgene insert in COT102 and the plasmid pCOT1 matched exactly.
Syngenta characterized the heritability of the transgene using standard Mendelian genetic tests. These genetic tests are detailed in the October 27, 2003 submission, and additional correspondence dated February 17, 2004. Syngenta performed Chi-square analysis of the segregants to show a stable pattern of inheritance as expected for a single integrated transgene.
Cottonseed, the by-product of cotton fiber production, is used in human food and animal feed. Cottonseed is processed into four major products: oil, meal, hulls, and linters. Cottonseed oil and to a lesser extent processed linters are routinely used in human food and have a long history of safe use. Cottonseed, cottonseed meal, and hulls are used in animal feed. Cottonseed contains natural toxicants, gossypol and cyclopropenoid fatty acids. As a result, cottonseed oil intended for human consumption is highly purified using a process that substantially reduces the content of gossypol and cyclopropenoid fatty acids. Whole cottonseed is used as a feed, supplying energy and fiber for dairy cows. Cottonseed meal is used as a protein supplement in animal feed. Hulls are used as a fiber component of livestock feeds due to their high cellulose and lignin content.
To compare the composition of COT102 with that of the non-transgenic parental cultivar Coker 312, Syngenta analyzed cottonseed samples from the 2001 and 2002 field trials. Syngenta hand harvested cottonseed from its research sites, ginned it in the laboratory, and acid delinted the fuzzy cottonseed by treatment with concentrated sulphuric acid. Syngenta described the preparation of the acid delinted cottonseed in its July 26, 2004 submission.
In 2001, cotton was grown at three geographical locations. A single sample of both the transgenic and non-transgenic cottonseed was collected for analysis from each location. Mean levels and standard deviations were calculated for all measured components using results from individual locations as replicates.
In 2002, cotton was grown at two locations using a replicated (four replicates), randomized block design. For each measured component, a mean value and standard deviation was calculated for each location and across both locations.
All analytical data from both 2001 and 2002 field trials were statistically evaluated using the F-test to determine whether there were statistically significant differences in the composition of the transgenic and non-transgenic lines.
Within the October 27, 2003 notification and in the submissions dated February 13, 2004, March 26, 2004, and July 26, 2004, Syngenta presents its evaluation of 49 separate components of acid delinted cottonseed in the transgenic and non-transgenic lines.
In addition, cottonseed obtained from one location in 2001 was processed into oil, non-toasted meal, and toasted meal. Total and free gossypol were measured in the resulting products.
Data presented by Syngenta for the 2001 crop show that there were no statistically significant differences between the transgenic and non-transgenic cottonseed, in the levels of all components except for lysine and tyrosine. The levels of these amino acids were higher in the transgenic than in the non-transgenic cottonseed. Additionally, the levels of eight (out of 17) amino acids and potassium in both transgenic and non-transgenic cottonseed and the level of linolenic acid in non-transgenic cottonseed were found to be outside the ranges reported for cottonseed in the International Life Sciences Institute (ILSI) Crop Composition Database.1
Samples collected from the replicated 2002 field trial were analyzed by a different commercial analytical laboratory. The statistical analysis of the analytical data combined from both locations showed no statistically significant differences between the transgenic and non-transgenic cottonseed in the levels of all measured components except for the minerals iron, zinc, and copper, and the amino acids serine and lysine. The levels of these components were lower in the transgenic cottonseed in comparison to the non-transgenic cottonseed. Nevertheless, the levels of all measured components were within ranges reported in published sources for acid delinted cottonseed including the ILSI Crop Composition Database accessed by Syngenta on May 14, 2004. Syngenta concludes that any difference in the composition between the transgenic and non-transgenic lines is not biologically significant.
Syngenta has concluded that insect-resistant cotton line COT102 is not materially different in safety, composition or any other relevant parameter from cotton now grown, marketed, and consumed. At this time, based on Syngenta's data and information, the Agency considers Syngenta's consultation on cotton line COT102 to be complete.
/s/
Susan J. Carlson, Ph.D.
(1)Syngenta explained that these discrepancies may be due to the fact that the 2001 trial was not replicated and only a single cottonseed sample was analyzed from each of the three locations. Syngenta also noted that an analytical error may have occurred in the determination of the amino acids glycine and glutamic acid.
