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Biotechnology Consultation
Note to the File
BNF No. 000081
Date: September 28, 2004
Subject: Bacillus thuringiensis Cry34Ab1/35Ab1 Corn Event DAS-59122-7
Keywords: Corn, Zea mays, insect-resistant corn, Cry34Ab1 protein, Cry35Ab1 protein, phosphinothricin acetyltransferase (PAT), Agrobacterium tumefaciens, Bacillus thuringiensis, insect resistance, western corn rootworm (Diabrotica virgifera), northern corn rootworm (D. barberi), Mexican corn rootworm (D. virgifera zeae).
In a submission dated December 11, 2003, Mycogen Seeds/Dow AgroSciences LLC (DAS) and Pioneer Hi-Bred International, Inc. (PHI) provided to the Food and Drug Administration (FDA) a summary of the safety and nutritional assessment they conducted on a new bioengineered insect-resistant corn line, corn event DAS-59122-7. The company provided additional information on March 5 and March 12, 2004. According to DAS/PHI the hybrids derived from corn event DAS-59122-7 demonstrate a different spectrum of activity against Coleopteran pests from other Bacillus thuringiensis (B. t.) proteins. DAS/PHI conclude that food and feed derived from the new insect resistant corn is as safe and nutritious as food and feed derived from conventional corn varieties currently being marketed.
The intended effect of this bioengineered corn is to confer resistance to Coleopteran insects such as the western corn rootworm, the northern corn rootworm, and the Mexican corn rootworm. To accomplish this objective, DAS/PHI introduced the genes cry34Ab1 and cry35Ab1, derived from B. t. strain PS149B1, and the pat gene, derived from Streptomyces viridochromogenes into the corn line Hi-II. The Cry34Ab1 and Cry35Ab1 proteins in corn event DAS-59122-7 confer resistance to Coleopteran insects. The pat gene confers tolerance to chemically synthesized phosphinothricin products such as glufosinate-ammonium herbicide and is used as a selectable marker.
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 Cry34Ab1 and Cry35Ab1 proteins in corn event DAS-59122-7 are considered pesticidal substances and the phosphinothricin acetyltransferase (PAT) protein is considered an inert ingredient. Therefore, the safety assessment of these proteins falls under the regulatory purview of EPA.
To generate the corn event DAS-59122-7, DAS/PHI used an Agrobacterium tumefaciens methodology. DAS/PHI transformed the public corn line Hi-II using the PHP17662 transformation vector. The vector is a binary T-DNA plasmid carrying the transgenes (cry34Ab1, cry35Ab1, and pat) for insertion into the plant genome. The T-DNA region of the vector contains the elements listed in Table 1 as well as T-DNA border segments.
| Genetic Element | Description |
|---|---|
| UBI1ZM PRO | Zea mays ubiquitin promoter (plus ubiquitin 5' UTR and intron) for cry34Ab1 gene expression. |
| cry34Ab1 | Maize-optimized synthetic cry34Ab1 gene conferring insect resistance to Coleopteran insects. |
| PINII TERM | Terminator for cry34Ab1 from Solanum tuberosum proteinase inhibitor II. |
| TA PEROXIDASE | Root-preferred promoter from Triticum aestivum peroxidase for cry35Ab1 gene expression. |
| cry35Ab1 | Maize-optimized synthetic cry35Ab1 gene conferring insect resistance to Coleopteran insects. |
| PINII TERM | Terminator for cry34Ab1 from Solanum tuberosum proteinase inhibitor II. |
| CaMV35S PRO | 35S promoter from Cauliflower Mosaic Virus for expression of pat. |
| pat | Synthetic plant-optimized phosphinothricin acetyltransferase coding sequence from Streptomyces viridochromogenes. Used as a selectable marker. |
| CaMV35S TERM | 35S terminator from Cauliflower Mosaic Virus for pat. |
The backbone of the vector contains the spectinomycin and tetracycline resistance genes to facilitate cloning and maintenance of the plasmid in bacterial hosts. DAS/PHI state that a lack of binding of DNA probes specific for spectinomycin and tetracycline resistance genes to the DNA isolated from the transformed corn indicates that the insertion in transgenic corn event DAS-59122-7 did not include antibiotic resistance genes or other genes outside the T-DNA region from plasmid PHP17662.
DAS/PHI characterized the DNA introduced into corn event DAS-59122-7, using restriction enzyme digestion of genomic DNA followed by Southern blot analysis. DAS/PHI state that based on the results obtained from these analyses, the T-DNA from plasmid PHP17662 integrated as a single intact copy. DAS/PHI further state that the integration was stable within and across generations. DAS/PHI also assessed Mendelian segregation of event DAS-59122-7 and concluded that DAS-59122-7 is inherited as a single dominant trait.
Corn grain (kernel) and its processed fractions are consumed as human food and animal feed. The majority of corn is used as animal feed; the remainder is exported, held as ending stock, processed into corn syrup, converted to ethanol, extracted for starch, used as processed food, and grown as corn seed. The kernel consists of pericarp, germ, and endosperm. The pericarp can be removed during processing to yield the germ (which contains 50% oil) and endosperm (which contains 70% starch). Corn can be processed by wet and dry milling. Wet milling produces starches, sweeteners (such as high fructose corn syrup), gluten feed, meal, dextrose, ethanol, and maltodextrins. Dry milling includes three processes, the stone grinding process, the dry-grind ethanol system, and the tempering degermination system. Stone grinding produces whole corn meal that is further processed to remove the oil. The dry-grind ethanol process produces ethanol through the fermentation of processed kernels, and a dried solubles by-product that is an important component of livestock feed. The tempering degermination system produces foods such as flaking grits, coarse - and fine grits, meal, flour, oil, and hominy feed.
Corn is also used for masa production, which is the starting material for tortillas, tortilla chips, taco shells, and corn chips. Masa is produced through the cooking of corn in alkali, followed by grinding to produce dough.
Corn grain and by-products of wet and dry milling are used as animal feed. The whole corn plant can also be used as feed for ruminant animals. Corn plants are harvested at an appropriate stage and fed to animals or stored as silage.
DAS/PHI analyzed the composition of forage and grain from transgenic corn event DAS-59122-7 and a non-transgenic control derived from the parental line Hi-II to assess whether the transgenic corn differs from non-transgenic corn. DAS/PHI collected plant material grown at six different field sites for this analysis. At each field site, DAS/PHI employed a randomized block design containing four blocks (replicates) with transgenic and non-transgenic seeds planted in two row plots located randomly within each block, and bordered by 12 rows of non-transgenic corn. At each location, blocks two, three, and four were designated for the collection of samples for compositional analysis. DAS/PHI used analysis of variance (ANOVA) to analyze the compositional data. DAS/PHI compared the mean level (from all field sites) of each component in transgenic and non-transgenic plants and identified statistically significant differences in the composition of non-transgenic control and transgenic plants at the five percent level of significance. DAS/PHI also conducted the statistical analysis of data from individual sites for those components that showed statistically significant differences in the mean levels.
DAS/PHI compared the composition of the transgenic corn to the composition of non-transgenic corn with a similar genetic background (near isoline) and to published literature values. DAS/PHI used the non-transgenic corn back cross 1 (BC1) hybrid material as the non-transgenic corn. The BC1 hybrid seed has a genetic background similar to that of corn event DAS-59122-7. To obtain BC1 hybrid material, DAS/PHI crossed the original transformant (T0) to Pioneer inbred A to generate T1 seed. The T1 plants were crossed with Pioneer inbred B twice to generate BC1 plants. The BC1 plants were crossed with Pioneer inbred C to generate BC1 hybrid seed that was planted in the composition study. The BC1 hybrid plants were segregating 1:1 for the Cry34Ab1/35Ab1 insert (i.e., 50 percent of the plants contain the Cry34Ab1/Cry35Ab1 insert and the other 50 percent do not contain the insert). Plants that were resistant to glufosinate-ammonium were used as test plants and the plants that were sensitive to glufosinate-ammonium were used as control plants for the analyses.
DAS/PHI measured the following groups of components:
A list of specific components contained in each group is shown in Table 2.
| Proximates | Minerals | Amino Acids | Fatty Acids | Anti-Nutrients | Secondary Metabolites | Vitamins |
|---|---|---|---|---|---|---|
| ash crude fat crude protein carbohydrates crude fiber acid detergent fiber (ADF) neutral detergent fiber (NDF) |
calcium copper iron magnesium manganese phosphorus potassium sodium zinc |
methionine cysteine lysine tryptophan threonine isoleucine histidine valine leucine arginine phenylalanine glycine alanine aspartic acid glutamic acid proline serine tyrosine |
palmitic (16:0) stearic (18:0) oleic (18:1) linoleic (18:2) linolenic (18:3) |
phytic acid trypsin inhibitor |
raffinose furfural p-coumaric acid ferulic acid |
A B1 B2 Folic acid E Tocopherols |
DAS/PHI determined the levels of the following components of forage of corn event DAS-59122-7 and non-transgenic control forage collected at the R41 development stage:
DAS/PHI report no statistically significant differences for forage in the mean levels of crude protein, crude fat, crude fiber, ADF and NDF between transgenic and non-transgenic plants. Statistically significant differences were observed in the levels of ash and carbohydrates between transgenic and control forage, but all values were within literature values. DAS/PHI report that the mean levels of calcium and phosphorous were higher in the forage of the transgenic plants compared to the forage from the non-transgenic plant, but that all values were within literature ranges.
DAS/PHI determined the levels of the following components of mature grain of corn event DAS-59122-7 and mature grain of the non-transgenic corn:
DAS/PHI report no statistically significant differences between transgenic and non-transgenic grain in the mean levels of proximate components, crude fat, crude fiber, ADF, and NDF. DAS/PHI observed statistically significant differences between transgenic and non-transgenic control grain in the mean levels of crude protein, ash, and carbohydrates, with the levels of crude protein and ash being higher, and the carbohydrate level being lower in the transgenic grain versus non-transgenic grain. However, all values were within the reported literature ranges.
DAS/PHI report statistically significant differences between transgenic and non-transgenic grain for the mean levels of all measured fatty acids, with the exception of oleic acid. Linoleic and linolenic acid levels were higher, and palmitic and stearic acid levels were lower in the transgenic versus the non-transgenic grain respectively. The levels of all measured fatty acid values were within literature ranges.
DAS/PHI report statistically significant differences in the mean levels of the amino acids tryptophan, isoleucine, histidine, valine, leucine, arginine, phenylalanine, proline and tyrosine, which were higher in the transgenic grain compared to the non-transgenic grain, but were within literature ranges. DAS/PHI report no statistically significant differences in the levels of minerals, except for calcium. The calcium level was higher in the transgenic grain compared to the non-transgenic grain but was within the literature range.
DAS/PHI reports no statistically significant differences in the levels of vitamin B1 and vitamin A (measured as β-carotene). Statistically significant differences were observed in the levels of folic acid and vitamin E (measured as α-tocopherol). The levels of all measured vitamins were within literature ranges, with the exception of vitamin B2 and β-carotene . Vitamin B2 was not detected and the levels of β-carotene in both transgenic and non-transgenic grain were higher than the levels reported in the literature. DAS/PHI attribute these higher values to the assay used to measure β-carotene, where other xanthophylls or carotenoid pigments may have been measured as β-carotene.
DAS/PHI also measured the naturally occurring secondary metabolites, furfural, a heterocyclic aldehyde, and two phenolic acids, p-coumaric acid and ferulic acid, as well as the anti-nutrients phytic acid, raffinose, and trypsin inhibitor. Furfural was not detected. DAS/PHI report no statistically significant differences between the transgenic and non-transgenic grain in the mean levels of the other secondary metabolites or anti-nutrients.
DAS/PHI have concluded that corn event DAS-59122-7 is not materially different in composition, safety, wholesomeness, or any relevant parameter from corn now grown, marketed, and consumed. At this time, based on DAS/PHI's summary of its data and information, the agency considers DAS/PHI's consultation on Cry34Ab1/35Ab1 corn event DAS-59122-7 to be complete.
/s/
Karin Ricker, Ph.D.
(1)The growth stage when the material within the kernel produces a doughy consistency. This stage can occur as early as 24 days after pollination.
