Biotechnology Consultation Note to the File BNF No. 000035
September 6, 1996
Subject: Roundup Ready (Glyphosate Tolerant) Corn & Insect Protected/Roundup Ready Corn
Corn, insect resistant, Bacillus thuringiensis subsp. kurstaki, cryIA(b), Insect control protein CryIA(b), Ostrinia nubilalis, European Corn Borer, hsp70 intron, Glyphosate herbicide tolerant (Roundup Ready), CP4 EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS protein), gox, glyphosate oxidoreductase (GOX protein), nptII, neomycin phosphotransferase II (NPTII protein).
In a submission dated July 2, 1996, Monsanto Company provided summary information to support their safety and nutritional assessment of new corn lines MON 802, 805, 830, 831 and 832.
Intended Effect and Food/Feed Use
The intended technical effects of these genetic modifications of corn are i) to confer resistance to the feeding of the European Corn Borer (ECB), Ostrinia nubilalis, a lepidopteran pest insect and/or ii) to confer tolerance to glyphosate, the active ingredient in the herbicide Roundup® (hence; Roundup Ready™). Monsanto states that insect resistance is conferred by the insecticidal protein cryIA(b), which is reported to be identical to that found in nature and in commercial insecticidal formulations obtained from Bacillus thuringiensis. Corn lines so modified, MON 802 and 805, also resist the feeding of another lepidopteran pest insect, the southwestern corn borer. The firm indicates that one protein, 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS), conferring tolerance to the herbicide glyphosate, is equivalent to that present in modified soybeans and cotton, about which Monsanto has previously informed the Agency. The GOX protein also confers glyphosate tolerance. Corn lines MON 830, 831, and 832 were modified to express only the herbicide tolerance trait (Roundup Ready: RR). Corn lines MON 802 and 805 are both insect resistant and Roundup Ready (IR/RR).
The firm reports that corn grain and its derivatives are used in many human foods, but 50 to 60% of the grain produced annually in the United States is used for animal feed. Human food products include starch, high fructose corn syrup, ethanol and corn oil. Whole plant corn silage, which makes up 10 to 12% of annual corn acreage, is a major ruminant feedstuff
Molecular Alterations and Characterization
The novel genetic material contained in lines MON 802, 805, 830, 831 and 832 was inserted into the corn genotype, Hi-II, using particle acceleration transformation. The transformation vectors included several plasmids containing one or more of the following genes: cryIA(b), CP4 EPSPS, and gox. CryIA(b) confers insect resistance, while the CP4 EPSPS and gox genes confer glyphosate tolerance. Herbicide tolerance was both a selection aid to identify modified plant cells and a desired commercial trait. Additionally, a nptII gene, encoding the enzyme neomycin phosphotransferase II (NPTII), was inserted under the control of its own bacterial promoter, enabling plasmid manipulation in bacterial systems.
Monsanto states that the cryIA(b) gene used to produce MON 802 and 805 IR/RR corn lines is a modification of the cryIA(b) gene from Bacillus thuringiensis subsp. kurstaki strain HD-1. For these lines and RR corn lines 830, 831 and 832, the CP4 EPSPS gene was derived from Agrobacterium sp. strain CP4, and the gox gene was cloned from Ochrobactrum anthropi (formerly Achromobacter) strain LBAA. All genes except nptII are reported to be under the control of the E35S promoter and the corn hsp70 intron. In addition, these genes contain the NOS 3' termination sequence.
The specific transformation vectors used were PV-ZMGT03 (gox) and PV-ZMBK15 (CP4 EPSPS, cryIA(b)) for MON 802 (IR/RR), PV-ZMBK10 (gox, CP4 EPSPS, cryIA(b)) for MON 805 (IR/RR), and PV-ZMBKO7 (cryIA(b)) and PV-ZMGT10 (CP4 EPSPS, gox) for MON 830, 831 and 832 (RR). All plasmids contained the nptII gene under the control of a bacterial promoter and an origin of replication (ori) from pUC, according to Monsanto.
To exhibit herbicide tolerance, the CP4 EPSPS protein must be translocated into the chloroplast. To achieve this, the chloroplast transit peptide coding sequence (CTP2) from the Arabidopsis thaliana EPSPS was fixed to the N-terminus of the CP4 EPSPS protein. The transit peptide is cleaved from the enzyme after arrival in the chloroplast. The GOX enzyme also requires translocation into the chloroplast for activity. Translocation is accomplished by use of another transit peptide, CTP1, derived from the ribulose-1,5-bisphosphate carboxylase (SSUIA) gene from A. thaliana. The hsp70 intron was used to increase transcription levels of both herbicide tolerance genes.
Monsanto determined the insert number and the genetic elements present in each insert by appropriate restriction enzyme digestion followed by Southern blot analysis. MON 802 contains two closely linked inserts: a 23 kilobase (Kb) insert with cryIA(b), CP4 EPSPS, gox, nptII, and ori-pUC, and a 8 Kb insert with gox, nptII, and ori-pUC. MON 805 contains a single 17 Kb insert with cryIA(b), CP4 EPSPS, gox, and nptII/ori-pUC. MON 830 has one 14 Kb insert containing CP4 EPSPS, gox, and the nptII/ori-pUC backbone. MON 831 has one 13.5 Kb insert containing CP4 EPSPS, gox and nptII/ori-pUC. MON 832 has one 16 Kb insert containing CP4 EPSPS, up to three copies of gox, and two nptII/ori-pUC sequences (one complete and one rearranged). Monsanto reports that segregation data obtained for three generations indicated stable introduction at a single site with Mendelian segregation for MON 805, 830, 831 and 832. The two inserts present in MON 802 are reported to be closely linked, since they segregated as a single locus through three generations. The firm indicates that the inserts were stable through at least four generations.
Expressed Proteins/Regulatory Considerations
Monsanto states that the cryIA(b) protein present in MON 802 and 805 is identical to that present in nature and commercial microbial preparations approved by the Environmental Protection Agency (EPA). The safe use of insecticidal proteins as pesticides and the use of selectable markers as pesticidal inert ingredients in the development of insect resistant plant varieties are regulated by EPA under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Federal Food, Drug, and Cosmetic Act (FFDCA). Therefore, we have not addressed the safe use of cryIA(b) as a pesticide or the safe use of CP4 EPSPS and GOX as pesticidal inert ingredients.
Monsanto examined the expression levels of introduced proteins in leaf, whole plant tissue and grain and reported that levels were low. The firm states that since the nptII gene is under control of a bacterial promoter, expression would not be expected in corn.
Compositional analyses by Monsanto for grain from the new corn varieties included proximate analyses (protein, fat, ash, and moisture), amino acid composition, fatty acid profile, calcium and phosphorus. Fiber content was determined using the crude fiber analysis for MON 802 and 805, while acid detergent fiber (ADF) and neutral detergent fiber (NDF) methodologies were utilized for varieties 830, 831 and 832. Values obtained for the new corn lines were comparable to appropriate control lines and to published literature values for corn varieties currently on the market.
Compositional analyses by Monsanto for forage from the new corn lines included proximate analyses (protein, fat, ash, carbohydrates, and moisture), ADF and NDF. Values obtained for the new corn lines were comparable to appropriate controls.
Monsanto has concluded that "Corn modified to be resistant to lepidopteran insect pests and/or tolerant to Roundup herbicide is not materially different in composition, safety, or any relevant parameter from corn now grown, marketed, and consumed." At this time, the Agency considers the consultation on corn lines containing transformation events MON 802, 805, 830, 831 and 832 to be complete.
V. Kelly Bunning, Ph.D.