Body

Response to Monsanto Company Petition 95-045-01p For a Determination of Nonregulated Status for Glyphosate Tolerant (Roundup Ready^TM) Cotton Lines 1445 and 1698

Prepared by

United States Department of Agriculture

Animal and Plant Health Inspection Service

Biotechnology, Biologics, and Environmental Protection

I. SUMMARY

In a petition dated February 9, 1995, Monsanto Company requested a determination from the Animal and Plant Health Inspection Service (APHIS) that glyphosate tolerant cotton lines 1445 and 1698, and any progeny derived from them, should no longer be considered regulated articles under APHIS regulations 7 CFR Part 340. The glyphosate tolerant cotton lines 1445 and 1698 (hereafter called 1445 and 1698) have been considered regulated articles because they were engineered with DNA sequences derived from the plant pathogens, caulimoviruses and Agrobacterium sp. strain CP4.

Lines 1445 and 1698 were developed by using recombinant DNA techniques to introduce a modified version of a EPSPS gene, which encodes the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The EPSPS introduced into these transgenic cotton lines is a resistant form of the similar enzyme present in all plants, bacteria and fungi, and thereby confer resistance or tolerance to the herbicide glyphosate. The EPSPS gene was originally isolated from the common soil borne plant pathogenic microorganism, Agrobacterium sp. strain CP4. After isolation, the EPSPS gene was modified by (1) attaching noncoding DNA regulatory sequences from a caulimovirus and (2) altering codon usage of the EPSPS coding region to enhance expression of the EPSPS gene in plants. In addition, the nptII (neomycin phosphotransferase II) gene isolated from bacterial transposon Tn5 that codes for resistance to the antibiotic kanamycin to function as a selectable marker in plants, and another selectable marker gene aad (3"(9)-O-aminoglycoside adenylyltransferase (AAD) to function in bacterial cells to confer resistance to streptomycin and spectinomycin antibiotics have also been introduced into pedigreed cotton Coker 312 lines 1445 and 1698, but not expressed in them. The transgenic cotton lines that are subject of the petition were developed by a widely used technique called Agro-infection which essentially involves using a plant pathogenic strain of Agrobacterium tumefaciens and its disarmed plasmid vector.

Based on a review of available scientific information, APHIS has determined that 1445 and 1698 do not present a plant pest risk and therefore are no longer regulated articles under the regulations found at 7 CFR Part 340. Because of this determination, regulatory oversight under these regulations will no longer be required from APHIS for field testing, importation, or interstate movement of 1445 and 1698 or their progeny.

This determination has been made based on an analysis that revealed that 1445 and 1698: (1) exhibit no plant pathogenic properties; (2) are no more likely to become weeds than cotton lines developed by traditional breeding techniques; (3) are unlikely to increase the weediness potential of any other cultivated plant or native wild species with which the organisms can interbreed; (4) will not harm other organisms, threatened and endangered organisms, or organisms such as bees, which are beneficial to cotton cultivation and agriculture in general; and 5) do not cause damage to processed agricultural commodities. APHIS has also concluded that there is no reason to believe that new progeny cotton varieties derived from 1445 and 1698 will exhibit new plant pest properties, i.e., properties substantially different from any observed for the 1445 and 1698 cotton lines already field tested, or those observed for cotton in traditional breeding programs.

II. BACKGROUND

APHIS Regulatory Authority. APHIS regulations found at 7 CFR Part 340 (hereafter referred to as the regulations) were promulgated pursuant to authority granted by the Federal Plant Pest Act (FPPA), (7 U.S.C. 150aa-150jj) as amended, and the Plant Quarantine Act (PQA), (7 U.S.C. 151-164a, 166-167) as amended. The regulations pertain to the introduction (importation, interstate movement, or release into the environment) of certain genetically engineered organisms and products.

A genetically engineered organism is considered a regulated article if the donor organism, recipient organism, vector, or vector agent used in engineering the organism belongs to one of the taxa listed in the regulation and is also a plant pest, or if there is reason to believe that it is a plant pest. Lines 1445 and 1698 have been considered "regulated articles" under Part 340 of the regulations because they have been engineered with certain noncoding regulatory and vector DNA sequences derived from the plant pathogenic organisms like the caulimoviruses, and an Agrobacterium species.

Section 340.6 of the regulations, entitled "Petition Process for Determination of Nonregulated Status," provides that a person may petition the Agency to evaluate submitted information and determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If APHIS determines that the regulated article is unlikely to pose a greater plant pest risk than the unmodified organism, the Agency can grant the petition in whole or in part. As a consequence of such a determination, APHIS permits would no longer be required for field testing, importation, or interstate movement of that article or its progeny.

APHIS decision on the regulatory status of 1445 and 1698 under regulations 7 CFR 340, does not release these cotton plants and their progeny from EPA and FDA regulatory oversight. The regulation of herbicide use on these cotton lines is under the jurisdiction of the EPA.

III. PUBLIC COMMENTS

APHIS received a total of ten comments on the petition from the following: a state department of agriculture (1); commodity group (1) and universities (8). All comments were strongly supportive of the petition. There was not a single negative comment received on the petition.

IV. ANALYSIS OF 1445 and 1698

Biology of Cotton: Cotton belongs to the genus Gossypium of the tribe Gossypeae of the family Malvaceae (Fryxell, 1979; Munro, 1987). Only four species of cotton are of any agronomic importance in the world; two diploid old world cotton or Asiatic cotton and two allotetraploid New World species. The old world cotton is restricted to India, Africa and Asia (Munro, 1987). The new World cotton comprises of 98% of cotton cultivated for fibre production. Wild species of cotton occur in arid parts of the tropics and subtropics. Fryxell (1984) has divided the wild diploid species into three geographical groups: the Australian group (11 species), the Afro-Arabian group (8 species), and the American group (12 species). Two species of the American group (tetraploid cotton) occur in Peru and in the Galapagos, and the remaining 10 occur in Western Mexico with one (G. thurberi Todaro) extending up to Arizona. G. tomentosum and G. hirsutum are two of the New World cottons that occur in Hawaii and middle America and drier areas of southern tip of Florida (Fryxell, 1984; Lee, 194). Wild populations of G. hirsutum are relatively rare and tend to be widely dispersed as beach strands or on small islands. There are examples of escaped cotton belonging to G. hirsutum and G. barbedense growing in the wild in Southern Florida and Hawaii. These escaped plants appear opportunistic toward disturbed land and appear not to be effective in inhabiting managed ecosystems.

Although natural outcrossing can occur in cotton, they are normally self-pollinating (Niles and Feaster, 1984). The pollen is heavy and sticky, and is heavily pollinated via bumble bees and honey bees. The range of natural crossing is very limited (100-200 feet) (McGregor, 1976).

APHIS considered whether the movement of the EPSPS gene from 1445 and 1698 to other cultivated cotton or wild relatives might result in offspring that would present problems as weeds. The genetic integrity of commercial cultivated cotton lines and varieties is strictly controlled through established plant breeding practices. These standard practices make it unlikely that this glyphosate tolerance trait will be inadvertently incorporated into the germplasm of cultivated cotton lines.

Rationale for Development of Glyphosate tolerant Cotton: Weeds are a severe constraint for cotton production specially when young cotton seedlings cannot compete with aggressive weeds in the early stages of seedling establishment. Current weed removal practices are inadequate at best to provide a weed free environment. In the United States alone, the cotton crop loss due to weeds is estimated to be $400 million annually. Weed management is a critical factor for cotton yield, and growers typically favor herbicide management strategies that control a broad spectrum of weed species, will not injure the crop, are cost effective, and have positive environmental attributes. Several classes of herbicides have effective broad spectrum weed control if used either singly or in combination; however, they may injure or kill some crops when used at the application rates suggested for weed control. For years, pre-emergence herbicides have been the major tool used for weed control in conventional cotton production. Pre-emergence herbicide treatments are applied prior to, or at the time of planting, before the crop and weed seedlings emerge from the soil.

With the advent of effective post-emergence herbicides and an increased use of no-tillage cotton, growers frequently seek to control weeds when and where they emerge. Depending on the incidence, timing, and density of weed species in a crop field, the grower can use only as much herbicide as necessary to achieve the desired level of weed control. The use of Roundup® as an effective post emergence herbicide with glyphosate tolerant cotton may make it possible to reduce the use of pre-emergence herbicides in cotton production.

Roundup® herbicide contains the active ingredient glyphosate which is a non-selective, post-emergent weed control agent. The target site of action of glyphosate is EPSPS that is present in all plants, bacteria, and fungi as a component of the Shikimate pathway of aromatic amino acid biosynthesis (Levin and Sprinson, 1964). The CP4 EPSPS which is naturally resistant to the inhibition by glyphosate (Padgette et al. 1993) has been introduced into 1445 and 1698 to confer tolerance to the foliar application of glyphosate. Roundup has the advantage of low residual activity, low soil leaching, and low toxicity to nontarget organisms or development of resistant weeds (Atkinson, 1985). Roundup toxicity recorded on certain fishes and invertebrates is due to the surfactant present in commercial formulations (Atkinson, 1985). Certain soil invertebrates show minor toxicity to glyphosate (Eijsackers, 1985). Although there are reported instances of certain cellular activities of a select group of microbes, they are of no undue concern as the commercial applications of glyphosate rarely exceed concentrations that cause the said effects (Carlisle and Trevors, 1988). EPA has classified glyphosate under Category E (evidence of non-carcinogenicity from humans) 57 FR 8739. Roundup is readily degraded by microorganisms in the soil.

Development of 1445 and 1698:

Lines 1445 and 1698 were developed by using recombinant DNA techniques to introduce a modified version of a EPSPS gene, which encodes the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The EPSPS introduced into these transgenic cotton lines is a resistant form of the similar enzyme present in all plants, bacteria and fungi, and thereby confer resistance or tolerance in the trnasgenic plants. The EPSPS gene was originally isolated from the common soil borne plant pathogenic microorganism, Agrobacterium sp. strain CP4. After isolation, the EPSPS gene was modified by (1) attaching noncoding DNA regulatory sequences from a Caulimovirus and (2) altering codon usage of the EPSPS coding region to enhance expression of the EPSPS gene in plants. In addition, nptII (neomycin phosphotransferase II) gene isolated from bacterial transposon Tn5 that codes for resistance to the antibiotic kanamycin to function as a selectable marker in plants, and another selectable marker gene aad (3"(9)-O-aminoglycoside adenylyltransferase (AAD) to function in bacterial cells to confer resistance to streptomycin and spectinomycin antibiotics have also been introduced into pedigreed cotton Coker 312 lines 1445 and 1698, but not expressed in them. The transgenic cotton lines that are subject of the petition were developed by a widely used technique called Agro-infection which essentially involves using a plant pathogenic strain of Agrobacterium tumefaciens and its disarmed plasmid vector (Umbeck et al. 1987; Trolinder and Goodin, 1987).

EPSPS is commonly found in plants, bacteria and fungi, and there has been no recorded instance of this enzyme causing toxicity to any living species of organism. The enzyme degrades rapidly in the environment upon natural decay of the vegetative parts of the plants.

Construction of the Binary Vectors Used in Transformation. The plasmids PV-GHGTO6 and PV-GHGTO7 used to transform the Coker 312 variety of cotton in tissue culture have Ti-plasmid backbone derived from A. tumefaciens. These plasmids are based on the incompatibility group P (IncP) plasmids referred to as RK2 (Stalker et al. 1981), and contain the synthetic EPSPS gene regulated by a caulimovirus promoter and terminator sequences derived from A. tumefaciens. In addition, the plasmid contains an streptomycin/spectinomycin resistance (aad 3") gene and a bacterial origin of replication. The aad3" gene has regulatory sequences recognized only in bacteria but not functional in the transgenic cotton cells. Therefore, the EPSPS and nptII are the only introduced genes which can be expressed in the plant cells. The vectors have only one of the border sequences of the Ti-Plasmid from integration into the plant DNA.

Protoplast Transformation System: The transgenic cotton lines that are subject of the petition were developed by a widely used technique called Agro-infection which essentially involves using a plant pathogenic strain of Agrobacterium tumefaciens and its disarmed plasmid vector (Umbeck et al. 1987; Trolinder and Goodin, 1987).

A. THE INTRODUCED GENES, THEIR PRODUCTS, AND THE ADDED REGULATORY SEQUENCES CONTROLLING THEIR EXPRESSION DO NOT PRESENT A PLANT PEST RISK IN 1445 and 1698.

Southern blot analyses indicate that 1445 and 1698 contain 1 and 2 copies of the EPSPS gene, respectively. Once inserted into the chromosome of the cotton plant, the introduced EPSPS gene is maintained and transmitted in the same manner as any other genes following the Mendelian laws of inheritance.

Expression of the EPSPS gene in 1445 and 1698 is modulated by noncoding DNA regulatory sequences derived from the plant pathogens. Specifically, these regulatory sequences are the promoter and 3'-nontranslated regions of the caulimoviruses (Gowda et al. 1989). These regulatory sequences are utilized widely in the expression of genes engineered into plants. Although these regulatory sequences are derived from plant pathogens, there is no evidence to suggest that they pose a plant pest risk.

B. 1445 and 1698 HAVE NO SIGNIFICANT POTENTIAL TO BECOME SUCCESSFUL WEEDS.

Cotton has been grown for centuries throughout the world without any reports that it is a serious weed pest, and it is unlikely to become a weed pest. In the United States, cotton is not listed as a weed in the major weed references (Crockett 1977; Holm et al. 1979; Muenscher 1980), nor is it present on the lists of noxious weed species distributed by the Federal Government (7 CFR Part 360).

The parent plant of 1445 and 1698 is a line of cotton (Gossypium hirsutum L.) known as Coker 312 that exhibits no appreciable weedy characteristics. The EPSPS gene is unlikely to increase weediness of 1445 and 1698. The glyphosate resistance of these plants will confer a selective advantage only when glyphosate is applied to the plants. No other attributes of 1445 and 1698 suggest that it be any more "weedy" than traditionally-bred cotton cultivars. Other than the resistance to the herbicide glyphosate, 1445 and 1698 have retained the agronomic characteristics of the parental cotton, including the sensitivity to other herbicides.

Monsanto Company has provided data regarding seed germination rates, yield characteristics, disease and pest susceptibilities, compositional analyses, and numerous other tests which support APHIS' conclusion that 1445 and 1698 are no more likely to become weeds than cotton developed by traditional breeding techniques. Cotton is not considered a weed.

A weed pest is a plant that grows persistently in locations where it is unwanted. Cotton has been grown for centuries throughout the world without any reports that it is a serious weed pest. In the United States, it is not a species listed under the Federal Noxious Weed Act. Cotton is not classified as a serious, principal, or common weed pest (Holm et al., 1979). Cotton is considered a highly domesticated, well-characterized crop plant that is not persistent in undisturbed environments without human intervention.

Evaluations of 1445 and 1698 in laboratory, greenhouse, and field tests support the conclusion that 1445 and 1698 have little potential to become weed pests. With the exception of the resistance to glyphosate, 1445 and 1698 have agronomic traits similar to those of traditionally bred cotton and do not exhibit traits that cause concern that they might become weed pests.

C. 1445 and 1698 WILL NOT INCREASE THE WEEDINESS POTENTIAL OF ANY OTHER PLANT WITH WHICH IT CAN INTERBREED.

Cotton belongs to the genus Gossypium of the tribe Gossypiae of the family Malvaceae (Fryxell, 1979; Munro, 1987). Only four species of cotton are of any agronomic importance in the world; two diploid old world cotton or Asiatic cotton and two allotetraploid new world species. The old world cotton is restricted to India, Africa and Asia (Munro, 1987). But, the new World cotton comprises of 98% of cotton cultivated for fibre production. Wild species of cotton occur in arid parts of the tropics and subtropics. Fryxell (1984) has divided the wild diploid species into three geographical groups: the Australian group (11 species), the Afro-Arabian group (8 species), and the American group (12 species). Two species of the American group occur in Peru and in the Galapagos, and the remaining 10 occur in Western Mexico with one (G. thurberi Todaro) extending up to Arizona. G. tomentosum and G. hirsutum are two of the New World cottons that occur in Hawaii and middle America and drier areas of southern tip of Florida (Fryxell, 1984; Lee, 194). Wild populations of G. hirsutum are relatively rare and tend to be widely dispersed as beach strands or on small islands. There are examples of escaped cotton belonging to G. hirsutum G. barbedense growing in the wild in Southern Florida and Hawaii. These escaped plants appear opportunistic toward disturbed land and appears not be effective in inhabiting managed ecosystems.

Although natural outcrossing can occur in cotton, it is normally self-pollinating (Niles and feaster, 1984). The pollen is heavy and sticky, and is heavily pollinated via bumble bees and honey bees. The range of natural crossing is very limited (100-200 feet) (McGregor, 1976).

D. Lines 1445 and 1698 WILL NOT HARM ORGANISMS BENEFICIAL TO AGRICULTURE OR ORGANISMS THAT ARE DESIGNATED AS THREATENED OR ENDANGERED.

APHIS evaluated the potential for 1445 and 1698 plants to harm organisms either directly or indirectly, particularly those organisms that are recognized as beneficial to agriculture. There is no reason to believe that the cultivation of 1445 and 1698 cotton or their progeny will exert any deleterious effects on organisms recognized as beneficial to agriculture. Likewise, cultivation of 1445 and 1698 will not harm any species designated as threatened or endangered. Lines 1445 and 1698 produce two enzymes, EPSPS and NPTII, that are not produced in nontransgenic cotton. There is no indication that this enzyme is toxic to beneficial organisms or results in the production of toxic constituents. In addition, APHIS can envision no mechanism whereby 1445 and 1698 would be injurious or pathogenic to beneficial organisms such as bees and earthworms.

The definition of 1445 and 1698 encompasses not only the cotton lines that already have been field tested, but also new cotton lines produced through conventional breeding using 1445 and 1698 as one or both parents. APHIS believes that the analysis applied to the 1445 and 1698 plants already field tested will apply equally well to these new cotton lines, and that the data provided by Monsanto Company justify the conclusion that such new lines derived from 1445 and 1698 will not present a plant pest risk. The variation in agronomic characteristics among the 1445 and 1698 plants that have been field tested does not differ significantly from that seen in commercial cultivars of cotton that have never been considered regulated articles. Therefore, there is no reason to believe that any of the progeny of 1445 and 1698 will possess plant pest properties.

E. 1445 and 1698 SHOULD NOT CAUSE DAMAGE TO PROCESSED AGRICULTURAL COMMODITIES.

The characteristics of 1445 and 1698 cotton have no apparent attributes that could have an indirect plant pest effect on any processed plant commodity. During extensive testing in the laboratory, greenhouse and in the field, plants of 1445 and 1698 exhibited the typical agronomic characteristics of the parent cotton. In the opinion of APHIS, the components and processing characteristics of 1445 and 1698 reveal no differences in any component that could have an indirect plant pest effect on any processed plant commodity.

IV. CONCLUSION

APHIS has determined that 1445 and 1698 that previously have been field tested under permits and notification system will no longer be considered regulated articles under APHIS regulations found at 7 CFR Part 340. Notifications under those regulations will no longer be required from APHIS for field testing, importation, or interstate movement of these cottons or their progeny. However, the importation of 1445 and 1698 cotton and vegetative plant material or seeds capable of propagation are still subject to the restrictions found in foreign quarantine notices in 7 CFR Part 319.

This determination has been made based on information from field trials, laboratory analyses, and literature references presented herein which demonstrate that:

1) Lines 1445 and 1698 exhibit no plant pathogenic properties;

2) Lines 1445 and 1698 are no more likely to become weeds than cotton developed by traditional breeding techniques;

3) Lines 1445 and 1698 are unlikely to increase the weediness potential for any other cultivated or wild species with which they can interbreed;

4) Lines 1445 and 1698 will not harm other organisms, including agriculturally beneficial organisms and threatened and endangered species; and

5) Lines 1445 and 1698 should not cause damage to processed agricultural commodities.

APHIS has also concluded that there is a reasonable certainty that lines 1445 and 1698 or varieties bred from these lines will not exhibit new plant pest properties, i.e., properties substantially different from any observed for 1445 and 1698 plants already field tested, or those observed for cotton in traditional breeding programs.

))))))))))))))))))))))))))))Q

John H. Payne, Ph.D.

Acting Director

Biotechnology, Biologics, and Environmental Protection

Animal and Plant Health Inspection Service

U.S. Department of Agriculture

Date

V. LITERATURE CITED

Atkinson, D. 1985. "Toxicological propeorties of glyphosate" in "The Herbicide Glyphosate," Grossbard, E. and Atkinson, D. (eds.) Butterworth and Company. Limited, Toronto, Canada.

Carlisle, S. M., and J. T. Trevors. 1988. Roundup in the environment, Water, Air, and Soil Pollution, 39:409-420.

Eijsackers, H. 1985. "Effect of glyphosate on soil fauna," in "The Herbicide Glyphosate," Grossbard, E. and Atkinson, D. (eds.) Butterworth and Company. Limited, Toronto, Canada.

Gowda, S., F. C. Wu, and R. J. Shepherd. 1989. Identification of promoter sequences for the major RNA transcripts of figwort mosaic and peanut chlorotic streak viruses (Caulimovirus Group). J. Cell. Biochem. 13D(supplement), 301.

Fryxell. P. A. 1979. The natural history of the cotton tribe (Malvaceae tribe Gossypiae), Texas A&M Press, College Station, Texas.

Holm, L., Pancho, J.V., Herbarger, J.P., Plucknett, D.L. (1979) A Geographical Atlas of World Weeds. John Wiley and Sons, New York. 391 pp.

Lee, J. A. 1979. Cotton, Agronomy No. 24, p.25, Soil Science Society of America, Inc. (Kohel, R. J. and C. F. Lewis, eds.), Wisconsin, USA

Levin, J. G. and D. B. Sprinson. 1964. The enzymatic formation and isolation of 5-enolpyruvylshikimate 3-phosphate. J. Biol. Chem. 239:1142-1150.

McGregor, S. E. 1976. Insect pollination of cultivated crop plants,

Agricultural handbook No. 496, United States Department of Agriculture, Agricultural Research Service, Washington, D.C.

Munro, J. M. 1987. Cotton. Second Edition. John Wiley & Sons, New York, NY.

Niles, G. A. and C. V. Feaster. 1984. Cotton, Agronomy No. 24, p.205, Soil Science Society of America, Inc. (Kohel, R. J. and C. F. Lewis, eds.), Wisconsin, USA.

Padgette, S. R., G. F. Barry, D. B. re, M. Weldon, D. A. Eicholtz, K. H. Kolacz, and G. M. Kishore. 1993. Purification, cloning, and characterization of highly glyphosate tolerant EPSP synthase from Agrobacterium sp. CP4. Monsanto technical Report MSL-12738, St. Louis.

Pietrzak, M., Shillito, D.S., Hohn, T., Potrykus, I. (1986) Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucleic Acids Research 14: 5857-5868.

Stalker, D. M., C. M. Thomas, and D. R. Helinski. 1981. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Mol. Gen. Gen. 181:8-12.

Trolinder, N. L., and J. R. Goodin. 1987. Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.) Plants. Bio/Technology. 5:263-266.

Umbeck, P., G. Herrerra-Estrella, M. Van Monatgu, and P. Zambryski. 1984. Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines the direction of DNA transfer from Agrobacterium to the plant genome. Cell 38:455-462.

United States Department of Agriculture. 1993. Agricultural Statistics. U.S. Government Printing Office, Washington, D.C. 517 pp.