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### PLANT PEST RISK ASSESS MENT FOR MON 87460 CORN

Monsanto Company has petitioned the Animal a nd Plant Health Inspectio n Service (APHIS) of the United States Department of Agriculture for a determination that genetically engineered (GE) corn ( Zea mays ) event MON 87460 (APHIS number 09-055-01p-a1) is unlikely to pose a plant pest risk and, therefore, should no longer be a regulated article under AP HIS’ regulations at 7 CFR part 340. APHIS administers 7 CFR part 340 under the authority of the plant pest provisions of the Plant Protection Act of 2000 1 . This plant pest risk assessment was conducted

to determine whether MON 87460 corn is unl ikely to pose a plant pest risk.

History of Development of MON 87460 Drought-Tolerant Corn
Corn is the second major food and feed crop in world. Recently corn is also considered as a major biofuel crop in the U.S.; therefore, it is exp ected that U.S. corn cultivation may continue to increase in the coming years (USDA 2007). Conventional breeding methods have made enormous progress in corn crop improvement (Sprague and Dudley 1988). Despite those improvements the demand for corn far exceeds the current production level, leading to a worldwide grain shortage.

As with many high yielding modern crop plants, drought is one of the major limiting factors in corn that prevents realization of optimum gr ain yield (Heisey and Ed meades 1999), and that problem may become a frequent feature across U. S. farmlands and elsewhere in the future. Abiotic stress is the primary cause of crop lo ss worldwide, reducing average yields for most major crop plants by more than 50% (Boyer 1982; Bray et al. 2000). In North America alone, it is estimated that 40% of annual crop losses are due to sub-optimal water availability (Boyer 1982). Although some of the recent corn cultivars are bred to e xhibit some degree of drought tolerance (Byrne et al. 1995; Tollenaar and Wu 1999; Bängiger et al. 2006), the current corn gene pool and conventional bree ding techniques may not be able to bring a significant improvement for drought tolerance in future corn cultivars. In this regard, new biotechniques combined with conventional breeding methods o ffer potential new tools and avenues to harness drought tolerant gene from different sources to increase corn yield st ability under water-limited conditions (Bänziger and Araus 2007).

Drought conditions vary in nature, severity, a nd impact (Jacobsen and Shaw 1989; Close et al. 1993; Campos et al. 2006). While extreme dr ought conditions prevent normal growth and development of plants leading to complete cr op failure, suboptimal soil water at key plant growth stages can also substantially reduce crop yield. Drought tolerant crops are designed to escape water limitation through both genetic and agronomic manipulations. The strength of drought tolerance is a relative qu antification measured over exis ting cultivars, which varies greatly among crop plants. Under no circumstan ces, however, it is likely that crop improvement alone can mitigate all economic losses under water-l imited conditions; rather it has the potential to play a key role in minimizing crop loss in drought-prone areas (H eisey and Morris 2006).

1 Section 403 (14) of the Plant Protection Ac t (7USC Sec 7702(14)) defines plant pest as: “Plant Pest - The term “plant pest” means any living stage of any of the following that can directly or indirectly injure, cau se damage to, or cause disease in any plant or plant product: (A) A protozoan. (B) A nonhuman animal. (C) A parasitic plant. (D) A bacterium. (E) A f ungus. (F) A virus or viroid. (G) An infectious agent or other pathogen. (H) Any article similar to or allied with any of the articles speci fied in the preceding subparagraphs.”2 Monsanto has developed MON 87460 corn event (hereafter referred to as MON 87460) that is expected to reduce yield loss under water-limited conditions compared to conventional corn. The ability to withstand the water limitation in MON 87460 comes from an introduced gene called cold shock protein B (CSPB ) derived from the bacterium Bacillus subtilis . The CSPB is a RNA chaperone. RNA chaperone s are ubiquitous and a bundant proteins found in all living organisms and viruses. RNA tends to be kinetically trapped in misfolded forms, and RNA binding proteins, acting as chaperones, can resolve these structures, ensuring accessibility for its biological function. In bacteria, RNA chaperones are believed to play a general role in sustaining growth by favoring active transcription, translation, and/or ribosome assembly (Castiglioni et al. 2008). In bacteria, the CSPB pr otein helps preserve normal ce llular functions during certain stresses by binding cellular RNA and unfolding nont ranslatable secondary structures affecting RNA stability and translation. As in bacteria , the CSPB protein in MON 87460 binds RNA and appears to help maintain plant cellular functions.

Data submitted by Monsanto suggest that M ON 87460 reduces yield loss, primarily through increased kernel number per ear, under water-l imited conditions by mini mizing the effect of water limitation on photosynthesis, stomatal conduc tance, and carbon fixation. On a plant level, corn yield losses associated with drought stre ss occur as a result of reduced synchrony between anthesis and silking, embryo loss, and/or reduced grain filling in viable kernel s (Claassen and Shaw 1970; Boyer and Westgate 2004; Campos et al. 2006). Studies on conventional germplasm with enhanced drought tolerance show that yield improvements are attained through improvements in all of these endpoints (Bolanos et al. 1993: Bolanos and Edmeads 1996: Barker et al. 2005). Therefore, the enhanced yield stability of MON 87460 under water-limited conditions, conferred by the expre ssed CSPB protein, appears to be the result of improvements in the natural stress response mech anisms over conventional corn.

MON 87460 corn has been field tested under APHIS regulations since 2002 and Monsanto provided data in the petition for field trials co mpleted prior to the petition submission. Because MON 87460 reduces yield loss under water-limited conditions, Monsanto de signed field studies to collect relevant data across a broad range of soil moisture (well watered and water-limited conditions) and environmental conditions releva nt to where commercial production would be expected. Data submitted by Monsanto shows th at under well-watered conditions, grain yield for MON 87460 is not signifi cantly different from conve ntional corn, yet MON87460 experienced reduced yield loss when water stre ss was around 20% less than normal. However, like conventional corn, MON 87460 is still subject to yield lo ss under severe water-limited conditions, particularly during flow ering and grainfill periods when corn yield potential is most sensitive to water stress. In the following pa ragraphs APHIS BRS summ arizes its plant pest

risks assessment of MON 87460 because of inserted genetic elements and/or their products.

Description of the Inserted Genetic Material MON 87460 was developed through a plant pathogenic bacterium Agrobacterium tumefaciens

mediated transformation of corn line LH59 em bryos (Armstrong and Phillips 1988). However,

the A. tumefaciens strain, ABI, that was used to de velop MON 87460, was made non pathogenic by removing the pathogenic sequences present in Ti (tumor inducing) plasmid originally present

in A. tumefaciens (Koncz and Schell 1986). The disarmed A. tumefaciens harbors a binary3 plasmid vector PV-ZMAP595 (Monsanto 2010, Figure III-1, p. 97). This vector was approximately 9.4 kb and contained a single T-DNA delineated by left and right border regions with in which there were two expression cassettes: a cspB gene expression cassette, which contained coding sequence for CSPB protei n, and a neomycin phosphotransferase II ( nptII ) expression cassette, which contained coding sequence fo r the NPTII protein.

The cspB expression cassette consiste d of the following genetic elements (Monsanto 2010, Table IV-1, p. 100):

 Promoter, leader, and intron from the rice ( Oryza sativa ) actin gene, Ract1 (McElroy et al. 1990).

 Full length coding sequences of Cold Shock Protein B ( cspB ) gene from the bacterium,

Bacillus subtilis (Willimsky et al. 1992).

 3' nontranslated termin ator sequence of the transcript 7 gene from Agrobacterium tumefaciens that directs polyadenylati on (Dhaese et al. 1983).

The nptII expression cassette consiste d of the following genetic elements (Monsanto 2010, Table IV-1, p. 100):

 loxP sequence from Bacteriophage P1 for the recombination site recognized by Cre recombinase (Russell et al. 1992). Cre-Lox recombination provides a way to knockout

genes.

 P- 35S promoter for the 35S RNA of the Caulif lower mosaic virus (Odell et al. 1985).

 CS- nptII coding sequence from Tn5 (Beck et al. 1982) in Escherichia coli encoding neomycin and kanamycin resistance (Fraley et al. 1983) that was used as a selectable marker during transformation selection.

 T- nos 3' nontranslated sequence of the nopaline synthase (NOS) gene from A. tumefaciens. This sequence terminates gene ex pression by a polyadenylation site (Depicker et al. 1982).

 loxP sequence from Bacteriophage P1 for the recombination site recognized by Cre recombinase (Russell et al. 1992). The loxP sites were inserted to facilitate the potential

excision of the nptII cassette using CRE recombinase.

In addition to the above-mentioned genetic el ements, the insert also contained noncoding intervening sequences of 1-73 base pair length and right and left border sequen ces that were used during DNA cloning.

Monsanto provided evidence demonstrating that:

 the final product does not contain any of the backbone sequences outside of the T-DNA borders from the transformation vector, PV-ZMAp595 (Monsanto 2010, Figure V-5, p. 117; Figure V-15, p. 134),

 the DNA inserted into the corn genome is present at a single locus and contains one functional copy of cspB and nptII genes (Monsanto 2010, Figur e V-4, p. 116; Figure V14, p. 133),4  the sequence and organization of inserted genes in MON 87460 are identical to their original sequences and arrangements in the donor plasmid PV-ZMAp595 (based on DNA sequence analysis),

 no novel open reading frames were created that spanned either the 5 ′ or 3 ′ junctions

between the T-DNA and corn genomic sequences, and

 the stability of introduced genes was demons trated by the presence of introduced genes via Southern blot fingerprint of MON 87460 (Monsanto 2010, Figures V-14 and V-15, p. 133-134) for seven generati ons tested (Monsanto 2010, Figure V-13, p. 132) in the breeding history. The stability was further confirmed by the Mendelian inheritance of the T-DNA in MON 87460 (Monsanto 2010, Table V-3, p. 136).

Two minor changes occurred during the MON 87460 transformation event; one in the inserted genetic element and one to the adjacent corn ge nomic DNA. A part (733 base pairs) of the promoter, rice actin gene ( Ract1 ), did not get incorporated into the transformed plant. According to Monsanto, this rearrangement probably result ed from double-strand break repair mechanisms in the plant during the Agrobacterium- mediated transformation pr ocess (Salomon and Puchta 1998). Furthermore, analysis of the T-DNA inserti on site indicated that a 22 base pair length of genomic DNA got deleted at the insert-to- plant DNA junction in MON 87460. Despite these minor genetic sequence modifications in MON 87460, neither the stability analysis of the inserted genetic elements over seven genera tions (Monsanto 2010, Figures V-14 and V-15, p. 133-134; Monsanto 2010, Table V-3, p. 136), nor th e forage and grain compositional assessment (p. 151-174 in Monsanto 2010) showed any biolog ically meaningful differences between MON 87460 and conventional corn control excep t for the drought tolerance traits.

PLANT PEST RISK ASSESSMENT

MON 87460 corn was produced by transformation of corn tissue using A. tumefaciens to introduce two genes: a gene ( cspB ) that confers drought to lerance and a gene ( nptII ) that

facilitates selection of tr ansformed plants. Because A. tumefaciens is a plant pest and some of the regulatory sequences (35S Promoter sequen ce from Cauliflower mosaic virus and nopaline synthase terminator sequence from A. tumefaciens ) used to facilitate expr ession of these genes in corn were derived from plant pest s, the engineered corn has been considered a regulated article under APHIS regulations at 7 CFR part 340.

Potential impacts to be addressed in this risk a ssessment are those that pertain to the use of MON 87460 corn and its progeny in the absence of conf inement. APHIS utilizes data and information submitted by the applicant, in addition to curr ent literature, to determine if MON 87460 is unlikely to pose a plant pest risk. If APHIS dete rmines that a GE organism is unlikely to pose a plant pest risk, then APHIS has no regul atory authority over that organism.

Of the information requested by APHIS for submi ssion of a petition for nonr egulated status (§ 340.6(c)(4)), APHIS examined information submitted by the applicant related to plant pest risk characteristics, expression of the gene product, changes to plant metabolism, disease and pest susceptibilities, impacts on the weediness of a ny other plant with whic h it can interbreed, weediness of the regulated article, and impact s on nontarget organisms. Issues related to5 agricultural or cultivation pract ices are discussed in the Environmental Assessment prepared by

APHIS for MON 87460 corn.

Potential Impacts of Genetic Modifications on Altered Disease and Pest Susceptibilities

USDA-APHIS assessed whether MON 87460 corn is likely to have sign ificantly increased disease and pest susceptibility. This assessm ent encompasses a thorough consideration of introduced traits, their impact on agronomic traits and plant composition, a nd interactions with pests and diseases.

Introduced Traits MON 87460 contains drought tolerance traits from the introduced gene product CSPB protein and a selectable marker gene product NPTII pr otein. The CSPB protei n in MON 87460 belongs to the cold-shock protein (CSP) family and is identical in amino acid sequence to the native CSPB protein produced in B. subtilis (Willimsky et al., 1992) with the exception of one amino acid change in the second position from leucine to valine. This amino acid change was implemented by Monsanto to facilitate the asse mbly of the plasmid vector PV-ZMAP595 for plant transformation. The structure of CSPB is well characterized (Sch indelin et al. 1993; Schindelin et al. 1994) and in bacteria CSPs ar e composed of 67-73 amino acids (Graumann et al. 1997). The CSPB protein in MON 87460 consis ts of 66 amino acids (Monsanto 2010, Figure IV-1, p. 102). Although Figure IV-1 shows 67 am ino acids for the CSPB protein in MON 87460, which were predicted based on cspB coding sequence, followi ng translation, however, the N-terminal methionine is cleaved by methio nine aminopeptidase leaving 66 amino acids in the final CSPB protein in MON 87460 (Monsan to 2010, Appendix B, p. 283-284). CSPs are a group of small proteins that contain a highly conserved RNA-binding sequence identified as a cold shock domain (CSD). Under environmental stress, bacterial CSD-c ontaining proteins have been shown to bind many types of RNA (Cristof ari and Darlix, 2002), leading to sustainable translation, maintenance of mRNA levels, and improved cellular f unction. Similar to bacteria, CSD-containing proteins in plants also bind RNA, unfold RNA secondary structures caused by environmental stress, and help maintain cellu lar functions under stress. These plant CSD proteins share a high level of similarity to th e bacterial CSPs and have been shown to share in

vitro and in vivo functions with bacteria l CSPs (Karlson and Imai 2003; Kim et al. 2007; Nakaminami et al. 2005 and 2006; Chaikam and Karlson 2008; Fusa ro et al. 2007). As with bacterial and other plant CSD-containi ng proteins, the CSPB protein from B.subtilis , which is expressed in MON 87460, binds R NA, unfolds RNA secondary structures, and accumulates in actively growing tissues (Monsanto 2010, Figures I-7 – I-9). CSPB accumu lation is highest in rapidly growing areas of the leaf and seedling and tends to decline as the tissue matures. CSPB

concentrations tend to increase ove r time in developing ears and d ecline over time in silks. Likewise, CSPB concentrations increase over time in immature tassels and either remain the same or decline in pollen. Taken together, the data on RNA binding, CSPB accumulation and

CSPB localization in MON 87460 are consistent with the pattern of RNA binding, accumulation, localization, and functions described for plan t CSD-containing proteins (Fusaro et al. 2007; Sasaki et al. 2007; Ch aikam and Karlson 2008).

Monsanto did not observe any abnormal or alte red mechanisms of plant response to drought stress in transgenic CSPB-containing corn plan ts. Greenhouse experiments were conducted with CSPB events that are genetica lly and phenotypically similar to MON 87460 to evaluate if these6 characteristics had been altered when compared with controls. The plants containing CSPB protein show broadly the same relative wate r content (Monsanto 2010, Table I-4), leaf water potential (Monsanto 2010, Figure I-22), and le af osmotic potential (Monsanto 2010, Figure I-23) as the control plants. CSPB-containing plants accu mulate similar levels of abscisic acid (ABA) (Monsanto 2010, Figure I-24) and os motically active solutes (sucrose , fructose, glucose, choline, proline, glycine betaine) as obs erved in controls (Monsanto 2 010, Figures I-25 - 28). Results from these experiments suggest that common mech anisms of plant respons e to drought stress are not altered in transgenic CSPB -containing corn plants. Thus , like endogenous CSD proteins found in plants, the CSPB protein in MON 87460 interacts with RNA, accumulates, and localizes to rapidly growing tissues and in de veloping reproductive organs, thereby helping to

maintain normal cellular function in those tissues critical to yiel d under water-limited conditions. Monsanto observed that the major component contributing to the im proved yield of MON 87460

under water-limited conditions was the increased number of kernels per plant (Monsanto 2010, Tables I-1 & I-2, p. 46-47), which is consistent with the current understanding of the effect of drought stress on corn yield pot ential (Barker et al. 2005).

NPTII protein is well characterized (Fuchs et al. 1993), and in MON 87460 NPTII protein consists of 264 amino acids (Monsanto 2010, Figu re IV-2, p. 102). The NPTII protein in MON 87460 confers resistance to kanamycin, so as to f acilitate the selection of transformed plants (Bevan et al. 1983). The effect of NPTII prot ein on plant growth and development has been extensively evaluated through se veral lines of experimental evidence, and several products containing NPTII have been approved by regulator y agencies on a global basis(e.g. so far NPTII

was used as a selectable marker in 28 petitions deregulated by APHIS BRS; http://www.isb.vt.edu/cfdocs/biopetitions1.cfm). NPTII is the most co mmonly used antibiotic resistance marker in several commercially grown biotechnology-de rived crops including YieldGard® Rootworm corn (MON 863), Bollg ard® cotton (MON 531), Bollgard®II cotton (MON 15985), and Roundup Ready cotton (MON 1445). NPTII protein was never found to facilitate plant pest characteristics to plants. Based on the known functions and mechanisms of these proteins (summarized in Monsanto 2010), neither of these proteins are expected to directly alter susceptibility to plant pathogens.

Compositional Analysis Compositional comparisons between MON 87460 and a conventional control were performed using the principles and analytes outlined in the OECD consensus documents for corn composition (OECD 2002 and 2006). The control substance was a conve ntional corn hybrid with genetic background simila r to MON 87460. The investig ation also included 15 other conventional commercial corn hybrids (Monsan to 2010, Appendix Tables E-1 and E-2, p. 311). The commercial reference hybrids selected by Mo nsanto for each study were adapted to the geographic region in which they were grown with selections based on agronomic characteristics such as relative maturity and drought tolerance ratings. The rati onale for the use of commercial references in various experiments was to provid e data for the development of a 99% tolerance interval for each variable analyzed. This interv al is expected to contain, with 95% confidence, 99% of the values obtained from the population of commercial corn hi ghlighting the existing natural variability in the commercial corn unde r cultivation. The knowledge of existing natural variability was used to draw biological meanings into those statistically significant differences between MON 87460 and controls. This comparativ e evaluation also considered natural ranges7 in corn component levels published in the literature or in the International Life Sciences Institute Crop Composition Database (ILSI 2006).

Monsanto’s compositional analyses of MON 874 60 were based on forage and grain harvested from two different growing seasons, the firs t during 2006 in the U.S. conducted under typical agronomic practices and water conditions, a nd the second during 2006/2007 in Chile under wellwatered and water-limited conditions. Component s evaluated in samples harvested from both studies included (1) moisture, protein, carbohydrates by calculati on, fat, fiber, and ash in a proximate analysis, (2) essential macro- and micr o-nutrients in a nutriti onal analysis, and (3) known endogenous toxicants and anti-nutrients. A supplementary anal ysis of secondary

metabolites associated with stress tolerance wa s also conducted by Monsanto for samples from Chile in 2006 and 2007 (see Appendix E for details). In total, there were 434 comparisons made for compositional analyses (7 se ts of comparisons x 53 components from grain) + (7 sets of comparisons x 9 components from forage).

Overall, a comprehensive evaluation of MON 87460 and the control sh owed no biologically meaningful differences for grain and forage co mpositions either for major nutrients (Monsanto 2010, Table VII-2, p. 157-158; Table VII-3, p. 1 64; Table VII-5, p. 168) or for secondary metabolites (Monsanto 2010, Table VII-6, p. 171; Monsanto 2010, Table VII-7, p. 173). The few detected differences were either exceedingl y small in magnitude or the mean component

values of MON 87460 and the control were within the 99% tolerance interval. Therefore, based on the data presented by Monsanto on forage and grain, it is reasonable to assume that the foods

and feeds derived from MON 87460 can be cons idered compositionally equivalent to those

derived from conventional corn.

Agronomic Properties Monsanto evaluated phenotypic and agronom ic characteristics of MON 87460 in a comparative manner to assess plant pest pot ential (OECD, 1993). These assessments included 14 plant growth and development characteristics, five seed germination parameters, two pollen characterist ics, several observations on plant-insect and plant-disease interactions, and plant responses to abiotic stressors (Monsanto 2010, Table VIII-1, p. 178). Monsanto provided phenotypic and agronomic data (see Section VIII. Phenotypic, Agronomic, and Environmental Interactions Assessment, p. 176-231) supporting th eir claims that MON

87460 is similar to unmodified control corn cultivar except for the intended drought tolerant trait and that MON 87460 does not possess characteristic s that would confer a plant pest risk compared to conventional corn. Detailed agr onomic and phenotypic charact eristics are discussed

further in relevant sections of this document, while the observed disease and pest susceptibility

of MON 87460 is discussed in the following paragraph.

Monsanto used well-established qua litative or quantitative technique s to measure field trials for damage due to diseases (Anthracnos e, Ear rot, Kernel rot, Eyespot, Fusarium , Gray leaf spot, Gray mold, Leaf blight, Maize dwarf mosaic virus, Northern leaf spot, Pythium , Rust, Seedling blight, Smut, Southern leaf blight, Stalk rot, Stew arts wilt, and Yellow leaf blight), insect pests (Aphids, Leafhoppers, Armyworms, Corn earworms, European corn borers, Southwestern corn borers, Grasshoppers, Corn rootworms, Flea be etles, Japanese beetles, Grape colaspis, Wireworms, White grubs, Spider mites, S eedcorn maggots) for two years conducted across8 different agroecological conditi ons. The data submitted by Monsanto indicated no meaningful

differences between MON 87460 corn and the non-transgenic counterparts for diseases (Monsanto 2010, Table H-2, p. 432), or insect pests (Monsanto 2010, Tables H-3 to H-7, p. 433- 441; Table H-4, p. 434: Tables H- 5 to H-6, p. p. 435-438). Thus MON 87460 corn is expected to be susceptible to the same plant pathogens and insect pests as conventional corn.

Monsanto’s aforementioned data (d escription of the inserted gene tic elements, expression of the gene product, compositional analysis, and agrono mic and phenotypic observations) indicates that MON 87460 corn is not biologically different from conventional co rn (with the exception of the CSPB3 and NPTII proteins), and MON87460 is no more susceptible to pests and diseases compared to conventional corn cultivars.

Potential Impacts from Outcrossing (Gene Flow ) to Sexually-compatible Wild Relatives
Gene flow is a natural biological process with si gnificant evolutionary importance. A number of angiosperm taxa are believed to be derived fr om hybridization or introgression between closely related taxa (Grant 1981; Soltis and Soltis 1993; Rieseberg 1997; He gde et al. 2006), and even in the existing floras, the occurrence of hybridization or introgression is reported to be widespread (Stace 1987; Rieseberg and Wendel 1993; Peterson et al. 2002). It has been a common practice by plant breeders to artificially introgress traits from wild relatives into crop plants to develop new cultivars. However, gene flow from crops to wild relatives is also thought of as having a potential to enhance the weedine ss of wild relatives, as observed in rice, sorghum, sunflower and few other crops (see Table 1 in Ellstrand et al. 1999).

Corn is a monoecious species with separate male and female inflorescences that enable cross pollination. Corn is predominantly a wind pollin ated outcrosser with occasional bee visitation for pollen. Bees rarely visit female infloresce nces (silk). Researcher s recognize that: (i) the percent gene flow will vary by popul ation, hybrid or inbred, (ii) the level of gene flow decreases with greater distance between the source and recipi ent plants; (iii) enviro nmental factors affect the level of gene flow, (iv) corn pollen is viable for a relatively short period of time under field conditions, (v) corn produces ample pollen over an extended period of time, and (vi) corn is not pollinated by insects (pollinating insects, especially bees, are occasional visitors to the tassels but rarely visit silks of corn) (Jemis on and Vayda, 2000; Luna et al., 2001).

APHIS evaluated the potential for gene introgr ession to occur from MO N 87460 corn to sexually compatible wild relatives and considered whethe r such introgression woul d result in increased weediness. Cultivated corn, or maize, Zea mays L. subsp. mays , is sexually compatible with several members of the genus Zea , and to a much lesser degree with members of the genus Tripsacum (OECD 2003; Monsanto 2010, Table II-1, p. 82 ). Wild diploid and tetraploid

members of Zea, collectively referred to as teosinte, ar e normally confined to the tropical and subtropical regions of Mexic o, Guatemala, and Nicaragua (W ilkes 1967; Matsuoke et al. 2002; Fukunaga et al. 2005). Corn and annual teosinte ( Zea mays subsp. mexicana ) are genetically compatible, wind-pollinated, and may hybridize when in close proximity to each other, e.g., in

areas of Mexico and Guatemala. While some te osinte may be considered weeds in certain instances, they are also used by some farmers for breeding improved maize (Sánchez and Ruiz 1997 and references therein). Teosinte is descri bed as being susceptible to many of the same pests and diseases that attack cultivated corn (Sánchez and Corral 1997). In the wild,9 introgressive hybridi zation from corn to teosinte is curr ently limited, in part, by several factors including geographic isolation, differing degrees of genetic in compatibility, differences in flowering time in some cases, developmen tal morphology and timing of the reproductive structures, dissemination, and dormancy (Doe bley 1990a and 1990b; Galinat 1988; Ellstrand 2007). Genetically based cross-incompatibil ity mechanisms are known to reduce hybrids

between corn and teosinte when corn is the pollen source (Baltazar et al. 2005; Kermicle and Evans 2005), which thus acts as a signif icant constraint to introgression.

Teosinte is not present in the U.S. other than as an occasional botanical garden specimen and a few small feral populations of Zea mexicana in Florida, Alabama, and Maryland and Zea perennis in South Carolina ( http://plants.usda.gov ). Introgression of genes from corn into

teosinte or Tripsacum species has not been described to occur in nature in the U.S. Furthermore, MON 87460 is unlikely to outcross w ith sexually compatible species in the U.S. as differences in factors such as flowering time, geographical se paration, and development factors make natural crosses in the U.S. highly unlikely.

Corn is also distantly related to the species in the genus Tripsacum, which contains up to 16 recognized species (Monsanto 2010, Table II-1, p. 82), most of which are native to Mexico, Central and South America, but three ( T. dactyloides, T. floridatum , and T. lanceolatum ) exist as wild and/or cultivated species in the continental U.S (OECD 2003); and two taxa ( T. fasciculatum and T. latifolium ) also occur in Puerto Rico ( http://plants.usda.gov ). In contrast with corn and teosinte which easily hybridize under certain conditions, special techniques are required to hybridize corn and Tripsacum and the offspring of the cros s show varying levels of sterility (Galinat 1988; Mangelsdorf 1974; Russell and Hallauer 1980). Furthermore, none of the sexually compatible relatives of corn in the U.S. are considered to be weeds in the U.S. (Holm et. al. 1979). Therefore, even in those instances of accidental gene flow between MON 87460 corn and wild relatives, the transgenes of MON 87460 corn are unlikely to transform corn wild relatives into more weedy species. Based on th e data presented in the petition, MON 87460 corn

does not exhibit characteristics that cause it to be any weedier than other cultivated corn (see the section “Potential Impacts Based on the Relative Weediness of MON 87460 Corn” below). Moreover, its potential impact due to the extremel y limited potential for gene introgression into teosinte is not expected to be any different than that of other cultivated corn varieties. Based on the above considerations, MON 87460 corn is unli kely to adversely impact sexually compatible

wild relatives or their weediness characters.

Potential Impacts Based on the Relative Weediness of MON 87460 Corn In the U.S., corn is not listed as a weed (C rockett 1977; Holm et al. 1979; Muenscher 1980), nor is it present on the Federal Noxious Weed List (7 CFR part 360; http://www.aphis.usda.gov/plan t_health/plant_pest_info/wee ds/downloads/weedlist2006.pdf ). Furthermore, corn is grown throughout the world w ithout any report that it is a serious weed or that it forms persistent feral populations. Co rn is poorly suited to survive without human assistance and is not capable of surviving as a weed (Baker 1965; K eeler 1989; Galinat 1988). Like many domesticated crops, corn seed from a previous year’s crop can overwinter and germinate the following year. For instance, the appearance of corn seedlings in soybean fields following a corn crop is a common occurrence. Manual or chemical measures are often applied to remove these volunteers, but the plants that are not removed do not typically result in feral10 populations in subsequent years. Corn does not possess su it of traits that ar e characteristics of successful weeds (Baker 1965; Keeler 1989).

APHIS assessed whether MON 87460 corn is any more likely to become a weed than the isogenic nontransgenic corn line, or other corn varieties cu rrently under cultivation. The assessment encompasses a thorough consideration of the basic biology of corn and an evaluation of the unique characteristics of MON 87460 corn evaluated under field conditions, including potential weedines traits su ch as seed dormancy and germination, rate of growth and development, flowering, seed dispersal, seed yield, and persistence of free living populations

outside cultivation. Monsanto co llected phenotypic and agronomic field data from field trials conducted at 27 U.S. locations during the 2006 a nd 2007 growing seasons th at included diverse agroecological regions representa tive of the major corn-growing areas of the upper mid-west in the U.S. Likewise, Monsanto also collected complim entary data from four field trials from Chile (Monsanto 2010, Table VIII-2, p. 189). In addi tion, data on abiotic stress tolerance from greenhouse and growth chamber assays were used to characterize the exte nt of stress tolerance imparted by the insertion of the cspB gene and determine whether any potential changes in

tolerance enhanced weedin ess potential in MON 87460.

For the majority of the traits assessed, there we re no statistically signifi cant differences between MON 87460 and nontransgenic control (Monsanto 2010, Tables VIII-4 to VIII-12, p. 192-204; Tables VIII-14 to VIII-16, p. 208-210). No biologi cally meaningful differences were detected in the germination and dormancy of seed from MON 87460. In particular, the absence of hard seed supports a conclusion of no increased weedin ess potential of M ON 87460 compared to conventional corn for germination and dormanc y characteristics (Monsanto 2010, Table VIII-14, p. 208). No differences were detected in pollen morphology or viab ility between MON 87460 and the control (Monsanto 2010, Table VIII-15 & VIII-16, p. 209-210). Results from the genotype-environmental interaction assessments also support the conclusion that MON 87460 has no increased susceptibility or tolerance to sp ecific diseases, arthropods, or abiotic stressors, with the exception of drought. Development of deep root system is typically one of the agronomic traits that has potenti al to enhance weediness in the natural environment. However that may not be the case with corn, as inbred lines with poor early root development exhibited higher yields under drought stress than ones with accelerated early development of roots (Bruce et al. 2002). It has been reas oned that the improved performan ce of corn cultivars to drought stress probably comes from better water use effi ciency (Messmer et al. 2009). Finally, MON 87460 was not altered in its ability to volunteer in cultivated fi elds or survive in areas not managed for agricultural production co mpared to conventional corn.

Based on the agronomic field data and literature survey concerning corn’s weediness potential, MON 87460 corn is unlikely to persist as a tr oublesome weed or to impact on current weed management practices. Furthermore, extensive post-harvest monitoring of field trial plots planted with MON 87460 under USDA-APHIS no tifications (Monsanto 2010, Appendix N, p. 519-523) did not reveal any differen ces in survivability or persis tence relative to other corn. These data suggest that MON 87460 is no more likely to become a weed than conventional corn.

Potential Impacts on Nontarget Organis ms, Including Bene ficial Organisms11 Monsanto is in consultation with FDA about f ood and feed derived from corn event MON 87460 and has submitted a comprehensive assessment of food and feed safety data on CSPB and NPTII proteins of MON 87460 to FDA. Monsanto cons idered the following factors to establish food and feed safety of introduced gene products.

 The donor organism of the CSPB protein, B. subtilis , is not pathogenic, is oftenused as a food additive, is present in many fermented food s, and has a history of safe consumption. In 1999, FDA designated enzyme preparations from this organism as GRAS (generally recognized as safe, FDA, 1999).

 The B. subtilis CSPB shares a high percent of identity with CSPs present in other bacterial species broadly used by the food i ndustry and with CSD-containing proteins in plant species used as a food. Many foods prepared with the help of B. subtilis have been consumed for a long time with no documented history of any adverse effects to human health. CSPB protein present in MON 87460 shar es amino acid identity to other naturally occurring CSD-containing proteins found in food and feed products. The amino acid identity of CSPB ranges from 35% to 98.5% across different plant and bacterial species. The CSPB protein is homologous to th e CSP proteins found in the genera Lactobacillus

, Lactococcus

, Bifidobacterium , and E. coli , which are normally present in gastrointestinal flora and, therefore, considered to be safe.

 A dietary safety assessment conducted to evalua te the risks to humans and animals from the CSPB and NPTII proteins present in the foods and feeds derived from MON 87460 did not cause any observed adverse effects ev en at the highest tested dose levels.

 CSPB protein represents no more than 0.00007% of the total protein in the grain of MON 87460. Digestive fate e xperiments conducted with the CSPB protein demonstrated that the full-length protein is rapidly digested in simulated gastric fluid, a characteristic shared among many proteins with a history of sa fe consumption. Proteins that are rapidly digestible in mammalian gastrointestinal systems are unlikely to be allergens when

consumed.

 CSPB and NPTII proteins do not share any am ino acid sequence simila rities with known allergens, gliadins, glutenins, or protein to xins which have adverse effects to mammals.

 The safety of the NPTII protein and its donor organism, E. coli , have been recognized by regulatory agencies and well documented. A ll existing data suggest that the NPTII protein represents a negligible hazard to human health and is safe for consumption.

The introduced gene products of MON 87460 do not contain pesticidal activity and are not aimed at any target organisms as a control meas ure. As mentioned ea rlier, MON 87460 is found to be safe for human and animal consumption. Therefore, Monsanto assessed the nontarget impact of MON 87460 on all those exposed orga nisms (e.g. pests, diseases, and beneficial arthropods) in the corn agroecosystem.

During the U.S. and Chile phenotypic fiel d studies at 31 locations in 2006 and 2007, each field site was rated at four time in tervals during the season for specific insects (pest and non-pests), and diseases (Monsanto 2010, Section VIII.F.1, p. 211-212) to evaluate whether the plant-disease or pl ant-insect interacti ons of MON 87460 were altered compared to commercial corn. Out of nearly 388 pest and non-pest arthropod evaluations, only two

differences were observed be tween MON 87460 and the control. Grasshopper damage was12 lower for MON 87460 compared to the control and European corn borer damage was higher for MON 87460 compared to the control. These differe nces were within the range of the references observed for corn cultivars. Likewise, out of th e more than 425 disease stressor observations, no

differences were detected be tween MON 87460 and the control. These results support the conclusion that no biologically meaningful inter actions were observed with the species exposed

to MON 87460 and that MON 87460 is not different in its environmen tal interactions relative to

other corn.

Potential Impacts from Transferring Ge netic Information from MON 87460 Corn to Organisms with which It cannot Interbreed APHIS examined the potential for the new genetic material inserted into MON 87460 corn to be horizontally transferred to other organisms w ithout sexual reproduction and whether such an event could lead directly or indi rectly to disease, damage, injury or harm to plants, including the creation of more virulent pathogens. The horizon tal gene transfer betw een unrelated organisms is one of the most intensively studied fields si nce 1940, and the issue gained extra attention with the release of transgenic plants into the envi ronment (Droge et al. 1998 ). Horizontal gene transfer has been implicated as a major contribu tor to the spread of an tibiotic resistance amongst pathogenic bacteria and the emergence of incr eased virulence in bact eria, eukaryotes, and viruses; and has contributed to major transitions in evolution.

Potential for Horizontal Gene Transfe r to Bacteria, Fungi, OR Viruses The MON 87460 contains two genes and two noncodi ng regulatory sequences from bacteria. Horizontal gene transfer and e xpression of DNA from a plant specie s to other bacterial species is unlikely to occur based on the following obser vations. Although ther e are many opportunities for plants to directly interact with fungi and bacteria (e.g. as co mmensals, symbionts, parasites, pathogens, decomposers, or in the guts of herbi vores), so far there are no reports of horizontal gene transfer to bacteria from eukaryotes or from plants to f ungi (as reviewed in Keese 2008). The only genes likely to be transferred successfu lly from genetically engineered plants to bacteria are other bacterial genes. Horizontal transfer from and expression in bacteria of the foreign DNA inserted into the nuclear genome of MON 87460 corn is unlikely to occur. First, many genomes (or parts thereof) have been sequen ced from bacteria that are closely associated with plants including Agrobacterium and Rhizobium (Kaneko et al. 2000; Kaneko et al. 2002; Wood et al. 2001). There is no evid ence that these organisms contain genes derived from plants. Second, in cases where review of sequence data implied that horizontal gene transfer occurred,

these events are inferred to occu r on an evolutionary time scale on the order of millions of years (Koonin et al. 2001; Brown 2003). Third, the FDA ha s evaluated horizontal gene transfer from

the use of antibiotic resistance marker genes and concluded that the likelihood of transfer of antibiotic resistance genes from plant genomes to microorganisms in the gastrointestinal tract of humans or animals, or in the environment, is remote ( http://vm.cfsan.fda.gov/~dms/opaarmg.html) . APHIS also considered whether horiz ontal transfer of DNA from MON 87460 corn to plant viruses was likely to occu r and would lead to the creation or selection of a more virulent plant pathogen through recombinati on with other plant viruses. Th is issue has been considered before by other science review panels and governme nt regulatory bodies (for a general review of the issue see Keese 2008). There are two noncoding regulatory sequences (35S and LoxP ) from virus present in MON 87460 corn. These two seque nces have not been implicated in viral recombination raising safety concerns (Hull et al. 2000). Therefore, APHIS concludes that13 horizontal gene transfer is unlik ely to occur and thus poses no significant environmental or plant pest risk.

Conclusion APHIS has reviewed and conducted a plant pest risk assessment on MON 87460 corn. Due to the lack of plant pest risk from the inserted ge netic material, the lack of atypical responses to disease or plant pests in the field, the lack of weediness characteristics of MON 87460 corn, the lack of deleterious effects on non-targets or bene ficial organisms in the agro-ecosystem, and the lack of horizontal gene transfer, APHIS conc ludes that MON 87460 corn is unlikely to pose a plant pest risk.14 References Armstrong, C.L., and Phillips, R. L.. 1988. Gene tic and cytogenetic variation in plants regenerated from organogenic and friable, embryogenic tissue cu ltures of maize. Crop Science 28: 363-369.

Baker, H. B. 1965. Characteristics and modes of origin of weeds. Pp. 147-169 in H. G. Baker and G. L. Stebbins (eds.), The genetics of colonizing species. Academic Press, London.

Baltazar, B. M., Sánchez González, J. J., de la Cruz-Larios, L., and Schoper, J. B. 2005. Pollination between maize and teosinte: an import ant determinant of gene flow in Mexico. Theoretical and Applied Genetics 110: 519–526.

Bänziger, M., and Araus, J. 2007. Recent adva nces in breeding maize for drought and salinity

stress tolerance. PP 587-601 in M.A. Jenks and P.M. Hasegawa and S.M. Jain (eds.), Advances in Molecular Breeding Toward Drought and Sa lt Tolerant Crops. Springer, Dordretch,

Netherlands.

Bängiger, M., Setimela, P. S., Hodson, D., a nd Vivek, B. 2006. Breeding for improved drought tolerance in maize adapted to southern Africa. Agricultu ral Water Management 80: 212-224.

Barker, T., Campos, H., Cooper, M. Dolan, D. Ed meades, G. O., Habben, J. Schussler, J. Wright, D., and Zinselmeoer, C. 2005. Improving drought tolerance in maize. Plant Breeding Review

25: 173-253.

Beck, E., Ludwig, G., Auerswald, E. A., Re iss, B. and Schaller, H. 1982. Nucleotide sequence and exact localizat ion of the neomycin phosphotransferase gene from

transposon Tn5 . Gene 19: 327-336.

Bevan, M.W, Flavell, R. B., and Chilton, M.D. 1983. A chimeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304: 184-187.

Bolanos, J., and Edmeades, G. O. 1996. The impo rtance of the anthesis -silking interval in breeding for drought tolerance in tropical maize. Field Crops Research 48: 65-80.

Bolanos, J., Edmeades, G. O., and Martinez, L. 1993. Eight cycles of selection for drought tolerance in tropical maize. II I. Responses in drought-adaptiv e physiological and morphological traits. Field Crops Research 31: 269-286.

Boyer, J. S. 1982. Plant productivity and environment. Science 218: 443–448.

Boyer, J. S., and Westgate, M. E. 2004. Gr ain yields with limited water. Journal of Experimental Botany 55: 2385-2394.

Bray, E.A. Bailey-Serres, J., and Weretilnyk, E. 2000. Responses to abiotic stresses. Pp. 1158-

1249 in W. Gruissem, B. Buchannan and R. Jones (e ds.) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD.15 Brown, J. R. 2003. Ancient horizontal gene transfer. Genetics 4: 121-132.

Bruce, W.B., Edmeades, G. O., and Barker , T. C. 2002. Molecular and physiological approaches to maize improvement for drought tolerance. Journal of Experimental Botany 53: 13-25.

Byrne, P.F., Bolanos, J., Edmeades, G. O. a nd Eaton, D. L. 1995. Gains from selection under drought versus multilocus te sting in related tropical mai ze populations. Crop Science 35:

63-69.

Campos, H., Cooper, M., Edmeades, G. O., Loffler , C., Schussler, J. R. and Ibanez. M. 2006. Changes in drought tolerance in ma ize associated with fifty year s of breeding for yield in the U.S. Corn Belt. Maydica 51: 369-381.

Castiglioni, P., Warner, D., Bensen, R. J., Anstro m, D. C., Harrison, J., Stoecker, M., Abad, M., Kumar, G., Salvador, S., Ordine , R. D., Navarro, S., Back, S., Fernandes, M., Targolli, J., Dasgupta, S., Bonin, C., Luethy, M. H., and H eard, J. E. 2008. Bacterial RNA chaperones confer abiotic stress tolerance in plants and improved grain yield in maize under water-limited conditions. Plant Physiology 147: 446-455.

Chaikam, V., and Karlson, D. 2008. Functi onal characterization of two cold shock domain proteins from Oryza sativa . Plant Cell and Environment 31: 995-1006.

Claassen, M. M., and Shaw, R. H. . 1970. Water deficit effect s on corn. II. Grain components. Agronomy Journal 62: 652-655.

Close, T. J., Fenton, R. D., Yang, A., Asghar, R., DeMason, D. A., Crone, D. E., Meyer, N. C., and Moonan, F. 1993. Dehydrin: the protein. Cu rrent Topics in Plant Physiology 10: 104-118.

Cristofari, G., and Darlix, J. L. 2002. The ubiquitous nature of RN A chaperone proteins. Progress in Nucleic Acid Research and Molecular Biology 72: 223-268.

Crockett, L. 1977. Wildly Successful Plants: North American Weeds. University of Hawaii Press, Hawaii.

Depicker, A., Stachel, S., Dhaese, P., Zambry ski, P., and Goodman, H. M. 1982. Nopaline synthase: transcript mapping and DNA se quence. Journal of Molecular and Applied Genetics 1: 561-573.

Dhaese, P., De Greve, H., D., Gielen, J., Seurin ck, L., Van Montague , M. and Schell, J. 1983. Identification of sequences involved in polyadenylation of hi gher plant nuclear transcripts using Agrobacterium T-DNA genes as models. EMBO Journal . 2: 419-

426.

Doebley, J. 1990a. Molecular evidence for gene flow among Zea species. BioScience

40: 443- 448.16 Doebley, J. 1990b. Molecular systematics of Zea (Gramineae). Maydica 35:143-50.

Dröge, M., Puhler, A., and Selbits chka, W. 1998. Horizontal ge ne transfer as a biosafety issue: A natural phenomenon of public concer n. Journal of Biot echnology 64: 75-90.

Ellstrand, N. C., Prentice, H. C., and Hancock, J. F. 1999. Gene flow and introgression from domesticated plants into their wild relatives. Annual Review of Ecology and Systematics

30: 539-563.

Ellstrand, N.C., Garner, L. C., Hegde, S., Guadagnuolo, R., and Blancas, L. 2007. Spontaneous hybridization between maize and teosinte. Journal of Heredity

98: 183-187.

FDA (Food and Drug Administratio n). 1999. Carbohydrase and protea se enzyme preparations

derived from Bacillus subtilis or Bacillus amyl oliquefaciens ; affirmation of GRAS status as direct food ingredients, Fi nal Rule. Federal Register 64:19887-19895, U.S. Food and Drug Administration, Washington, D.C.

Fraley, R.T., Rogers, S.. G., Horsch, R. B., Sanders, P. R., Flick, J. S., Adams, S. P., Bittner, M. L., Brand, L. A., Fink, C. L., Fry, J. S., Galluppi, G. R., Goldberg, S. B., Hoffmann, N. L., and Woo, S. C. 1983. Expre ssion of bacterial gene s in plant cells. Proceedings of the National Acad emy of Sciences (USA) 80: 4803-4807.

Fuchs, R.L., R.A. Heeren, M.E. Gustaf son, G.J. Rogan, D.E. Bartnicki, R.M. Leimgruber, R.F. Finn, A. Hershman, and S.A. Berberich. 1993. Purification and characterization of microbially expr essed neomycin phosphotransferase II (NPTII) protein and its equivalence to the plant expressed protein. BioTechnology 11: 1537-1542.

Fukunaga, K., Hill, J., Vigouroux, Y., Matsuoka, Y., Sanc hez, J. S., Liu, K., Buckler, E. S., and Doebley, J. 2005. Genetics 169: 2241–2255.

Fusaro, A.F., Bocca, S.N, Ramos, R.L.B., Barroca, R.M., Magioli, C., Jorge, V.C., Coutinho, T.C., Rangel-Lima, C.M., De Rycke, R., Inze, D., Engler, G., and Sachetto-Martins, G. 2007. AtGRP2, a Co ld-induced Nucleo-cytoplasmic RNAbinding Protein, has a role in flower and se ed development. Planta. 225: 1339-1351

Galinat, W.C. 1988. The origin of corn. Pp.1-31 in Sprague, G.F. and J.W. Dudley (eds.) Corn and Corn Improvement. 3 rd ed., American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Madison, WI.

Grant, V. 1981. Plant speciation. Columbia University Press, New York.17 Graumann, P., T.M. Wendrich, M.H.W. Weber, K. Schroder, and M.A. Marahiel. 1997. A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesi s at optimal and low temperatur es. Molecular Microbiology 25:

741-756.

Hegde, S.G., Nason, J. D., Clegg, J. M., and Ellstrand, N. C. 2006. The evolution of

California’s wild radish has re sulted in the extincti on of its progenitors. Evolution 60:1187-

1197.

Heisey, P. W., and Edmeades, G. O. 1999. Ma ize production in drought-stressed environments: Technical options and research resource a llocation. Part I of CIMMYT 1997/1998 World Facts

and Trends: Maize Production in Drought-Stressed Environments: Technical Options and

Research resource Allocati on. Mexico, D. F.: CIMMYT.

Heisey, P.W., and Morris, M.L. 2006. Economic impact of water limited conditions on cereal grain production. Pp. 17-48 in J.M. Ribaut (ed.) Drought Ad aptation in Cereals. Haworth Press Inc., Binghamton.

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

Hull, R., Covey, S. N., and Dale, P. 2000. Gene tically modified plants and the 35S promoter: assessing the risks and enhancing the debate. Microbial Ecology in Health and Disease 12: 1-5.

ILSI (International Life Sciences Institute). 2006. International Life Sciences Institute Crop Composition Database, version 3.0. http://www.cropcomposition.org/ (Last modified: 10/1/2008)

Jacobson, J. V., Shaw, D. C. 1989. Heat-stabl e protein and abscisic acid action in barley aleurone cells. Plant Physiology 91: 1520-1526.

Jemison, J.M., and Vayda, M. 2000. Pollen transport from genetically modified corn. Page 145 in 2000 Agronomy Abstracts. Am erican Society of Agronomy (ASA), Madison, Wisconsin.

Kaneko, T., Nakamura, Y., Sato, S., Asamizu, E., Kato, T., Sasamoto, S., Watanabe, A., Idesawa, K., Ishikawa, A., Kawashima, K., Kimu ra, T., Kishida, Y., Kiyokawa, C., Kohara, M., Matsumoto, M., Matsuno, A., Mochizuki, Y., Nakayama, S., Nakazaki, N., Shimpo, S., Sugimoto, M., Takeuchi, C., Yamada, M., and Taba ta, S. 2000. Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti . DNA Research 7: 331-338.

Kaneko, T., Nakamura, Y., Sato, S., Minami sawa, K., Uchiumi, T., Sasamoto, S., Watanabe, A., Idesawa, K., Iriguchi, M., Ka washima, K., Kohara, M., Matsumoto, M., Shimpo, S., Tsuruoka, H., Wada, T., Yamada, M ., and Tabata, S. 2002. Complete genomic sequence of nitrogen-fix ing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA

Research 9: 189-197.18 Karlson, D., and Imai, R. 2003. Conservation of the cold shock domain protein family in plants. Plant Physiology 131: 12-15

Kermicle, J. L., and Evans, M. M. S. 2005. Pollen-pistil barriers to crossing in maize and teosinte result from incongruity rather th an active rejection. Sexua l Plant Reproduction 18:

187–194.

Keeler, K. 1989. Can genetical ly engineered crops become weeds? Bio/Technology 7: 1134-

1139.

Keese, P. 2008. Review Article: Risks from GMOs due to horizontal gene transfer. Environmental Biosafety Research 7: 123-149.

Kim, J.Y., Park, S. J., Jang, B. S., Jung, C. H ., Ahn, S. J., Goh, C. H., Cho, K., Han, O., and Kang, H. S. 2007. Functional characterizati on of a glycine-rich RNA-binding protein 2

in Arabidopsis thaliana under abiotic stress conditions. Plant Journal 50: 439-451.

Koncz, C., and Schell, J. 1986. The promoter of Tl-DNA gene 5 contro ls the tissue specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Molecular

and General Genetics 204: 383-396.

Koonin, E. V., Makarova,K. S., and Aravind, L. 2001. Horizontal gene transfer in prokaryotes: quantification and classification. Annual Review of Microbiology 55: 709-742.

Luna, S.V., Figueroa, J. M., Baltazar,B. M., Go mez, R. L., Townsend, R., and Schoper, J. B. 2001. Maize pollen longevity and distance isolation requirement for effective pollen control. Crop Science 41: 1551-1557.

Mangelsdorf, P.C. 1974. Corn: its Origi n, Evolution, and Improvement. Harvard University Press, Cambridge, MA.

Matsuoka, Y., Vigouroux, Y., Goodman, M. M., Sanchez, J., Buckler, E., and Doebley, J. 2002. Proceedings of National Academy of Sciences (USA) 99: 6080–6084.

McElroy, D., Zhang, W., Cao, J., a nd Wu, R. 1990. Isolation of an efficient actin promoter

for use in rice transforma tion. Plant Cell 2: 163-171.

Messmer, R., Fracheboud, Y., Banziger, M., Varg as, M., Stamp, P., and Ribaut, J-M. 2009. Drought stress and tropical mai ze: QTL-by-environment interac tions and stability of QTLs across environments for yield components and secondary traits. Theoretical and Applied

Genetics 119: 913–930.

Monsanto (2010). Petition for the Determina tion of Nonregulated Status for MON 87460. Submitted by W. R. Reeves, Regulatory Affa irs Manager. Monsanto Company (See Table http://www.aphis.usda.gov/ biotechnology/not_reg.html)

.19 Muenscher, W. C. 1980. Weeds. 2nd Edition. Cornell University Press, New York.

Nakaminami, K., Karlson, D.T., and Imai, R. 2006. Functional conservation of cold shock domains in bacteria and higher plants. Proceedings of the National Academy of Sciences (USA) 103: 10122-10127

Nakaminami, K., Sasaki, K., Kajita, S., Takeda, H., Karlson, D., Ohgi, K., and Imai, R. 2005. Heat stable ssDNA/RNA-binding activ ity of a wheat cold shock domain protein. FEBS Letters 579: 4887-4891.

OECD (Organization for Economic Co-ope ration and Development). 1993. Safety considerations for biotechnology: Scale-up of crop plants. Organization for Economic Cooperation and Development, Paris, France.

OECD (Organization for Economic Co-operati on and Development). 2002. Consensus document on compositional considerati ons for new varieties of maize ( Zea mays ): key food and feed nutrients, antinutrients and secondary pl ant metabolites. Organisation for Economic Cooperation and Development. Paris, France.

OECD (Organization for Economic Co-operation and Development). 2003a. Consensus document on the biology of Zea mays subsp. mays (maize). Series on Harmonisation of Regulatory Oversight in Biot echnology, No. 27, OECD, Paris.

OECD (Organization for Economic Co-operation and Development). 2003b. Considerations for the safety assessment of animal feedstuffs deri ved from genetically modified plants. Series on the Safety of Novel Foods and Feeds. Paris, France

OECD (Organization for Economic Co-operation and Development). 2006. An Introduction to the Food/Feed Safety Consensus Documents of th e Task Force. Organisation for Economic Cooperation and Development, Paris, France.

Odell, J.T., Nagy, F., and Chua, N-H. 1985. Id entification of DNA sequences required for activity of the cauliflower mosaic vi rus 35S promoter. Nature 313: 810-812.

Peterson, C.D., Pearman, D. A., and Dines, T. D. 2002. New Atlas of the British flora. Oxford University Press, London, U.K.

Rieseberg, L. H. 1997. Hybrid origins of plan t species. Annual Review of Ecology and Systematics 28: 359-389.

Rieseberg, L.H., and Wendel, J. F. 1993. Intr ogression and its conseque nces in plants. Pp.

70–109 i n R.G. Harrison (ed.) Hybrid Zones and the Evolutionary Process. Oxford University Press, Oxford, U.K.20 Russell, W.A., and Hallauer, A. R. 1980. Corn. Pp. 299-312 in W. R. Fehr and H.H. Hadley (eds.) Hybridization of Crop Plants. American Society of Agronomy an d Crop Science Society of America, Madison, Wisconsin.

Russell, S.H., Hoopes, J. L., and Odell, J. T. 1992. Directed excision of a transgene from the plant genome. Molecular and General Genetics 234: 49-59.

Sánchez, G. J. J. and Ruiz, C. J. A. 1997. Teosinte distribution in Mexico. Pp

18-39 in J. A. Serratos, M. C. Willcox, and F. Cast illo-Gonzalez (eds.) Gene Flow among Maize Landraces, Improved Maize Varieties, and Teosin te: Implications for Transgenic Maize. CIMMYT, Mexico, D.F.

Sasaki, K., Kim, M. H., and Imai, R. 2007. Arabidopsis cold shock domain protein 2 is a RNA chaperone that is regulated by cold and developmental signals. Biochemical and Biophysical Research Co mmunications 364: 633-638.

Schindelin, H., Marahiel, M. A., and Heinema nn, U. 1993. Universal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein.

Nature. 364:164-168.

Schindelin, H., Jiang, W., Inouye, M. and Heinemann, U. 1994. Crystal structure of CspA, the major cold shock protein of Escherichia coli . Proceedings of the National Academy of Sciences (USA) 91: 5119-5123.

Salomon, S. and Puchta, H. 1998. Captur e of genomic and T-DNA sequences during double-strand break repair in somatic pl ant cells. EMBO Journal 17: 6086-6095.

Sprague, G. F. and Dudley, J. F. (editors). 1988. Corn and Corn Improvement, 3 rd Edition. ASA-CSSA-SSSA, Madison, WI.

Soltis, D.E., and Soltis, P. S. 1993. Molecular data and the dynamic nature of polyploidy.

Critical Reviews in Plant Sciences 12: 243–273.

Stace, C.A. 1987. Hybridization and the plant species. Pp. 115–127 in K.M. Urbanska (ed.) Differentiation Patterns in Higher Plants. Academic Press, New York.

Tollenaar, M., and Wu, J. 1999. Yield improvement in tropical maize is attributable to greater stress tolerance. Crop Science 39: 1597-1604.USDA-APHIS. 2000.

USDA (U.S. Department of Agriculture). 2007. USDA Agricultural Projections to 2016

Office of the Chief Economist

, Washington, DC

.

Wilkes, H. G. 1967. Teosinte: The closest relati ve of maize. The Bussey Institute, Harvard University, Cambridge, MA.

Willimsky, G., H. Bang, G. Fischer and M. A. Marahiel. 1992. Characterization of cspB , a21 Bacillus subtilis inducible cold shock gene affecting cell viability at low temperatures. Journal of Bacteriology 174: 6326-6335.

Wood, D. W., Setubal, J. C., Kaul, K., Monks, D. E., Kitajima, J. P., Okura, V. K., Zhou, Y., Chen, L., Wood, G. E., Almeida Jr., N. F., Woo, L., Chen, Y., Paul sen, I. T., J. Eisen, J. A., Karp, P. D., Bovee Sr., D., Chapman, P., Clendenni ng, J., Deatherage, G., Gillet, W., Grant, C., Kutyavin, T., Levy, R., Li, M.-J., McClelland, E., Palmieri, A., Raymond, C., Rouse, G., Saenphimmachak, C., Wu, Z., Rome ro, P., Gordon, D., Zhang, S., Yoo, H., Tao, Y., Biddle, P., Jung, M., Krespan, W., Perry, M., Gordon-Kamm, B ., Liao, L., Kim, S., Hendrick, C., Zhao, ZY., Dolan, M., Chumley, F., Tingey, S. V., Tomb, J-F., Gordon, M. P., Olson, M. V., and Nester, E. W. 2001. The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

Science 294: 2317-2323.