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<title>Pioneer has developed a process called ‘Seed Production Technology’ (SPT) that facilitates production of male-sterile corn lines that used as female inbred parents for subsequent hybrid seed production</title>
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<body><p><b>Plant Pest Risk Assessment for DP-32138-1 Corn</b></p>
<p>Pioneer Hi-Bred International, has petitioned APHIS (APHIS number 08-338-01p) for a determination that genetically engineered (GE) corn (<i>Zea mays </i>L. subsp. <i>mays</i>) event DP-32138-1 is unlikely to pose a plant pest
 risk and, therefore, should no longer be a regulated article under APHIS’ 7 Code of Federal Regulations (CFR) part 340. APHIS administers 7 CFR part 340 under the authority of the Plant Protection Act (PPA) of 2000. This plant pest risk assessment was conducted to determine if DP-32138-1 is unlikely to pose a plant pest risk. 
</p>
<p class="normal_(Web),Normal_(Web)_Char">Potential impacts to be addressed in this risk assessment are those that pertain to the use of DP-32138-1 and its progeny in the absence of confinement. APHIS utilizes data and information submitted by the applicant, in addition to current literature, to determine if DP-32138-1 is unlikely to pose a plant pest risk. If APHIS determines that a GE organism is not a plant pest, then APHIS has no regulatory authority over that organism. </p>
<p class="normal_(Web),Normal_(Web)_Char">Based on the information requested by APHIS for submission of a petition for determination of nonregulated status (§ 340.6(c), USDA-APHIS 2009) and in order to determine whether DP-32138-1 is unlikely to pose a plant pest risk, BRS considered information such as: plant pest risk characteristics, disease and pest susceptibilities, expression of the gene product, new enzymes<i>, </i>changes to plant metabolism, weediness of the regulated article, impact on the weediness of any other plant with which it can interbreed, agricultural or cultivation practices, effects of the regulated article on non-target organisms, and transfer of genetic information to organisms with which it cannot interbreed.
</p>
<p class="normal_(Web),Normal_(Web)_Char">The Environmental Assessment (EA) for this petition included an analysis of agricultural or cultivation practices for DP-32138-1. Potential impacts addressed in this risk assessment are those that pertain to plant pest risk characteristics. The genes inserted in DP-32138-1 were evaluated to determine if they cause plant disease. In addition, morphological characteristics of this corn line were analyzed to determine if this variety would become weedy or invasive. Gene flow and introgression of the inserted genes into weedy and wild relatives was evaluated to determine the potential of increased weedy or invasive characteristics and the likelihood of transfer of genetic information to organisms with which corn cannot interbreed. Finally, APHIS evaluated and compared DP-32138-1 to conventional corn in regards to disease and pests susceptibility and conducted an analysis of the effects of the regulated article on non-target organisms. A thorough assessment of the effects of the determination on non-target and beneficial organisms, and threatened and endangered species is included in the Environmental Assessment.
</p>
<h3>Development of Male Fertility/ Male Sterility DP-32138-1 Corn</h3>
<p>Since the 1930s, corn productivity in the U.S. has been greatly enhanced by the use of hybrid corn seed (Sleper 2006). Corn hybrids are characterized by increased resistance to diseases and enhanced agronomic characteristics compared with the parental lines (Brewbaker 1964). The production of hybrid corn seed involves a cross between two inbred lines
, where the pollen from the tassel/male parent is used to fertilize the ear/female parent. Because corn is mostly self pollinated (OECD 2003), hybrid corn seed is typically produced by the removal of male flowers (tassels) from the female parent either mechanically or by hand. However, mechanical detasseling can result in up to 40 percent reduction in seed yield when compared to that of hand detassel treatments (Wych 1988). In addition, female plants may be blown over by a storm and escape detasseling, allowing secondary tassels to develop after completion of the manual detasseling. In either case, some of the female plants will be self-pollinated resulting in seed of the female inbred being harvested along with the hybrid seed. </p>
<p>As an alternative to tassel removal, numerous genetic strategies have been attempted to achieve male sterility
 (Skibbe 2005). Over 40 genetic elements are associated with male sterility. Some of these elements are located on the nuclear chromosomes while others are located on the mitochondrial chromosomes (Skibbe 2005). A major drawback to male sterile genetic approaches is the difficulty in generating male inbred lines because no functional pollen is produced in a male sterile plant line. </p>
<p>Pioneer has developed a novel method (petition Appendices 1 through 7) that uses DP-32138-1 to produce male sterile/female inbred plants for the generation of hybrid corn seed that is non-transgenic (known as “Seed Production Technology”, petition pp. 23- 26, figures 3, 4 & 5). As detailed in the petition, the carefully controlled expression of a seed color marker gene and pollen fertility and sterility genes facilitates the generation of red transgenic seed for seed increase of male sterile-female inbred lines, and for the production of non-transgenic fertile pollen for use in non-transgenic hybrid seed production (petition figure 3, 4 & 5). </p>
<p>DP-32138-1 lines are not intended for commercial production (petition pp. 23, 101, 122-123). The total acreage in the U.S. planted each year with DP-32138-1 is expected to be less than 5,000 acres (petition table 20). If the DP-32138-1 is licensed to third parties and adopted across the entire U.S. seed industry, the total acreage is not expected to exceed 20,000 acres (petition table 20). </p>
<p>FDA has completed review of the new protein consultations for DsRed2 and ZM-AA1 on January 29, 2010 and has no further questions.
</p>
<p><i>Description of the Modification</i></p>
<p>DP-32138-1 corn was produced by transformation using disarmed <i>Agrobacterium tumefaciens </i>(petition pp. 35 through 37, figures 11 and 12)<i>.</i> Immature embryos of Hi-II (<i>ms45/ms45</i>) (Cigan 2001) were infected with <i>Agrobacterium</i> strain LBA4404 containing plasmid PHP24597. Plants containing the introduced DNA were selected based on the production of a red color marker and then screened for male fertility (petition p. 35, figure 11). </p>
<p>The plasmid PHP24597 contained three gene expression cassettes flanked by the right and left border of T-DNA from the <i>Agrobacterium</i> Ti-plasmid (petition figure 13 & 14, table 2):</p>
<p><u>Pollen fertility restorer <i>Ms45</i></u> - expression cassette consists of two genetic elements (petition p. 43):
</p>
<p>· The 5126 anther-preferred
 promoter from <i>Z. mays</i> localizes the expression to the anther (Cigan 1997).</p>
<p>· The pollen fertility restoration gene (<i>Ms45</i>) from <i>Z. mays </i>restores development of the microspore cell wall that gives rise to pollen
 (Cigan 2001). The presence of the transgene <i>Ms45</i> allows for production of viable pollen. </p>
<p><u>α-Amylase <i>zm-aa1</i></u> - expression cassette consists of four genetic elements (petition p. 43):
</p>
<p>· The polygalacturonase promoter (PG47) from <i>Z. mays</i> localizes expression to pollen (Allen 1993).</p>
<p>· Transit peptide <i>brittle-</i>1 (<i>zm-bt1</i>)<i> </i>from <i>Z. mays </i>targets expression to the amyloplast
 (Sullivan 1991). The <i>zm-aa1 </i>gene was truncated to remove the native transit peptide sequence and replaced by the <i>zm-bt1</i> amyloplast-targeting sequence.</p>
<p>· The alpha-amylase (α-amylase)
 <i>zm-aa1 </i>gene from <i>Z. mays </i>(petition figure 43)<i> </i>encodes the ZM-AA1 protein which breaks down starch (Janeček 1994). When the ZM-AA1 protein is expressed in immature pollen, the depletion of starch reserves renders the pollen infertile. </p>
<p>· The <i>In2-1 </i>terminator from <i>Z. mays</i> from the <i>In2-1 </i>gene (Hershey 1991).</p>
<p><u>Red color marker</u> <i><u>DsRed2(AltI)</u> - </i>expression cassette consists of four genetic elements (petition p. 43):
</p>
<p>· A 35S enhancer from the cauliflower mosaic virus (CaMV) (Odell 1985, Odell 1988).
</p>
<p>· The lipid transfer protein (<i>Ltp2</i>)<i> </i>promoter from barley localizes expression to the aleurone layer of the seed (Kalla 1994).</p>
<p>· The <i>DsRed2</i> gene from a marine coral-like anemone <i>Discosoma</i> sp. (Matz 1999, Wasson-Blader 2001) was modified by a single base pair substitution (in which an internal <i>Bst</i>EII restriction site was removed without changing the amino acid sequence in the DsRed2 protein) to produce <i>DsRed2(AltI</i>). </p>
<p>DP-32138-1 seeds expressing the DsRed2 protein are colored pinkish-red (petition figure 10, Wasson-Blader 2001) and can be sorted from yellow seed (that does not express DsRed2) using a color sorting machine (petition figures 4, 5 and 10, Appendices 2 through 7). The DsRed2 protein enables detection and sorting of transgenic red corn seeds from non-transgenic yellow seeds.</p>
<p>· The proteinase inhibitor II (<i>pinII</i>) terminator from <i>Solanum tuberosum</i> (An 1989, Keil 1986).
</p>
<p>Data from Southern blot analyses demonstrate that DP-32138-1 contains: (1) a single copy of the 5126 promoter and <i>Ms45</i> gene (petition table 4, figure 19); (2)<i> </i>a single copy of the PG47 promoter, <i>zm-bt1</i> transit peptide, <i>zm-aa1</i> gene and <i>In2-1 </i>terminator (petition table 4, figures 20 and 21); and (3) a single copy of the 35S enhancer, the <i>Ltp2</i> promoter, <i>DsRed2(AltI)</i> and <i>pinII </i>terminator (petition table 4, figures 22 and 23).</p>
<p><b>Potential of DP-32138-1 to Become Invasive and/or a Weed </b></p>
<p>In the U.S., corn is not listed as a weed (Crockett 1977, Holm 1979, Muenscher 1980), nor is it present in the Federal noxious weed list (7 CFR part 360; <a href="http://www.aphis.usda.gov/plant_health/plant_pest_info/weeds/downloads/weedlist-2010doc.pdf"><u>http://www.aphis.usda.gov/plant_health/plant_pest_info/weeds/downloads/weedlist-2010doc.pdf</u></a>). Furthermore, corn is grown throughout the world without any report that it is a serious weed or that it forms persistent feral populations (Gould 1968). 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 feral populations in following years. Corn also possesses few of the characteristics of plants that are notably successful weeds (Baker 1965, Keeler 1989).
</p>
<p>APHIS assessed whether DP-32138-1 is any more likely to become a weed than the
</p>
<p>isogenic nontransgenic corn line, or other corn varieties currently under cultivation. The assessment encompasses a thorough consideration of the basic biology of corn and an evaluation of the unique characteristics of DP-32138-1 under field conditions. Pioneer has been conducting agronomic field trials of DP-32138-1 since 2005 in the U.S. corn belt at over 43 locations (petition Appendix 12). Agronomic data were collected from DP-32138-1 and its control counterparts in six locations that provide a range of environmental conditions representative of where the DP-32138-1 is expected to be grown for seed production (petition figure 47). Field trial data (petition pp. 101- 107, tables 11 and 12) indicated that DP-32138-1 does not exhibit characteristics that would cause it to be weedier than the parental corn line. No differences in phenotypic characteristics that might contribute to enhanced weediness were observed between DP-32138-1 and control lines for the wide range of phenotypic endpoints assessed in these field trials or in greenhouse or laboratory experiments (petition table 11). There was no increase in weediness potential as measured by differences observed in the field for germination, seedling vigor, plant height, ear height, stalk lodging, root lodging, final population, plant health, time to silking, time to pollen, pollen viability, and seed germination (petition tables 10 and 12). None of the measured attributes showed any differences relative to its comparator, suggesting that DP-32138-1 is not weedier than current corn cultivars. </p>
<p>These results on growth characteristics, seed production and germination indicate that the DP-32138-1 is not significantly different from its comparators. There is no indication that DP-32138-1 possesses a selective advantage that would result in increased weediness. DP-32138-1 lacks the ability to persist as a troublesome weed, and there would be no significant impact on current weed management practices for corn cultivation.</p>
<h3>Potential for Gene Flow and Gene Introgression from DP-32138-1 into Sexually-Compatible Relatives</h3>
<p>Gene flow is a natural biological process with significant evolutionary importance. A number of angiosperm taxa are believed to be derived from hybridization or introgression between closely related taxa (Soltis 1993, Rieseberg 1997) and even in existing floras, the occurrence of hybridization or introgression is reported to be widespread (Stace 1987, Rieseberg 1993). 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 the potential to enhance the weediness of wild relatives, as observed in rice, sorghum, sunflower, and a few other crops (Ellstrand 1999). </p>
<p>APHIS evaluated the potential for gene introgression to occur from DP-32138-1 to sexually compatible wild relatives and considered whether such introgression would result in increased weediness. Cultivated corn is sexually compatible with other members of the genus <i>Zea</i>, and to a much lesser degree with members of the genus <i>Tripsacum</i> (OECD 2003). The closest wild relatives of corn, various <i>Zea</i> species referred to as teosinte, are normally confined to the tropical and subtropical regions of Mexico, Guatemala, and Nicaragua. In the U.S. a fairly rare, sparsely dispersed feral population of teosinte has been reported in Florida (USDA Plant database: <a href="http://plants.usda.gov/java/county?state_name=Florida&statefips=12&symbol=ZEME"><u>http://plants.usda.gov/java/county?state_name=Florida&statefips=12&symbol=ZEME</u></a>). 
</p>
<p>The genus <i>Tripsacum </i>contains up to 16 recognized species, most of which are native to Mexico, Central and South America.  Three of these species (<i>T. dactyloides, T. floridatum</i>, and <i>T. lanceolatum</i>) exist as wild and/or cultivated species in the continental U.S.; and two taxa (<i>T. fasciculatum</i> and <i>T. latifolium</i>) also occur in Puerto Rico (USDA PLANTS Database, accessed 11/9/2009). Though many of these species occur where corn might be cultivated, gene introgression from DP-32138-1 under natural conditions is highly unlikely. Hybrids of <i>Tripsacum </i>species with <i>Zea </i>are difficult to obtain outside of a laboratory and are often sterile or have greatly reduced fertility and none of them typically withstand even the mildest winters (Galinat 1988, Magelsdorf 1939). Furthermore, none of the sexually compatible relatives of corn in the U.S. are considered to be weeds in the U.S. (Holm 1979). Therefore, even in those instances of incidental gene flow between DP-32138-1 and wild relatives, the transgenes of DP-32138-1 are unlikely to transform corn wild relatives into more weedy species.
</p>
<p>Introgression of genes from corn into teosinte or <i>Tripsacum</i> species has not been described to occur in nature in the U.S. While some teosinte may be considered weeds in certain instances, they are also used by some farmers for breeding improved maize (Sánchez 1997 and references therein). Teosinte is described as being susceptible to many of the same pests and diseases that attack cultivated corn (Sánchez 1997). In the wild, introgressive hybridization from corn to teosinte is currently limited, in part, by several factors including geographic isolation, differing degrees of genetic incompatibility, differences in flowering time in some cases, developmental morphology and timing of the reproductive structures, dissemination, and dormancy (Doebley 1990a and 1990b, Galinat 1988, Ellstrand 2007). First-generation hybrids are generally less fit for survival and dissemination in the wild and show substantially reduced reproductive capacity, which thus acts as a significant constraint to introgression. Even if gene flow to a wild relative of corn did occur, an engineered trait that does not offer any adaptive advantage will probably not persist in the weed population (Ellstrand 1990). DP-32138-1 contains a male fertility gene that is normally present in corn, a male sterility gene, and a color selectable marker gene. None of these genes would be expected to confer a selective advantage if gene flow were to occur. Data included in the petition demonstrated that there were no significant differences in viability and morphology (as measured by size shape and color) of pollen collected from DP-32138-1 plants and near-isoline reference plants (petition figure 9, table 12); therefore, the outcrossing rate of DP-32138-1 is not expected to be any different from other corn. Moreover, its potential impact due to the extremely limited potential for gene introgression into teosinte is not expected to be any different than that of other cultivated corn varieties. Therefore, USDA has determined that any adverse consequences of gene flow from DP-32138-1 to wild or weedy species in the U.S. are highly unlikely.</p>
<p><b>Potential for Transfer of Genetic Information to Organisms with which DP32138-1 Cannot Interbreed
</b></p>
<p>Horizontal gene transfer and expression of DNA from a plant species to bacteria is unlikely to occur (Keese 2008). First, many bacteria (or parts thereof) that are closely associated with plants have been sequenced, including <i>Agrobacterium</i> and <i>Rhizobium </i>(Kaneko 2000, Kaneko 2002, Wood 2001). There is no evidence 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 occur on an evolutionary time scale on the order of millions of years (Brown 2003, Koonin 2001). Third, FDA has 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 (FDA 1998). Therefore, APHIS concludes that horizontal gene transfer is unlikely to occur and thus poses no significant environmental or plant pest risk.</p>
<h3>Potential for DP32138-1 to have Altered Disease and Pest Susceptibilities 
</h3>
<p>APHIS assessed whether DP-32138-1 is likely to have significantly altered disease and pest susceptibility. This assessment encompassed a thorough consideration of the introduced trait and disease and pest susceptibility data from DP-32138-1 field trials. </p>
<p>Corn is not a plant pest in the U.S. (<a href="http://www.aphis.usda.gov/plant_health/plant_pest_info/weeds/downloads/weedlist-2010doc.pdf"><u>http://www.aphis.usda.gov/plant_health/plant_pest_info/weeds/downloads/weedlist-2010doc.pdf</u></a>). The <i>Agrobacterium </i>transformed plants used in the generation of DP-32138-1 were treated with an antibiotic to kill the <i>Agrobacterium </i>cells. Furthermore, DNA sequences derived from plant pests that were incorporated in DP-32138-1 do not result in the production of infectious agents or disease symptoms in plants, and so it is unlikely that DP-32138-1 could pose a plant pest risk. The description of the genetic modifications, including genetic elements, expression of the gene product and their functions for DP-32138-1 has been summarized above. </p>
<p>DP-32138-1 has been field tested in the U.S. since 2005 (petition p. 101, Appendix 12). Agronomic data was collected at six different locations in 2007 that provided a range of environmental conditions representative of where DP-32138-1 is expected to be grown (petition figure 47). Pioneer routinely monitors their corn field trials for the following disease agents: <i>Aspergillus</i> sp., <i>Aureobasidium zeae, Cercospora zeae-maydis,</i> <i>Colletotrichum graminicola,</i> <i>Exserohilum turcicum, Fusarium</i> spp., <i>Gibberella zeae, Pantoea stewartii, Phytopthora </i>spp., <i>Puccinia polysora, Puccinia sorghi </i>and <i>Ustilago zeae </i>(petition Appendix 13, table 2).<i> </i>The corn insects monitored include Aphididae<i>, Adoretus sinicus, Chaetocnema pulicaria, Chrysoperla carnea Cicadellidae, </i>Coleoptera<i>, Diabrotica </i>spp<i>., Glischrochilus quadrisignatus, Helicoverpa zea, </i>Lepidoptera<i>, Ostrinia nubilalis, Popillia japonica, Richia albicosta, Spodoptera frugiperda, Syrphidae, Tetranychidae, Thripidae</i> (petition Appendix 13, table 1). The data submitted by Pioneer indicated no meaningful differences between DP-32138-1 and non-transgenic counterparts for disease or insect damage (petition p. 107, Appendix 13, tables 1 and 2). </p>
<p>The data presented in the petition indicate no difference in compositional and nutritional quality of DP-32138-1 compared to conventional corn, apart from the presence of the MS45, ZM-AA1 and DsRed2 proteins. None of the values for seed composition characteristics were outside the range of natural variability of conventional corn (petition tables 13, 14, 15, 16, 17, 18 and 19). Therefore, the composition of DP-32138-1 is not biologically different than conventional corn (with the exception of the MS45, ZM-AA1 and DsRed2 proteins). Based on the known functions and mechanisms of actions of these proteins (summarized in the petition), none of these proteins are expected to directly alter susceptibility to plant pathogens. Thus DP-32138-1 is expected to be susceptible to the same plant pathogens as conventional corn.</p>
<p><b>Conclusion
</b></p>
<p class="normal_(Web),Normal_(Web)_Char">APHIS has prepared this plant pest risk assessment in order to determine if event DP-32138-1 is unlikely to pose a plant pest risk. Based on the information provided by the applicant and the lack of plant pest risk from the inserted genetic material, weedy characteristics, atypical responses to disease or plant pests in the field, effects on non-targets or beneficial organisms in the agro-ecosystem, and horizontal gene transfer, APHIS has concluded that event DP-32138-1 is unlikely to pose a plant pest risk. 
</p>
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<p>� 7 CFR part 340.1 defines a plant pest as:
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<p>“Any living stage (including active and dormant forms) of insects, mites, nematodes, slugs, snails, protozoa, or other invertebrate animals, bacteria, fungi, other parasitic plants or reproductive parts thereof; viruses; or any organisms similar to or allied with any of the foregoing; or any infectious agents or substances, which can directly or indirectly injure or cause disease or damage in or to any plants or parts thereof, or any processed, manufactured, or other products of plants” (USDA-APHIS-BRS). 
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<p>� Inbred lines are populations of identical or nearly identical plants used as stocks for the creation of hybrid lines.
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<p>� Male sterility - the inability to produce functional pollen.
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<p>� Pollen development takes place within the anther (Bedinger 1992). The 5126 promoter localizes expression to an inner cell layer of the anthers called the tapetum (petition figures 8A & 8B). 
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<p>� Mutations in the Ms45 gene causes male sterility because there is no functional pollen produced. A mutation in the Ms45 gene is designated as ms45. 
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<p>� Amyloplast is an organelle in the cytoplasm of plant cells where starch is stored.
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<p>� Catalyze the hydrolysis of α-1,4- D-glucosidic bonds in polysaccharides such as starch.
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