1



## Plant Pest Risk Assessment for DP

- 32138

- 1 Corn

Pioneer Hi

- Bred International, has petitioned APHIS (APHIS number 08

- 338

- 01p) for a determination that genetically engineered (GE) corn ( Zea mays L. subsp. mays ) event DP

- 32138

- 1 is unlikely to pose a plant pest risk ( Pioneer 2009 ) 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 pes t provisions of the Plant Protection Act of 2000 1

. This plant pest risk assessment was conducted to determine if DP

- 321 38

- 1 is unlikely to pose a plant pest risk.

DP

- 32138

- 1 was produced by transformation of a developing corn embryo of line H

- II using A . tumefacien s. Because A. tumefaciens is a plant pest and some of the regulatory sequences ( Cauliflower Mosaic Virus enhancer ) used to facilitate expression of these genes in corn were derived from plant pests, this corn has been considered a regulated

ar ticle under APHIS regulations at 7 CFR part 340.

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 unlikely to pose a plant pest risk , then APHIS has no regulatory authority over that organis m.

Potent ial impacts in this Plant Pest Risk Assessment are those that pertain to plant pest risk characteristics. APHIS regulation 7 CFR 340.6(c) specifies the information needed for consideration in a petition for nonregulated status. APHIS will evaluate informat ion submitted by the applicant related to plant pest risk characteristics, disease and pest susceptibilities, expression of the gene product, new enzymes, or changes to plant metabolism, weediness of the regulated article, any impacts on the weediness of a ny other plant with which it can interbreed , and transfer of genetic information to organisms with which it cannot interbreed.

T he Environmental Assessment (EA ) for this petition considered whether agricultural or cultivation practices for DP

- 32138

- 1 ma y result in impacts on the environment . 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

. Development of Male Fertility/ Male Sterility 

### DP

- 32138

- 1 Corn

1 Section 403 (14) of the Plant Protection Act (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, cause 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 fungus. (F) A virus or viroid. (G) An infectious a gent or other pathogen. (H) Any article similar to or allied with any of the articles specified in the preceding subparagraphs.”2

Since the 1930s, corn productivity in the U.S. has been greatly enhanced by the use of hybrid corn seed (Sleper 2006). Corn h ybrids are characterized by increase d resistance to diseases and enhanced agronomic characteristics compared with the parental lines (Brewbaker 1964). The production of h ybrid corn seed involves a cross between two inbred lines 2 , where the pollen from the tassel /male parent is use d to fertilize the ear/ f emale paren t

. 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, m echanical detasseling can result in up to 40 percent red uction in seed yield when compared to that of hand detassel treatments (Wych 1988). In addition, f

emale 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 plant s will be self

- pollinated resulting in seed of the female inbred being harvested along with the hybrid seed.

As an alternative to tassel removal , n umerous genetic stra te gies have been attempted to achieve male sterility 3 (Skibbe 2005). Over 40 genetic elements are associated with male sterility

. S ome of these elements are located on the nuclear chrom osomes while others are located on the mitochondria l chromosomes (Skibbe 2005)

. A major draw back to male sterile genetic appr oaches is the difficulty in generating male inbred lines because no functional pollen is produced in a male sterile plant line.

Pioneer has developed a novel method ( Pioneer 2009

, Appendices 1 through 7) that uses DP

- 32138

- 1 to produce male sterile /femal e inbred plants for the generation of hybrid corn seed that is non

- transgenic ( known as “Seed Production Technology”, Pioneer 2009

, pp. 23

- 26, f

igure s 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 increa se of male sterile

- female inbred lines

, and for the production of non

- transgenic fertile pollen for use in non

- transgenic hybrid seed production ( Pion eer 2009

, f igure 3, 4 & 5)

.

DP

- 32138

- 1 lines are used to produce male sterile female inbred lines and are not used for commercial product ion of commodity corn ( Pioneer 2009

, pp. 23, 101, 122

- 123). The
t otal acreage in the U.S. planted each year with DP

- 3 2138

- 1 is expected to be less than 5,000 acres ( Pioneer 2009

, t able 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 ( Pioneer 2009

, t able 20).

FDA has completed review of the new protein consultations for DsRed2 and ZM

- AA1 on January 29, 2010 and has no further questions.

Descript ion of the Modification

2 Inbred lines are populations of identical or nearly identical plants used as stocks for the creation of hybrid lines.
3 Mal e sterility

- the inability to produce functional pollen.3

DP

- 32138

- 1 corn was produced by transformation using disarmed Agrobacterium tumefaciens ( Pioneer 2009

, pp. 35 through 37, figure s 11 and 12)

. Immature embryos of Hi

- II ( ms45/

ms45 ) (Cigan 2001) were infected with Agrobacterium strain LBA4 404 containing plasmid PHP24597. Plant s containing the introduced DNA were selected based on the production of a red color marker and then screened for male fertility ( Pioneer 2009

, p. 35, figure 11).

T he plasmid PHP24597 contained three gene expression cassettes flanked by the right and left border of T

- DNA from the Agrobacterium Ti

- plasmid ( Pi oneer 2009

, figure 13 & 14, table 2) :

P ollen fertility restorer Ms45

- expression cassette consists of two genetic elements ( Pioneer 2009

, p. 43) :

The 5126 anther

- preferred 4 promoter from Z. mays localizes the expression to the anther (Cigan 1997 ).

The pollen fertility restoration gene ( Ms45 ) from Z. mays restores development of the microspore cell wall that gives rise to pollen 5 (Cigan 2001)

. The presence of the transgene Ms45 allows for production of viable pollen

.

α

- A mylase zm

- aa1

- expression cassette consists of four genetic elements ( Pioneer 2009

, p. 43) :

The pol ygalacturonase promoter ( PG47 ) from Z. mays localizes expression to pollen (Allen 1993).

Transit peptide brittle

- 1 ( zm

- bt1 ) from Z. mays targets expression to the amyloplast 6 (Sullivan 1991). The zm

- aa1 gene was truncated to remove the native transit peptide sequence and replaced by the zm

- bt1 amyloplast

- targeting
sequence.

The alpha

- amylase (α

- amylase ) 7 zm

- aa1 gene from Z. mays ( Pioneer 2009

, f igure 43) encode s the ZM

- AA1 pr otein 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.

The I n2

- 1 terminator from Z. mays from the I n2

- 1 gene (Hershey 1991)

.

4 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).
5 Mut ations in the Ms45 gene causes male sterility because there is no functional pollen produced. A mutation in the Ms45 gene is designated as ms45

. 6 Amyl oplast is an organelle in the cytoplasm of plant cells where starch is stored.
7 Catalyze the hydrolysis of α

- 1,4

- D

- glucosidic bonds in polysaccharides such as starch.4

R ed color marker DsRed

2 (AltI)

- expression cassette consists of four genetic elements ( Pioneer 2009

, p. 43) :

A 35S enhancer from the cauliflower mosaic v irus (CaMV) (Odell 1985, Odell 1988).

The lipid transfer protein ( Ltp2 ) promoter from barley localizes expression to the aleurone layer of the seed (Kalla 1994).

The DsRed2 gene from a marine coral

- like anemone Discosoma sp. (Matz 1999, Wasson

- Bla der 2001) was modified by a single base pair substitution (in which an internal Bst

EII restriction site was re moved without changing the amino acid sequence in the DsRed2 protein) to produce DsRed2 (AltI )

.

DP

- 32138

- 1 seeds expressing the DsRed2 protein are colored pinkish

- red ( Pioneer 2009

, figure 10, Wasson

- Bla der 2001) and can be sorted from yellow seed (that d oes not express DsRed2 ) using a color sorting machine ( Pioneer 2009

, f igure s 4, 5 and 10 , Appendices 2 through 7 ). The D s Red2 protein enable s detection and sorting of transgenic red corn seeds from non

- transgenic yellow seeds.

The proteinase inhibitor II ( pinII ) terminator from Solanum tuberosum (An 1989, Keil 1986).

Data from Southern blot analyses demonstrate that DP

- 32138

- 1 contain s : (1) a single copy of the 5126 promoter and Ms45 gene ( Pioneer 2009

, t able 4, f igure 19) ; (2) a single copy of the PG47 promoter, zm

- bt1 transit peptide, zm

- aa1 gene and I n2

- 1 terminator (petition t able 4, f

igure s 20 and 21) ; and (3) a single copy of the 35S enhancer, the Lt p 2 promoter, DsRed

2 (AltI) and pinII terminator ( Pioneer 2009

, t able 4, f igure s 22 and 23).

Potential of DP

- 32138

- 1 to Become Invasive and/or a Weed

Corn possesses few of the characteristics of those plants that are notably successful as weeds (Baker 1965, Keeler 1989). In the U.S., corn is not listed as a weed (Crockett 1977, Holm 1979, Muenscher 1980), nor is it present i n the Federal noxious weed list (7 CFR part 360; 8 . 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 domest icated crops, corn seed from a previous year’s crop can overwinter and germinate the foll owing 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 applie d to remove these volunteers, but the plants that are not removed do not typically result in feral populations in following years.

APHIS assessed whether DP

- 32138

- 1 is any more likely to become a weed than the

8 http://www.aphis.usda.gov/plant_health/plant_pest_info/weeds/ downloads/weedlist2006.pdf5

isogenic nontransgenic corn line, or other c orn 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 tr ials of DP

- 32138

- 1 since 2005 in the U.S. corn belt at over 43 locations ( Pioneer 2009

, Appendix 12). Agronomic data were collected from DP

- 32138

- 1 and its control counterparts in six locations that provide a range of environmental conditions representativ e of where the DP

- 32138

- 1 is expected to be grown for seed production ( Pioneer 2009

, figure 47). Field trial data ( Pioneer 2009

, 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 ( Pioneer 2009

, 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 ( Pioneer 2009

, tables 10 and 12). None of the measure d attributes showed any differences relative to its comparator, suggest ing that DP

- 32138

- 1 is not weedier than
current corn cultivars.

These results on growth characteristics

, seed production and germination indicate that the DP

- 32138

- 1 is not significan tly 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

. Potential for Gene Flow and Gene Introgression from DP

- 32138

- 1 into Sexually

Compatible Relatives

G ene flow from crops to wild relatives may have the potenti al to enhance the weediness of wild relatives, as observed in rice, sorghum, sunflower

, and a few other crops ( Ellstrand 1999). Gene flow is a natural biological process with significant evolutionary importance. A number of angiosperm taxa are believed to be derived from hybridization or int rogression 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 t o artificially introgress traits from wild relatives into crop plants to develop ne w cultivars.

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 Zea , and to a much lesser degree with members of the genus Tripsacum (OECD 2003). The closest wild relatives of corn, various Zea species referred t o as teosinte, are normally confined to the tropical and subtropical regions of Mexico,6

Guatemala, and Nicaragua. In the U.S. a fairly rare, sparsely dispersed feral population of teosinte has been reported in Florida (USDA Plant database 9 )

. The genus T

ripsacum contains up to 16 recognized species, most of which are native to Mexico, Central and South America

. T hree of these species ( T. dactyloides, T. floridatum , and T. lanceolatum ) exist as wild and/or cultivated species in the continental U.S

. ; and two taxa ( T. fasciculatum and T. latifolium ) 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 unli kely. Hybrids of Tripsacum species with Zea 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.

Introgression of genes from corn into teosinte or Trips a cum 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 al so 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, introgress ive hybridizatio n 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 str uctur es, 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 a s a significan t 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 mal e 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 ( Pioneer 2009

, figure 9, table 12 ); therefore, the outcrossing rate of DP

- 3 2138

- 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. Therefo re, 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.

9 http://plants.usda.gov/java/county?state_name=Florida&statefips=12&symbol=ZEME7

Potential for Transfer of Genetic Information to Organisms with which DP32138

- 1 Cannot Interbreed

APHIS assessed whether h orizontal gene transfer might occur between DP

- 32138 corn and inserted genes with other organisms. However, such transfer and expression of DNA from a plant species to bacteria is unlikely to occur (Keese 2008). First, many bacteria (or parts the reof) that are closely associated with plants have been sequenced, including Agrobacterium and Rhizobium (Kaneko 2000, Kaneko 2002, Wood 2001). There is no evidence that these organisms contain genes derived from plants. Second, in cases where review of se quence data implied that horizontal gene transfer occurred, these events we re inferred to have occur red on an evolutionary time scale on the order of millions of years (Brown 2003, Koonin 2001). Third, FDA has evaluated horizontal gene transfer following p lant transformation with 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 rem ote (FDA 1998). Therefore, APHIS concludes that horizontal gene transfer is unlikely to occur and thus poses no significant environmental or plant pest risk.

Potential for DP32138

- 1 to have Altered Disease and Pest Susceptibilities

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.

DP

- 321 38

- 1 has been f

ield tested in the U.S. since 2005 ( Pioneer 2009

, 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 exp ected to be gr own ( Pioneer 2009

, f

igure 47). Pioneer routinely monitors their corn field trials for the following disease agents: Aspergillus sp., Aureobasidium zeae, Cercospora zeae

- maydis, Colletotrichum graminicola, Exserohilum turcicum, Fusarium sp p ., Gibberella zea e , Pantoea stewartii, Phyt opthora spp., Puccinia polysora, Puccinia sorghi and Ustilago

zeae ( Pioneer 2009

, Appendix 13, t able 2). The corn insects monitored include Aphididae , Adoretus sinicus, Chaetocnema pulicaria, Chrysoperla carnea Cicadellidae, Coleoptera

, Diabrotica spp ., Glischrochilus quadrisignatus, Helicoverpa zea, Lepidoptera

, Ostrinia nubilalis, Popillia japonica, Richia albicosta, Spodoptera frugiperda, Syrphidae, Tetranychidae, Thripidae (petition Appendix 13, t able 1). The data submitte d by Pioneer indicated no meaningful differences between DP

- 32138

- 1 and

non

- transgenic counterparts for disease or insect damage ( Pioneer 2009

, p. 107, Appendix 13, t able s 1 and 2).

Furthermore, DNA sequences derived from plant pests that were incorporat ed in DP

32138

- 1 d id not result in the production of infectious agents or disease symptoms in field trials with the 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.8

The use o f Agrobacterium for transforming DP

- 32138

- 1 is unlikely to propagate infectious agents. Transformed plants used in the generation of DP

- 32138

- 1 were treated with an antibiotic to eliminate the Agrobacterium from plant culture using well known protocols ( Pi oneer 2009 )

.

APHIS considered whether corn constituents that differed between DP

- 32138

- 1 corn and other corn varieties might be sufficient to lead to increased susceptibility to pathogens or pests. 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 ( Pioneer 2009 )

. N one of the values for seed composition characteristics were outside the range of natural variabili ty of conventional corn ( Pioneer 2009

, 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 Pioneer 2009 ), 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.

Corn is not a plant pest in the United States 10 , and the introduced DNA in DP

- 32138 is unlikely to pose a plant pest risk . Based on the analysis of genetic modifications and their functions and field testing data submitted by p etitioner, APHIS conclude s that there

are no significant differences between DP

- 32138

- 1 corn and the non

- transgenic counterparts relative to pest and disease susceptibility.

Conclusion

APHIS has prep ared 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 characteristi cs, 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.

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