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Herculex® RW Detailed Safety Assessment

1.   Molecular and Protein Analysis

1.1   Genes Used and Their Molecular Analysis in HXRW

Three genes, cry34Ab1 (referred to as cry34), cry35Ab1 (referred to as cry35), and pat were used for Agrobacterium mediated transformation to generate Herculex RW (HXRW; event DAS-59122-7).

The source of the cry34 and cry35 genes is the common soil bacterium Bacillus thuringiensis strain PS149B1 (ARS culture collection # NRRL B-21553). The cry genes are expressed in HXRW as two separate parasporal crystal proteins, Cry34 and Cry35, with respective molecular weights of 14 kDa and 44 kDa. Both insecticidal crystal proteins (ICPs) are required to provide commercial levels of activity on western (Diabrotica virgifiera virgifera), northern (Diabrotica berberi) and Mexican corn rootworm (Diabrotica virgifera zeae, CRW) larvae. Both cry genes were optimized for expression in corn, by substitution of alternative codons to bias the codon usage to that preferred by the target plant (Adang et al., 1987). Genes for cry34 and cry35 were synthesized for the 14 kDa and 44 kDa coding sequences and transformed together into corn plants. The proteins encoded by the synthetic transgenes are identical in sequence to the native Bt proteins (Moellenbeck et al. 2001).

The source of the pat gene is the ubiquitous soil bacterium Streptomyces viridochromogenes. The gene encodes for a phosphinothricin N-acetyltransferase of 183 amino acids that converts L-PPT (L-phosphinotricin), the active ingredient in glufosinate-ammonium herbicide, to its inactive form N-acetyl-L-PPT (De Block et al., 1988; Wehrmann et al., 1996; OECD, 1999). L-PPT, binds to, and inactivates, the enzyme glutamine synthetase in plants, preventing the detoxification of excess ammonia, which ultimately results in plant death. Plants expressing the PAT enzyme are therefore tolerant to the herbicide, enabling treatment of surrounding weeds without harm to the crop. The herbicide tolerance trait was also used as a selectable marker to facilitate generation of transgenic plants. The pat gene was optimized for expression in corn by modifying the G+C codon bias to a level more typical for plant DNA.

To drive gene expression, the ubiquitin promoter from maize (UB1ZM) (Christensen et al., 1992) is used for the cry34 gene and the promoter from the Triticum asestivum (wheat) peroxidase gene (TA peroxidase pro) with its native leader sequence (Hertig et al., 1991) for cry35 gene. Transcription of both cry genes is stopped by the terminal sequence from Solanum tuberosum (potato) proteinase inhibitor II gene (PINII; An et al., 1989). The promoter and transcription stop sequences for the pat gene are from the 35S transcript of the cauliflower mosaic virus (CaMV35S) (Pietrzak, et al., 1986). The DNA segment that was used to generate HXRW trait is shown in Figure 1.

Figure 1: DNA segment (7515 bp) used to produce the HXRW trait

Southern blot studies indicate that a DNA fragment, as described above, is integrated into the corn genomic DNA with no indication that rearrangements of the DNA had occurred.

1.2   Genetic Stability of Inserted DNA Fragment and Agronomic Performance

The presence and integrity of the transferred genes in HXRW was investigated over multiple generations to ascertain genetic stability. The results from several rounds of backcrossing and self-crossing demonstrate that the cry34, cry35 and pat genes are stable. Observations of the phenotype indicated that the transgenes are inherited as a single dominant element according to Mendelian segregation patterns.

General performance for a broad range of agronomic traits of HXRW was evaluated in thousands of field trials in corn growing regions within the U.S and abroad. Several characteristics were measured, including root damage, plant height, tassel size, leaf color and ear shape. The results indicated no difference between HXRW and its non-biotech counterpart, except for the introduced trait.

1.3   Cry34/35 and PAT Protein Expression in Plant Tissues

Field studies representative of the conditions and growth stages corresponding to commercial corn production were undertaken. The protein expression data presented below are obtained from trials conducted during the 2002-2003 growing season in Chile.

The expression levels of Cry34, Cry35 and PAT proteins in leaf, pollen, root, stalk, whole plant, forage, grain and senescent whole plant tissues from HXRW and non-biotech corn with comparable background genetics were measured using ELISA (Enzyme Linked Immunosorbent Assay) specifically developed for each protein. Western immunoblot analysis was used to confirm the integrity of the newly expressed proteins.

Cry34 protein concentrations were measurable in plant tissues from mature plants with average levels of 76.5 ng/mg tissue dry weight in whole plant, 163 ng/mg in leaf, 49.7 ng/mg in root and 49.7 ng/mg in grain. The levels for Cry35 protein were 13.9 ng/mg in whole plant, 54.4 ng/mg in leaf, 3.1 ng/mg in root and 0.99 ng/mg in grain.

PAT protein was found in leaf and root tissue samples at measurable levels up to approximately 0.38 ng/mg tissue dry weight. These levels are sufficient to confer tolerance to glufosinate-ammonium herbicide at the level of the whole plant. In whole plant and grain tissue, the levels of PAT protein were below the limit of detection.

As expected, expression of the Cry34, Cry35 and PAT proteins was not detected in any samples from the control plants.  » More

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2.   Food and Feed Safety Analysis

Following rigorous testing in accordance with federal agency guidelines, the transgenic proteins expressed in HXRW were found to possess no similarities to known toxic or allergenic proteins. Additionally, in feeding trials, the performance of HXRW grain was comparable to non-biotech corn, indicating that it is nutritionally equivalent.

To obtain the quantities of the Cry34, Cry35 and PAT proteins necessary for biochemical characterization and toxicological as well as allergenicity tests, the proteins were produced using a bacterial expression system. A range of analyses was subsequently undertaken to establish that the microbially produced proteins were equivalent to the proteins produced in HXRW. Several characteristics were analyzed, including molecular weight, immunoreactivity, post-translational modification, N-terminal amino acid sequence and bioactivity on susceptible insect larvae.

2.1   Toxicity Potential

Two tools that were used to assess the potential for the transgenic proteins in HXRW to cause toxicity are bioinformatic analysis and direct evaluation of the acute toxicity of the individual proteins.

In the bioinformatic analysis, the amino acid sequences of the Cry34, Cry35, and PAT proteins were compared to the amino acid sequences of the sequences of proteins in publicly available databases using computer search tools. No significant amino acid sequence similarities were observed between the sequences of known protein toxins and allergenic proteins.

Proteins that are toxic are believed to act via acute mechanisms of action (Sjoblad et al., 1992). Acute toxicology studies were conducted with the transgenic proteins expressed in HXRW. Groups of mice received an oral dose containing 2700 mg Cry34 protein/kg body weight (bw), 1850 mg Cry34 protein/kg bw, or a combination of 482 mg/kg bw Cry34 plus 1520 mg/kg bw Cry35 proteins. A dose equivalent to 5000 mg/kg bw was used for the PAT toxicity study. These doses are equivalent to many thousand times the estimated dietary intake for human consumption. Following these doses, mice were observed for mortality and clinical or behavioral signs of toxicity as well as individual body weights over a period of 14 days. No signs of toxicity were seen in any of the groups of mice tested.  From these studies it was concluded that the Cry34, Cry35, and PAT proteins in HXRW are not acutely toxic.

2.2   Allergenicity Potential

The prevalence of food allergy is approximately 1-2% in adults and 6-8% in children (Metcalfe et al., 1996, Sampson, 1997). A food allergy is a reaction of the immune system to an otherwise harmless food or food component. Most food allergies are mediated by IgE and are characteristic of type-I reactions. The percentage of proteins that are allergenic is very small. Only approximately 200 of the hundreds of thousands of proteins humans consume in food are food allergens (Day, 1992). Hefle et al. (1996) surveyed the literature and identified approximately 180 foods reported to be allergenic. Of those, eight food groups (peanuts, soybeans, crustacea, fish, cow's milk, eggs, tree nuts and wheat) account in general for 90% of all reported food allergies worldwide (Metcalfe, et al., 1996). The remaining 10 percent of food allergies are caused by less commonly allergenic proteins or minor allergens and affect a relatively small number of people (Hefle et al, 1996).

The process by which the allergy assessment has been conducted has involved guidance from a number of expert scientific bodies, including the U.S. FDA (1992), FAO/WHO (2001), and Codex (2003). The objectives of the recommendations provided by these expert bodies for assessing the potential allergenicity of foods derived from biotech crops are two-fold: 1) protect allergic consumers from accidental exposure to allergens or cross-reactive proteins that may trigger an adverse reaction in those already allergic to such proteins and 2) protect the general population from risks associated with the introduction of genes encoding proteins that are likely to become food allergens.

The approach by which allergy assessment has usually been conducted involves the use of a step-wise, decision tree (Metcalfe et al., 1996; FAO/WHO, 2001). However, to date, no single factor has been recognized as the primary identifier for allergenicity.  Therefore, a weight-of-evidence approach, which accounts for a variety of factors and experimental approaches for an overall assessment of the allergenic potential of the new protein, is utilized (Codex, 2003). These assessments are currently based on what is known about food allergens such as history of exposure and safety of the gene(s) (i.e., Is the source of the gene allergenic?); molecular structures (e.g., amino acid sequence homology and structural similarity to known human allergens); physicochemical properties such stability to pepsin digestion in vitro; an estimate of the exposure of the novel protein(s) to the gastrointestinal tract where absorption occurs (e.g., digestibility,  protein abundance in the crop, and food/feed processing effects); and where appropriate, human and clinical tests (e.g., skin prick test, food challenge studies).  » More

Cry34, cry35 and pat genes were derived from common soil microorganisms, Bacillus thuringiensis and Streptomyces viridochromogenes, respectively. Cry proteins and PAT, as novel protein in plants, have a history of safe use in agricultural crop commodities and are not associated with allergenicity. In accordance with suggested recommendations for assessing potential allergenicity (FAO/WHO, 2001; Codex, 2003), Cry proteins in general have been demonstrated to have no amino acid sequence similarity to known allergens, are rapidly digested utilizing in vitro pepsin resistance assays, heat labile, have a low prevalence in food, and low-to-no glycosylation (Betz et al., 2000). Recently, Batista et al. (2005) performed skin prick tests with protein extracts prepared from a number of biotech and conventional corn and soybean samples and reported no differences in terms of allergenicity between those samples. In addition, the U.S. EPA states that "after decades of widespread use of Bt as a pesticide, there have been no confirmed reports of immediate or delayed allergic reactions to the delta-endotoxin itself despite significant oral, dermal and inhalation exposure to the microbial product"(USEPA, 2000). The lack of allergenic concern for cry proteins was recently reconfirmed in an April 2005 U.S. EPA Scientific Advisory Panel (SAP) Report (US EPA, 2005) in which the panel states it “…was not aware of any report of Bt being an allergen.” Taken together, these data indicate a lack of allergenic concern for Cry proteins in general.

As part of the ‘weight-of-evidence’ assessment, the amino acid sequence of the Cry 34/35 and PAT proteins were compared to a database containing the amino acid sequences of known allergens using established criteria (Metcalfe et al., 1996; FAO/WHO, 2001; Codex, 2003).  It is recommended that IgE cross-reactivity between a novel protein and a known allergen be considered a possibility when there is more than 35% identity over a segment of 80 amino acids. To further exclude the possibility of cross-reactivity with a known allergen, a stepwise contiguous identical amino acid segment search is recommended with known allergens to identify amino acid sequences that may represent linear IgE binding epitopes. Eight contiguous amino acid matches between a novel protein and a known allergen(s) have been recommended to identify sequences that may represent linear epitopes (Metcalfe et al., 1996). The results of this comprehensive search demonstrated that neither the Cry34 nor Cry35 or PAT proteins shared significant amino acid sequence similarity with known allergenic proteins.

The resistance of a protein to pepsin in vitro (Astwood et al., 1996; Thomas et al., 2004) has been correlated with protein allergenicity (Astwood et al., 1996), although the relationship is not absolute (Fu et al., 2002). The in vitro pepsin digestibility of bacterially derived Cry34 and Cry35 proteins was determined in laboratory experiments. In the experiments, the proteins were exposed to pepsin for various periods of time up to 60 minutes.  Based on the results of the digestibility tests and the other weight-of–evidence data discussed in this section, the U.S. EPA concluded that Cry 34/35 is unlikely to be a food allergen (USEPA, 2005).

As some protein allergens are glycosylated, studies were carried out to determine whether Cry34 or Cry 35 is a glycoprotein. The potential glycosylation of both proteins in planta and from a microbial source was examined by a sensitive immuno-blot technique used for glycoprotein detection. The studies indicated that that Cry34 or Cry35 are not  glycosylated.

2.3   Heat Lability (Stability)

Two studies on heat lability of the Cry34/35 proteins were conducted.  In the first study aqueous formulations of the proteins were incubated at 60, 75 or 90oC for 30 minutes.  After incubation, neonate southern corn rootworm larvae (SCR) were exposed for 6 days to artificial dietary substrates containing the heat incubated proteins. A second study was conducted to examine the heat lability of the individual proteins Cry34 and Cry35 by fortifying heated samples with non-heated samples of each protein. Based on these studies, the Cry 34/35 proteins were determined to be heat labile after exposure to 90°C for 30 minutes, as defined by inactivation of biological activity.

In summary, Cry 34/35 proteins originate from a non-allergenic source, exhibit no amino acid sequence similarity with known allergens, are not glycosylated, are rapidly to moderately digested in vitro, heat inactivated and present at very low levels in food.  Based on these data, the U.S. EPA concluded that Cry 34/35 proteins are unlikely to be food allergens (USEPA, 2005).

2.4   Compositional Analysis

A key component in the regulatory approval process is the demonstration that a crop derived through biotechnology is “substantially equivalent” to a conventional crop. The key constituents in corn grain have been evaluated in order to compare them to corresponding data from HXRW expressing Cry34, Cry35 and PAT proteins, as well as a non-biotech counterpart and published literature values obtained for non-biotech varieties of corn. As a reference tool, the Organization for Economic Co-operation and Development (OECD) has produced a consensus document on compositional considerations for new varieties of maize, which looks at nutrients and plant metabolites (OECD, 2002).

This evaluation includes a study of the major constituents that are characteristic of whole corn grain, taking into account the natural variation in composition that is known to occur due to genetic variability and geographical or environmental factors.  » More    » More

Several major studies were conducted at different geographical areas to determine the compositional profile of key corn tissues collected from HXRW and non-biotech control lines with comparable genetic background.

A nutrient analysis focused on a number of components from corn forage and grain, including fat, protein, fiber content, moisture and ash. In addition, grain was measured for fatty acid content, amino acid profile, mineral content, vitamin content and potential antinutrients.

The analyte values measured in HXRW were compared with those obtained from a control line (near isoline).  Differences of p<0.05 were considered as statistically different. Since it is not unexpected that some analytes reveal a certain statistically difference between test and control line, means across locations were also compared with the range of analyte value as reported in the literature. An analyte mean value that falls within the reported range, strongly suggests that statistical differences are of no biological significance.

The following nutrient analysis information is based on data collected from 2002/03 Chile field trials.


2.5   Nutritional Performance

In assessing the safety of a food or feed derived through biotechnology, it is important to demonstrate that it is nutritionally equivalent to an appropriate non-biotech counterpart. In most cases, this can be achieved through an understanding of the genetic modification and its consequences, coupled with an extensive compositional analysis of the food. Carefully designed feeding studies in animals may provide further reassurance that the food is nutritionally equivalent to non-biotech counterpart.

To evaluate the potential for long-term consumption of HXRW to cause adverse effects, a thirteen-week (90-day) feeding study was conducted in rats. Rat diets were prepared with a 35% concentration of HXRW grain in accordance with a standard rodent laboratory diet (Purina Test Diet 2002?).  For comparison, separate control diets were prepared with the same concentration of the near-isogenic control corn grain or a commercially available non-biotech reference corn grain. Two additional control groups consumed commercial diets containing 35% non-biotech corn grains.

Over the course of the in-life phase of the feeding study, nutritional performance was evaluated by determination of body weights, feed consumption, and feeding efficiencies.   At the end of the feeding trial, the overall health of all animals was evaluated using standard indicators for trials of this type including hematology and serum chemistry and an evaluation of all major organ systems in the body for indications of weight changes or microscopic signs of pathology. Compared to rats consuming the control diets, there were no biologically significant, diet-related differences observed in rats consuming diets containing HXRW.

A similar study was conducted to compare the nutritional performance of HXRW grain with non-biotech control grains in broiler chickens. Rapidly growing broiler chickens are sensitive to changes in nutrient quality in diets, and, therefore, serve as a useful model species to evaluate the wholesomeness of food and feed. As in the rat feeding study, there were no significant differences in nutritional performance metrics observed between broilers consuming diets prepared with HXRW grain and those consuming diets with non-biotech control corn grains.

2.6   Human Dietary Risk Assessment

Compositional analysis of grain, as well as toxicity and allergenicity analysis of the novel proteins, indicated that HXRW is as safe as non-biotech corn for human and animal consumption. Another data point to assess the risk for human consumption is the analysis of potential dietary risk.

One way to characterize dietary risk is to calculate the amount of a food that would have to be eaten to expose a person to the same level of protein that was utilized in toxicology studies of the protein. The estimated amount of food is then evaluated in terms of how feasible it would be to eat that amount of food in one day. For example, Cry34 protein expression in HXRW grain is approximately 55 ng/mg dry weight. This means that one milligram of grain contains 0.000055 mg of protein. Mice were dosed using 2700 mg of microbially produced protein per kilogram body weight. To receive an equivalent dose of Cry34 protein in grain, each mouse would have had to eat 48.7 kg of grain per kg body weight (bw). Similar estimates were made as well for Cry35.  Following these assumptions, one can calculate the amount of raw grain that would have to be eaten by an infant, child, or an adult to match the protein levels used in the mouse toxicity studies.


  Cry34
Alone
Cry35
Alone
Cry34/Cry35
Combination
   Expression (ng/mg) 55.39 0.95 55.39/0.95
   Tox Test Dose (mg/kg bw) 2700 1850 482/1520
   Amount (kg/day) of grain needed to be consumed to match dose in toxicology tests:
   Child (10 kg) 487 19474 44/8000
   Adult (60 kg) 2925 116842 522/96000

The actual exposure to Cry34/35 proteins in the diet is expected to be much lower due to:


  • grain containing the Cry34/35 proteins will be mixed with other grain not containing those proteins;
  • reductions in protein concentrations will occur during processing to produce high fructose corn syrup and vegetable oils (commodities that contain negligible levels of protein)
  • protein degradation due to exposure to heat during food preparation.

These worst-case calculations indicate a clear margin of safety for these proteins. The actual margin of safety will be much greater when the effects of factors such as market share and processing are taken into account.

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3.   Environmental Impact Analysis

Following federal agency guidelines, HXRW was extensively tested to evaluate its environmental safety. All tests indicated that HXRW has no adverse effects on soil organisms, animals and non-target insects.

3.1   Estimated Environmental Concentration (EEC)

Non-target organisms may be exposed to Cry34/35 proteins expressed in HXRW through either direct or indirect routes. Exposure estimates for organisms directly feeding on corn tissues expressing Cry34/35 proteins are based on the high-end expression for the relevant plant tissue to which a non-target organism of concern may be exposed through direct ingestion. High-end exposure estimates (HEEE; USEPA, 1997) were used in comparison with the results from the ecotoxicology studies.  These values range from 32 to 235 μg/g tissue dry weight for Cry 34 and from 0.03 to 87 μg/g for Cry35 for all plant tissues from HXRW throughout the growing season.

3.2   Soil Degradation

Some of the Cry34 and Cry35 proteins exposed to the soil or soil organisms in the field will be from post-harvest crop debris in the field. A bioassay based on the insecticidal activity of Cry34/35 against southern corn rootworm was used to determine the biodegradation in the soil. Untreated soil and soil treated with 5mg/g soil each of Cry34 and Cry35 protein were exposed to southern corn rootworm neonate larvae. Over a 28-day period, larval growth inhibition was measured and a DT50 (time to 50% reduction of protein activity) of 3.2 days was obtained. This value is comparable with the degradation in soil of other Cry proteins published in the literature (Sims and Holden, 1996; Herman et al., 2003).

3.3   Exposure to Non-Target Organisms

Feeding studies with both purified Cry34 and Cry35 proteins mixed into prescribed diet material or with plant tissue derived from HXRW were performed on EPA-recommended representative animals. The amount of Cry34 and Cry35 proteins used in these studies was generally well above the estimated amount in soil and water.

Non-target organisms may be exposed to the Cry34/35 proteins in two ways:  direct consumption of the plant material or indirect consumption (multi-trophic interactions:  animals feeding on animals that have ingested Cry34/35 proteins). Numerous laboratory and field monitoring studies have been conducted to assess the impact of Cry34/35 expressing maize on non-target organisms.

3.4   Non-Target Laboratory Studies

Feeding studies with microbially derived Cry34/Cry35 purified proteins mixed into artificial diet or with plant tissue derived from corn expressing Cry34/35 proteins were performed on EPA-recommended representative animals. The amount of Cry34 and Cry35 proteins used in these studies was generally above the expected environmental concentration. Organisms tested included:


  • Convergent ladybird beetle adults
  • Pink spotted (aka twelve spotted) ladybird beetle larvae
  • Ground beetle larvae
  • Springtails
  • Green lacewings
  • Parasitic wasps
  • Monarch butterfly larvae
  • Honeybee larvae
  • Earthworms
  • Aquatic invertebrates

No adverse effects were detected in the feeding studies of these organisms at environmental relevant concentrations.

3.5   Non-Target Field Studies 

Field studies monitoring the environmental impact of HXRW on non-target arthropod populations have been conducted annually since 2001. Several methods of sampling have been employed throughout the growing seasons including: visual observations, sticky traps, pitfall traps, soil samples and litterbag traps. The results were compared with non-biotech corn as well as conventional insecticides used for rootworm control. To date, no adverse effects have been observed when comparing the abundance of non-target organisms in HXRW field plots compared to non-biotech corn field plots.

3.6   Endangered Species Risk Assessment

Because of the selectivity of Cry34/35 proteins for coleopteran species, endangered species concerns primarily focus on the order Coleoptera. From the 16 Coleopteran insect species either classified as endangered or threatened by the U.S. Fish and Wildlife Service, only the American burying beetle exists in corn production areas. After reviewing the preferred habitats and feeding behavior of the American burying beetle, the EPA concluded that there would be no exposure to significant levels of Cry34/35 proteins (Both larvae and adult insects feed exclusively on carrion with some limited adult predation on corn).

3.7   Weediness Potential

Corn does not exhibit any significant weediness characteristics and is non-invasive in natural environments (Canadian Food Inspection Agency, 1994). Corn hybrids have been domesticated for such a long period of time that the seeds cannot be disseminated without human intervention, nor can corn readily survive in the U.S. from one growing season to the next because of the poor dormancy. Volunteer corn plants are, in any case, easily identified and controlled through manual or chemical means.    

In addition, there are no wild, weedy relatives of Zea mays known to exist in the United States. Therefore, outcrossing of the cry34/35 genes or pat gene does not pose a plant pest risk due to the enhancement of weediness of wild relatives of corn.  » More

3.8   Horizontal Gene Transfer

There is no known mechanism for, or definitive demonstration of, DNA transfer from plants to microbes (Nap et al., 1992; Redenbaugh et al., 1994).  Even if such a transfer were to take place, transfer of the cry34/35 or pat genes from HXRW is not expected to present a significant risk to human health or the environment. Genes encoding the PAT enzyme and similar acetyltransferases are ubiquituously present in nature. Similarly, the cry34/35 genes were isolated from an ubiquitous soil bacterium and therefore already present in nature.  Cry34/35 itself has been shown to occur in geographically diverse settings associated with agricultural and non-agricultural lands. Recipients would, therefore, not pose a greater risk to humans, animals and the environment than those organisms in which the genes are naturally present.  » More

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4.   Post Marketing Stewardship

DuPont is committed to rigorous stewardship of products derived through biotechnology through the development process and after a product has become commercialized.

4.1   Availability of Diagnostics

Validated methods to detect the cry34 and cry35 genes and Cry34 and Cry35 proteins in biotech plant material are developed by independent companies and are available before planting of the first HXRW crop (2006 season). Two methods enable the detection of the respective proteins (lateral flow test kit, or dip stick; and an ELISA assay). A qualitative as well as a quantitative PCR-based test kit provides detection methods of product specific DNA.

4.2   Insect Resistance Management (IRM)

Companies registering biotech corn products expressing a Bt trait for corn rootworm protection, such as HXRW, are required by the EPA to develop and implement an insect resistance management (IRM) plan. The IRM plan is designed to prevent or delay the development of insects resistant to Bt proteins.

A key component of IRM plan for HXRW is the planting of a refuge, which is corn that does not produce a protein for controlling corn rootworms.  The refuge can be planted in number of configuration, but it must be positioned in the same field or adjacent field to HXRW corn. The refuge provides a habitat where corn rootworms will not be exposed to HXRW plants. Thus the refuge produces Bt susceptible corn rootworm beetles which can mate with potentially resistant beetles that could emerge from the near-by HXRW corn. The mating of susceptible and resistant corn rootworm beetles results in offspring that are susceptible to the HXRW corn. These offspring will die as larvae when they feed on roots of a HXRW plant. Planting a refuge is one strategy to slow the evolution of resistance by target insect to Bt corn rootworm proteins and preserve the efficacy of Bt corn rootworm technology.

Pioneer Hi-Bred International, Inc., utilizes a comprehensive, multi-faceted approach to IRM education that targets our customers and sale force. Pioneer develops and distributes informational and training material on proper refuge management.  These materials include the HXRW Product Use Guide, brochures, articles in Growing Point magazine and communications from the sales force to growers.

In addition, as a condition of registration and to assist in ensuring IRM compliance, Pioneer is required to assess adherence to refuge requirements by corn growers. The activities associated with monitoring refuge compliance by growers are described in the HXRW Compliance Assurance Program (CAP) submitted to and approved by EPA. The CAP lays out the process for assessing IRM compliance as well as the corrective actions to take with growers who have not complied with refuge requirements.  These actions include notification, follow-up assessments and addition IRM education. Repeated non-compliance can result in the inability of a grower to access Bt products for at least a year.

Research continues in both, the public and private sector, to further define practical and effective long-term management strategies for corn expressing insect control proteins.  Results from these efforts will be periodically incorporated into existing IRM plans as appropriate.

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Herculex Insect Protection technology by Dow AgroSciences and Pioneer Hi-Bred. ®Herculex is a registered trademark of Dow AgroSciences LLC.