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Published online 28 March 2005
Published in Crop Sci 45:931-938 (2005)
© 2005 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

A Method of Controlling Corn Rootworm Feeding Using a Bacillus thuringiensis Protein Expressed in Transgenic Maize

Ty Vaughn*, Tracey Cavato, Gurdip Brar, Timothy Coombe, Todd DeGooyer, Stephanie Ford, Mark Groth, Arlene Howe, Scott Johnson, Kathryn Kolacz, Clinton Pilcher, John Purcell, Charles Romano, Leigh English and Jay Pershing

Monsanto Company, 700 Chesterfield Parkway West, Chesterfield, MO 63017

* Corresponding author (ty.t.vaughn{at}monsanto.com)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The corn rootworm (CRW; Diabrotica spp.) is one of the most serious pests of corn in the USA. Chemical insecticides and crop rotation have been the only two options available to growers for managing CRW. Unfortunately, both of these tactics can be ineffective as a result of either resistance or behavioral modifications. In this paper, we describe transgenic maize (Zea mays L.) hybrids that control CRW. These hybrids were created with a Cry3Bb1 Bacillus thuringiensis (Bt) variant that is approximately eight times more lethal to corn rootworm larvae than the wild-type protein. A DNA vector containing the modified cry3Bb1 gene was placed under control of a root-enhanced promoter (4-AS1) and was introduced into embryonic maize cells by microprojectile bombardment. Described here is the molecular genetic characterization, protein expression levels, and field performance of the recently commercialized MON863 hybrids.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CORN ROOTWORM is the most destructive insect pest of corn in the USA. When last assessed in 1995, CRW was responsible for the largest expenditure by growers for insect management in corn production systems (Pike, 1995). CRW larvae inflict damage to corn plants by feeding on the root tissues, which reduces the ability of the plant to take up water and nutrients from the soil (Reidell, 1990). Damaged plants are also prone to lodging, resulting in reduced yield (Spike and Tollefson, 1991) and adding significant time to harvesting operations. In addition to the financial costs associated with yield loss, chemical control tactics for CRW also have significant environmental costs. Organophosphates, carbamates, pyrethroids, and phenyl pyrazoles insecticides are typically used to manage this pest. Further, certain CRW species have also recently overcome crop rotation control strategies through behavioral adaptations. Thus, a pressing need exists for new and effective options for growers to manage CRW.

Recently, Moellenbeck et al. (2001) described a CRW protected corn hybrid that was developed following the introduction of a gene encoding a binary delta-endotoxin from the Bt strain PS149B1. Their research demonstrated that plants expressing the PS149B1 endotoxin provided protection from CRW larval feeding damage.

The first commercial transgenic maize hybrid designed to control CRW larval feeding was introduced in 2003 in the USA and is described in this paper. This hybrid uses a variant Bt Cry3Bb1 insecticidal protein (Donovan et al., 1992). Cry3Bb1 is known to be biologically active against several species within the Coleopteran family Chrysomelidae, including the western corn rootworm, Diabrotica virgifera virgifera LeConte (Rupar et al., 1991). The biological activity of this protein against D. virgifera virgifera suggested its potential use in creating transgenic plants expressing Cry3Bb1 that would confer protection to corn root tissue from larval feeding damage. To further augment protection of the root system from larval feeding damage, modifications were introduced in the cry3Bb1 gene that gave rise to an amino acid variant Cry3Bb1 protein with an eight-fold increase in biological activity when compared to the wild-type Cry3Bb protein against D. undecimpunctata howardi Barber (English et al., 2000a, 2000b, 2000c, 2000d). Diabrotica virgifera virgifera and D. barberi Smith & Lawrence were subsequently tested in artificial diet bioassays and indicated similar levels of activity; the LC50 for all three Diabrotica species was approximately 10.4 µg protein cm–2 of diet surface area (unpublished data). This modified cry3Bb1 gene was optimized for expression in monocots via modifications of the codon sequence (Klein et al., 1989) and driven by the 4-AS1 promoter, which was known to confer enhanced expression in root tissues in tobacco (Lam and Chua, 1990). This paper provides an in-depth description of the recently commercialized CRW protected transgenic maize and includes the molecular characterization of the transgene insert, growth stage related changes in, root expression levels of the variant Cry3Bb1 protein, as well as both field and growth chamber evaluations of CRW control.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transformation and Molecular Analysis
A transgenic plant was generated by particle acceleration technology (Gordon-Kamm et al., 1990) and NPTII selection (D'Halluin et al., 1992) to transform corn cells with a 4.7-kb linear DNA fragment derived from plasmid PV-ZMIR13 (Fig. 1A) . The resulting transgenic plant identified was called MON863. The linear DNA fragment of this event, obtained by digestion with the restriction endonuclease MluI, contained two gene expression cassettes consisting of (i) the cry3Bb1 coding region regulated by the root-enhanced 4-AS1 plant promoter, the wheat chlorophyll a/b-binding protein (wtCAB) leader, rice actin intron, and the wheat heat shock protein (tahsp17 3') transcriptional terminator and (ii) the nptII (Beck et al., 1982) coding region regulated by the 35S promoter, and the NOS 3' transcriptional terminator (Table 1). Genomic DNA was extracted from event MON863 and analyzed using Southern blot analysis (Southern, 1975) to determine the number of transgenic insertions, the copy number of the transgenes at each insertion site, the integrity of the inserted cry3Bb1 gene, the nptII transgene cassettes, and the absence of plasmid backbone sequence.



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  		Fig. 1. Plasmid map of PV-ZMIR13 and the inserted DNA in event MON863. (A) The MluI restriction fragment of plasmid PV-ZMIR13 was used for transformation to generate event MON863. This region is highlighted by the curved line while the remaining section is the plasmid backbone and elements therein used for bacterial selection. (B) A map of the characterized transgene insert in event MON863. The arrows depict the directionality of the genetic elements within the insert.

 

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  		Table 1. Summary of genetic elements in plasmid PV-ZMIR13 used to generated event MON863.

 
The 5' and 3' insert-to-plant genomic DNA junctions were delimited by polymerase chain reactions (PCR) followed by DNA sequencing. The 5' junction was verified with one primer complimentary to the 5' genomic flanking sequence, paired with a second primer in the E35S promoter covering 312 bp. The 3' junction was verified with one primer complimentary to the 3' end of the Cry3Bb1 coding sequence paired with a second primer complimentary to the 3' genomic flanking sequence spanning 363 bp. The negative controls consisting of nontransgenic maize (MON846) and distilled water did not generate either PCR product. Only the predicted size PCR products were generated from the 5' and 3' ends of event MON863, demonstrating the specificity of the primer pairs to the MON863 event. The PCR products from event MON863 were subjected to DNA sequencing to further confirm the 5' and 3' ends of the insert as well as the genomic DNA which flanks both ends of the insert.

A Southern blot analysis was performed with plant genomic DNA extracted from processed grain tissue utilizing a modified CTAB-based method (Rogers and Bendich, 1985). Ten micrograms of total genomic DNA were digested in parallel with positive control samples (plasmid PV-ZMIR13 DNA spiked into 10 µg of nontransgenic corn DNA) and subjected to Southern blot analyses. Probe templates were labeled with 32P-dCTP (6000 Ci/mmol) by the RadPrime DNA Labeling System (Gibco BRL, Gaithersburg MD), and purified on a Sephadex G-50 column (Roche, Indianapolis, IN).

PCR and Sequence Verification of the 5' and 3' Genomic DNA Flanking Sequences
The PCR-based technique GenomeWalker (Clontech, Palo Alto, CA) was used to generate PCR products containing DNA at the 5' and 3' ends of the transgene insert, as well as the corn genomic DNA flanking the 5' and 3' ends of insert in corn event MON863. The 5' and 3' insert-to-plant genomic DNA junctions were then verified by means of nested PCR primers followed by DNA sequencing. The PCR amplifications were conducted with 100 ng of genomic DNA as a template in a 50-µL reaction volume containing a final concentration of 2.5 mM Mg2+, 0.4 µM of each primer, 200 µM each dNTP, and 2.5 units of Taq DNA polymerase. The amplification of the reactions was performed under the following cycling conditions: 94°C for 1 min; 38 cycles at 96°C for 30 s, 70°C for 30 s, 72°C for 1.5 min; 1 cycle at 72°C for 10 min. The PCR products were isolated from the agarose gel with the QIAquick gel extraction kit (Qiagen, Valencia, CA) and subjected to DNA sequencing by dye-terminator chemistry.

Protein Expression
MON863 plants, homozygous for the cry3Bb1 gene, were crossed with five elite inbred maize genotypes to create hybrid plants. Cry3Bb1 protein synthesis was estimated by an enzyme-linked immunosorbent assay (ELISA) from 14 plants for each of the five resultant hybrid lines and was measured from the entire root system at the V4 and V9 growth stage. Hybrid plants used in the quantification of the Cry3Bb1 protein were grown individually under controlled environmental conditions (temperature: 24°C night, 28°C day; RH: 50%; photoperiod: 14 h of light:10 h or dark). Roots were thoroughly washed and frozen before homogenization and storage. Samples were stored at –80°C. Homogenized root tissues were extracted by means of PBST [phosphate buffered saline plus 0.07% (v/v) Tween-20] containing 0.1% (w/v) BSA (bovine serum albumin) at a 1:100 tissue weight to buffer volume ratio with a Wheaton overhead stirrer. The solubilized protein supernatant was isolated by centrifugation and loaded alongside a Cry3Bb1 pure protein standards on 96-well polystyrene capture plates coated with high titer polyclonal Cry3Bb1 capture antibodies. Concentrations of Cry3Bb1 were determined in each sample by extrapolating an optical density reading against the standard curve of pure Cry3Bb1 protein and reported in part per million (ppm, µg g–1), fresh weight basis (i.e., µg Cry3Bb1 per gram fresh weight tissue extracted). Expression results were averaged for each hybrid at both sample times and reported on a ppm, fresh weight basis. Analysis of variance (ANOVA) was performed by SAS-JMP (version 4.0) statistical software (SAS Institute, Inc., Cary, NC) for variability in expression between the hybrid lines and between the growth stages. The main effects used in the ANOVA model were "Hybrid" and "Growth Stage."

Insect Bioassay
Two CRW efficacy protocols were employed to test for CRW larval feeding control. The first insect assay was conducted under controlled conditions in a growth chamber. Fifteen potted plants for the five hybrids described in the ELISA analysis, plus negative control lines, were infested with 1200 eggs into the root zone at the V3 growth stage. Plant roots systems were evaluated 6 wk after infestation. All roots in both studies were evaluated by the Iowa 1-6 root damage rating system (Hills and Peters, 1971) and evaluated using SAS-JMP 4.0. Mean separations were conducted by Tukey's HSD test (Tukey, 1953) with a P level of 0.05.

A single MON863 transgenic hybrid line was field tested at nine locations in 1999, 10 locations in 2000, and 12 locations in 2001. These trials were conducted throughout the corn belt including Illinois, Iowa, Nebraska, and Missouri. In these trials, the transgenic hybrid was compared to a nontransgenic line of similar genetic background and to Force3G (Syngenta, Basel, Switzerland) [tefluthrin: 2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1RS,3RS)-3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate], a common insecticide used by corn growers for control of larval CRW damage. Each field trial was artificially infested with approximately 1400 D. virgifera virgifera eggs per 30.5 cm of row at the V2 toV3 growth stage. Tefluthrin was applied in-furrow at the labeled rate (5 oz/1000 ft) simultaneously with the nontransgenic seed. A nontransgenic line served as the negative control. Each plant root was rated at the V10 growth stage by the Iowa root damage rating (RDR) system (Hills and Peters, 1971).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Molecular Characterization
The number of introduced insertion sites in event MON863 was evaluated by digesting genomic DNA from both MON863 and the negative control (MON846) with NdeI, a restriction endonuclease that does not cleave within the DNA fragment from plasmid PV-ZMIR13 used for transformation. The Southern blot, probed with MluI fragment (Fig. 1, Panel A), indicated that MON863 contained one band at approximately 5.0 kb (Fig. 2A , Lanes 2 and 5). As the linear DNA fragment from plasmid PV-ZMIR13 used for transformation was 4.7 kb, this result established that MON863 contained one DNA insertion located on a 5.0-kb NdeI restriction fragment.



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  		Fig. 2. Southern blot analysis for insert and copy number. Ten micrograms of MON846 DNA (A1xA634) (lane 1) and MON863 DNA (Lanes 2 and 5) were digested with NdeI (Panel A) or EcoRV (Panel B). Plasmid PV-ZMIR13 DNA (Lanes 3 and 4) was spiked into 10 µg of MON846 DNA and digested with NdeI and EcoRV (Panel A) or EcoRV (Panel B). High molecular weight DNA ladder (Gibco BRL, Gaithersburg, MD) was loaded on the long run and molecular weight markers II and IX (Roche, Indianapolis, IN) were loaded on the short run in both panels. The arrows denote the sizes obtained from the molecular weight markers on the ethidium-stained gel.

 
The single linear DNA insert contained only one copy of the transformation vector harboring the Cry3Bb1 gene. The number of copies of the transgenic cassette from plasmid PV-ZMIR13 was determined by digesting event MON863 genomic DNA with the restriction endonuclease EcoRV, an endonuclease that cuts only once in the linear DNA fragment from plasmid PV-ZMIR13 used to generate the event (Fig. 1, Panel A). The Southern blot was probed with the entire PV-ZMIR13 plasmid. The blot results showed that event MON863 produced two bands at approximately 3.7 and 9.6 kb (Fig. 2B, Lanes 2 and 5). EcoRV cleaved only once within the portion of PV-ZMIR13 used for transformation and thus only two bands were predicted in a single copy event. Since only two bands were produced in this analysis, the transgenic event contains only one copy of the transformation cassette from plasmid PV-ZMIR13 at the locus of integration.

The fully integrated nptII and cry3Bb1 genes in MON863 were found to be high fidelity copies of the transformation cassette. The integrity of the nptII and cry3Bb1 coding regions and transgene cassettes were evaluated by digestion with the restriction endonuclease HindIII, which cleaves at the 5' and 3' ends of each of the transgene cassettes. Individual Southern blots with DNA cut by HindIII were probed with the nptII (Fig. 3 , Panel A) or the cry3Bb1 (Fig. 3, Panel B) coding regions. The predicted 1.6-kb band was observed in the transgenic event when probed with the nptII coding region (Fig. 3, Panel A, Lanes 2 and 5) indicating that the nptII transgene cassette and coding region were intact. A band of approximately 3.2 kb was observed in the genomic DNA when digested with HindIII and probed with the cry3Bb1 coding region (Fig. 3, Panel B, Lanes 2 and 5). This band was slightly larger than the predicted 3.0-kb band. Subsequent sequencing of the 3' end of the insert and the flanking genomic DNA indicated that the polylinker sequence containing a HindIII site was truncated by 10 bp. A plant genomic HindIII restriction site was identified approximately 175 bp from the 3' end of the insert (data not shown) accounting for the larger sized band.



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  		Fig. 3. Southern blot analysis for nptII and cry3Bb1 cassette and coding region intactness and plasmid PV-ZMIR13 backbone. Ten micrograms of MON846 DNA (A1xA634) (lane 1) and MON863 DNA (Lanes 2 and 5) were digested with HindIII. Plasmid PV-ZMIR13 DNA (Lanes 3 and 4) was spiked into 10 µg of MON846 DNA and digested with HindIII. The membranes were probed with 32P-labeled full-length nptII coding sequence (Panel A), full-length cry3Bb coding sequence (Panel B), or two backbone probes encompassing the entire backbone sequence except for the nptII coding region (Panel C). High molecular weight DNA ladder (Gibco BRL, Gaithersburg, MD) was loaded on the long run and molecular weight markers II and IX (Roche, Indianapolis, IN) were loaded on the short run in all three panels. The arrows denote the sizes obtained from the molecular weight markers on the ethidium-stained gel.

 
The remaining genetic elements present in the nptII and cry3Bb1 cassettes (Fig. 1A) were also used as probes in Southern blot analyses. All probes generated the predicted size bands of 1.6 or 3.2 kb for the nptII or cry3Bb1 transgene cassettes, respectively (data not shown). The presence of the expected size bands and the lack of additional bands on Southern blots when assessing the integrity of the nptII and cry3Bb1 transgene cassettes supported the conclusion that MON863 contained single, intact nptII and cry3Bb1 transgene cassettes. To further confirm the integrity of the inserted DNA in MON863, the entire insertion of transgenic DNA was sequenced and was determined to exactly match the plasmid sequence.

The plasmid backbone MluI-MluI restriction fragment was not used in the transformation process and was not found in the MON863 event. The backbone consisted of ori-pUC and a second nptII coding region (Fig. 1A). Genomic DNA was digested with the restriction endonuclease HindIII and probed with two overlapping fragments, which encompassed the entire backbone sequence except for the nptII coding region. While the expected size band at 2.6 kb, on the basis of PV-ZMIR13, was present in the plasmid control lanes (Fig. 3C, Lanes 3–4), no hybridizing band was detected in the plant genomic DNA (Fig. 3, Panel C, Lanes 2 and 5), indicating that the MON863 event did not contain any detectable PV-ZMIR13 plasmid backbone sequence.

Cry3Bb1 Expression
The ELISA values generated for the five hybrids at the V4 and V9 growth stages are shown in Table 2. The effect due to "Hybrid" and the interaction of "Hybrid" x "Growth Stage" were not significant (F < 1.90; df = 4; p > 0.11), indicating that all five hybrids exhibited similar expression patterns between the two growth stages. The effect due to "Growth Stage" was significant (F = 66.08, df = 1; p > 0.01), indicating that the expression level of Cry3Bb1 between V4 and V9 varied significantly. There was a general trend for a decrease in root expression as the plant matured from V4 to V9. The average expression at V4 for all five hybrids was 69.8 µg g–1 and the average expression at V9 was 44.0 µg g–1 for an average difference of 25.8 µg g–1.


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  		Table 2. Root damage ratings and Cry3Bb1 expression of MON863 hybrids in a growth chamber study.

 
Insect Bioassays
The same five hybrid lines that were analyzed for Cry3Bb1 expression as described above were also evaluated for root damage in a growth chamber assay in 2000. The efficacy results are also shown in Table 2 (Growth Chamber Test). All five of the transgenic hybrid lines protected the root system significantly better than the unprotected, nontransgenic lines. Thus, the trend for Cry3Bb1 expression to decline from the V4 to V9 growth stage does not negatively affect the larval feeding protection provided by the MON863 event.

The average root damage rating (RDR) across all field locations is summarized in Table 3 by year. The transgenic hybrid and tefluthrin provided significant larval feeding protection when compared to the untreated, nontransgenic line in all 3 yr. In 2001, MON863 performed statistically better than both the untreated, nontransgenic line and tefluthrin. In addition to the root ratings summarized in Table 3, visual differences from CRW larval feeding can also be noted in terms of root damage and plant phenotype (Fig. 4a and 4b , respectively).


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  		Table 3. Summary of root damage ratings for MON863 over 3 years of field trials.

 



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  		Fig. 4. Damage inflicted by CRW larval feeding on MON863 hybrids and conventional corn hybrids. Figure 4a shows the level of plant stunting on the conventional corn hybrid (left three rows) compared to MON863 protected hybrids (right three rows). In Fig. 4b, the left root is a conventional corn hybrid and has been severely damaged while the root on the right side of the frame is protected by event MON863.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Currently, there are several genetically enhanced crop cultivars that resist insect damage by expressing one or more genes from the common soil bacterium, Bacillus thuringiensis. Bacillus thuringiensis produces parasporal inclusions (i.e., crystals) during the stationary and sporulation phases of growth, which contain proteins that are toxic to selected lepidopteran, coleopteran, and dipteran insect species. Thousands of Bt strain isolates have been collected from around the world and several of these strain isolates have been extensively studied and commercialized as active ingredients for biopesticidal products (Baum et al., 1996). These products display selective activity against a number of pests, including dipteran, coleopteran, and lepidopteran insects. Biopesticidal products based on recombinant Bt strains have also been commercialized for use in agriculture since the 1960s. Typically, commercial quantities of these microbes are prepared in large-scale cultures in which the bacteria are allowed to sporulate. The spores and proteins are then formulated for application on crop plants (Bernhard and Utz, 1993).

In 1991, Rupar et al. reported discovery of a novel Bt subspecies kumamotoensis strain that produced a crystal protein with insecticidal activity against the southern corn rootworm (D. undecimpunctata). Donovan et al. (1992) isolated and sequenced the gene encoding this crystal protein, which was designated as CryIIIB2. Following the adoption of standardized nomenclature for identifying Bt crystal proteins, the protein isolated from this strain was renamed Cry3Bb1 (Crickmore et al., 1998). This strain of Bt was later isolated and commercially produced as a biopesticidal foliar spray (Raven Biological Insecticide, Ecogen Inc., Langhorne PA) for the control of Colorado potato beetle [Leptinotarsa decemlineata (Say)] in potato (Solanum tuberosum L.) crops.

This paper describes the development of a transgenic event expressing a variant of the wild-type cry3Bb1 gene from Bt subspecies kumamotoensis that encodes a protein with enhanced insecticidal activity against corn rootworms and is codon-optimized for expression in monocotyledonous plants (English et al., 2000a, 2000b, 2000c, 2000d). The resulting Cry3Bb1 variant is approximately eight times more lethal to corn rootworm larvae than the wild-type protein. A DNA vector containing the variant cry3Bb1 gene was linked to a constitutive plant-expressible promoter and was introduced into embryonic maize cells by microprojectile bombardment (Klein et al., 1989; Gordon-Kamm et al., 1990). Plants were regenerated from the transformed callus tissue and assayed for the presence of Cry3Bb1 protein by ELISA. Maize event MON863 was selected from hundreds of transformation events produced and developed for commercialization. Additional transgenic maize cultivars that produce Bt proteins for corn rootworm control are under development by Dow AgroSciences LLC (Zionsville, IN), and Pioneer Hi-Bred International, Inc. (Johnston, IA) (Moellenbeck et al., 2001).

In this paper, we provide a detailed molecular description of MON863. MON863 was shown to possess the intended genomic elements in their entirety and on further characterization, revealed a single, intact copy of the cry3Bb1 gene located at a single insertion point in the plant genomic DNA. Identification of transgenic events containing a simple, single insert are more easily introgressed and can greatly reduce the technical breeding challenges that occur during segregation and their eventual conversion into elite hybrid germplasm.

The commercial use of transgenic crops expressing Bt insecticidal proteins has increased in recent years because of the numerous advantages they offer over traditional chemical insecticides. These benefits include convenience and reductions in insecticide usage and labor (Payne et al., 2003). The results of a market survey indicate that the operational, yield risk reduction and environmental benefits offered by MON863 hybrids are of value to growers (Alston et al., 2002). These nonpecuniary benefits were valued to at US$17.89/ha by likely adopters of this technology. The use of MON863 also fits well within the integrated pest management (IPM) context, the premise of which relies on growers using all available technologies to manage their agroecosystem (Gray and Steffey, 2002). For example, crop rotation will remain a key cultural strategy for managing rootworm populations in many regions and larval insecticides or seed treatments may be needed on refuge acres. Thus, scouting will be a prudent tactic for growers that wish to optimize CRW management.

This report shows that maize hybrids containing MON863 are more efficacious than soil and seed-applied insecticides in protecting roots from larval feeding damage because the protein is expressed throughout the plant rather than only in a confined area surrounding the root zone. The Cry3Bb1 protein is plant-incorporated, it does not require activation (as many conventional insecticides do), and its performance is more consistent than current commercially available options. Varieties containing the Cry3Bb1 protein can also be combined through conventional breeding with other genetically enhanced maize varieties, such as those with herbicide tolerance or lepidopteran insect protection, providing an additional option for insect control.


   ACKNOWLEDGMENTS
 
We thank Bei Zhang, James Roberts, Barbara Isaac, Greg Brown, Michael Pleau, and Ellen Rigden for their valuable assistance in the characterization of event MON863.

Received for publication May 19, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


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