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Development and Characterization of a CP4 EPSPS-Based, Glyphosate-Tolerant Corn Event

Monsanto Co., 700 Chesterfield Parkway West, Chesterfield, MO 63017

^* Corresponding author (gregory.r.heck{at}monsanto.com)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
5-Enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4 (CP4 EPSPS) confers tolerance to the nonselective herbicide glyphosate (marketed under the trade name Roundup¹) when sufficiently expressed in transgenic plants. Dual CP4 EPSPS transgene cassettes were transformed into corn (Zea mays L.) under the transcriptional regulatory control of the rice (Oryza sativa L.) actin 1 (P-Ract1) and the enhanced Cauliflower mosaic virus 35S (P-e35S) promoters, respectively, to impart fully constitutive expression in corn. Resulting events were tested for lack of chlorosis and malformation injury after two sequential applications of 1.68 kg acid equivalents (a.e.) ha^–1 glyphosate. Agronomic parameters, male fertility, appropriate Mendelian segregation of the trait, plus characteristics of the transgenic integration site were also evaluated. From this selection process, the NK603 event was chosen for commercialization as the event that embodied the most optimal profile of tolerance, agronomics, and molecular characteristics. The NK603 event exhibited high glyphosate tolerance from one transgenic locus bearing a single copy of the dual cassettes integrated into the corn genome with a minimum of target sequence disruption. Trait expression in the NK603 event has remained stable over more than eight generations as shown through tolerance testing, western blots of CP4 EPSPS accumulation, and Southern blot analysis of the transgene.

Abbreviations: a.e., acid equivalents • bp, base pair • CP4 EPSPS, 5-enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4 • ELISA, enzyme-linked immunosorbant assay • fwt, fresh weight • kb, kilo base • PAGE, polyacrylamide gel electrophoresis • PCR, polymerase chain reaction • UTR, untranslated region

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
AGRICULTURAL CROPS tolerant to the herbicide glyphosate (N-phosphonomethyl-glycine) have been planted on increasing numbers of hectares since the introduction of the glyphosate-tolerant 40-3-2 soybean event in 1996 (Padgette et al., 1996; marketed under the Roundup Ready brand¹). By 2000, transgenic glyphosate-tolerant soybean [Glycine max (L.) Merr.] use expanded to more than 16000000 ha in the USA (Carpenter and Gianessi, 2001) plus additional hectares in Argentina and other geographies. Similarly, the introduction of glyphosate-tolerant cotton (Gossypium spp.) and canola (Brassica napus L.) has met with a high level of adoption in the USA. This was attributable to a number of characteristics of the glyphosate weed management system, including nonselective postemergent weed control, crop safety, low toxicity on nontarget organisms, lack of soil mobility, flexibility of application, and cost (Franz et al., 1997). Another feature of this weed management system has been the slow development of resistance in wild plant populations despite glyphosate use for over 28 yr. Currently, only three resistant weed biotypes have been identified [Conyza canadensis (L.) Cronq., Eleusine indica L., and Lolium rigidum Gaud., with a recent report of Lolium multiflorum Lam., which is under investigation], in contrast to herbicides such as acetolactate synthase inhibitors (e.g., chlorsulfuron), where more than 70 resistant species have developed (Heap et al., 2003). The overall effectiveness of glyphosate for weed management in conjunction with herbicide tolerant crops species has also promoted environmentally sound practices such as conservation tillage for the control of soil erosion.

Two key elements needed for the development of commercially viable glyphosate-tolerant crops are a resistant target enzyme and sufficient expression of that enzyme within the transgenic plant—a multifaceted challenge that necessitates understanding quantitative, spatial, and developmental components of gene expression. 5-Enol-pyruvylshikimate-3-phosphate synthase (EPSPS) is the target enzyme for glyphosate inhibition within the aromatic amino acid biosynthetic pathway. Disruption of this pathway not only creates a deficiency in protein synthetic precursors, it also affects many other plant cell components that are derived from intermediates and derivatives of this pathway (e.g., auxins, lignans, flavonoids, anthocyanins, and quinones). Therefore, a crop plant must be engineered with a resistant enzyme to maintain flux through this pathway for uninhibited growth and development. Numerous candidate EPSPS enzymes from native and mutagenized microbial and plant sources were examined in an effort to select an enzyme with high catalytic efficiency in the presence of glyphosate (Ruff et al., 1991; Barry et al., 1992). Agrobacterium sp. strain CP4 EPSPS (CP4 EPSPS) was found to be an exceptional enzyme during this screening process (Barry et al., 1992; Padgette et al., 1995) and is the transgenic EPSPS protein produced in glyphosate-tolerant Roundup Ready soybean, cotton, and canola. Transgenic expression of CP4 EPSPS within these crops provided the appropriate support to the aromatic amino acid biosynthetic pathway without negative impact on yield, compositional qualities, and nutritional value of the harvested product (Delannay et al., 1995; Hammond et al., 1995; Harrison et al., 1996; Padgette et al., 1995; Nida et al., 1996b).

The second component to successfully engineer herbicide tolerance is expression of a resistant enzyme in all cell types that receive a significant dose of the herbicide and require a functional aromatic amino acid pathway. Glyphosate is a phloem-mobile herbicide that translocates from source tissues such as mature leaves to sink tissues in much the same way that photosynthates such as sucrose are mobilized within the plant. When corn was treated with glyphosate, the herbicide was delivered to meristematic and young developing organs of the apex, roots, and inflorescences (Hetherington et al., 1999). As a result, high glyphosate concentrations occurred where a fully functional aromatic amino acid pathway was most needed. Heterologous promoters and other regulatory elements (i.e., nonnative sequences which can direct transcription and transcript accumulation) have typically been used to provide constitutive expression at stoichiometrically higher levels than the endogenous target enzymes. This ensures a broad margin of safety for the crop plant bearing the tolerance trait. Expression must also be stable under many different environmental regimes that include a variable matrix of stress and favorable growth parameters. Failure to meet these expression criteria in even a few cell types could result in morphological defects, chlorosis, sterility, and/or perceptible yield loss in the engineered crop in response to glyphosate application.

In this report, we present data on the development of the glyphosate-tolerant corn event, NK603. This represents the extension of CP4 EPSPS transgene utility demonstrated in other crops into this major agricultural species. Selection of the NK603 event was based on a combination of factors including glyphosate tolerance, agronomic characteristics, segregation, and molecular integration profile resulting in a event which meets rigorous field use requirements and grain quality standards of the harvested product (USDA, 2000, and references therein). Data used in the selection process are presented. Additional yield and tolerance trial data taken at multiple locations from the NK603 event (manuscripts in preparation) will be presented in a separate manuscript to complement the early characterization work presented here.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Production of Glyphosate Tolerant Corn Events
As with glyphosate tolerant soybean (Padgette et al., 1995) and cotton (Nida et al., 1996a), transgenic cassettes were created for monocot expression from a combination of promoter, transit peptide sequence, CP4 EPSPS, and transcriptional termination sequences. For the PV-ZMGT32 plasmid used to generate the NK603 event, two cassettes were tandemly joined in a head-to-tail orientation within a pUC119-derived (Vieira and Messing, 1987) high-copy plasmid backbone bearing a kanamycin selectable marker (neomycin phosphotransferase II, nptII) for maintenance in bacteria. The first transgenic cassette combined the rice actin 1 promoter and intron (McElroy et al., 1990) to transcriptionally regulate a single open reading frame consisting of the Arabidopsis thaliana EPSPS chloroplast transit peptide (Shah et al., 1986) linked to the Agrobacterium sp. CP4 EPSPS sequence (Barry et al., 1992). The 3' nontranslated region of the nopaline synthase gene from Agrobacterium tumefaciens T-DNA borders the 3' end of the first transgenic cassette to direct appropriate processing of RNA transcripts (Fraley et al., 1983). The structure of the second transgene cassette is the same as the first, except the rice actin 1 promoter and intron have been replaced by the enhanced Cauliflower mosaic virus promoter (Kay et al., 1987) and maize hsp70 intron (Brown and Santino, 1994), respectively.

PV-ZMGT32 was digested with MluI to liberate an approximately 6.7-kb fragment containing both CP4 EPSPS cassettes. The vector backbone was purified away from this fragment by preparative gel electrophoresis. Precultured maize embryos (Songstad et al., 1996) from a Hi-II (A188 x B73 derivative cross, Armstong et al., 1991) x FBLL (commercial inbred) cross were bombarded with gold particles coated with the purified MluI fragment of PV-ZMGT32 with a helium-driven particle accelerator. DNA was precipitated onto the gold particles using a calcium chloride–spermidine protocol, essentially as described by Klein et al. (1988). Selection of glyphosate-tolerant regenerating calli was accomplished on N6 medium containing 3 mM glyphosate (described in Howe et al., 2002). As plantlets regenerated and rooted, they were removed to soil and transferred to the greenhouse. One hundred-seven resistant callus events were produced that were regenerated into 87 plantlets for further testing under greenhouse conditions. Once in the greenhouse, plants were sprayed with 1.68 kg a.e. ha^–1 glyphosate (twice the typical field use rate) at approximately the V4 to V5 leaf stage (staging equivalent to seed-grown plants with four to five leaves sufficiently mature to each have an exposed ligule and flared auricles). Plants were visually scored for chlorosis and vegetative malformation 10–14 d after treatment. Forty-two events which exhibited <10 to 15% visually estimated leaf chlorosis and vegetative malformation were advanced to an out-cross with the public maize inbred, B73, to generate B73 BC0F1 testing material.

Field Evaluations
Trials were planted in a split block design (6-m-long rows spaced 0.76 m apart; approximately 6 seeds/m) with two replications per location. Main plots received the indicated rates of glyphosate using the Roundup Ultra formulation applied in a carrier volume of approximately 94-187 L ha^–1. Subplots were defined by event. Tolerance was scored 10 to 14 d after each glyphosate treatment. Glyphosate injury was evaluated as chlorosis and malformation monitored relative to untreated control plants grown under weed free conditions. Visual estimation of these agronomic characteristics at <10% of untreated controls was considered a minimum criterion for further advancement in the event selection process. Other agronomic characters were monitored at appropriate times to evaluate equivalency of the advancing events with and without glyphosate treatment.

Immunolocalization
Anthers were dissected from immature corn tassels at the microspore mother cell stage and fixed in 4% formaldehyde overnight. Tissue was dehydrated through a graded ethanol series (35, 50, 75, 80, 95, and 100%, v/v) and then imbedded in plastic resin according to manufacturer's instructions (ImmunoBed Kit; Polysciences, Inc., Warrington, PA). Five-micrometer sections were cut and fixed on glass slides coated with poly-glycine. General morphology was examined by staining with Toluidine Blue O. Immunolocalization of CP4 EPSPS proteins in tissue sections was performed by established conditions (Ryerse et al., 1997) with goat antiserum to CP4 EPSPS as the primary antibody (1: 100000 dilution). Nonimmunized goat serum, also diluted 1:100 000, was used as the equivalent of a pre-immune primary antibody control. Positive detection of CP4 EPSPS was accomplished by development with biotin–conjugated rabbit antigoat antibodies followed by peroxidase-conjugated streptavidin, and finally color development with freshly made AEC substrate (Zymed, San Francisco, CA) (Ryerse et al., 1997). Sections were examined with a Zeiss Axioplan microscope (Carl Zeiss, Inc., Thornwood, NY) and images were captured with a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI).

Molecular Characterization
Corn genomic DNA was isolated (Saghai-Maroof et al., 1984) and quantified by fluorimetry. Restriction digestion, gel electrophoresis, Southern blotting, and hybridization to radiolabeled probes were completed according to standard procedures (Sambrook et al., 1989). Both short and long electrophoresis runs were done to allow optimal resolution of small and large restriction fragments, respectively, before blotting. Templates for radioactive probe synthesis were prepared from gel-purified restriction fragments of PV-ZMGT32 or generated by PCR using oligonucleotides that anneal at the ends of the respective element. Probes were labeled by means of the Klenow fragment of DNA polymerase I, random oligomers, and ³²P-dCTP (RadPrime DNA Labeling System, Invitrogen Life Technologies, Carlsbad, CA). After hybridization, blots were washed at increasing stringency with the final wash being 0.1x SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0), 0.5% (w/v) sodium dodecyl sulfate (SDS) at 68°C. Multiple exposures of the blot were generated with Kodak Biomax MR film in conjunction with a Kodak Biomax MS intensifying screen (Eastman Kodak; Rochester, NY) including many-fold overexposure to permit detection of faint signals.

Genomic sequence flanking the NK603 integration site was cloned using the GenomeWalker kit (BD Biosciences Clonetech, Palo Alto, CA) according to manufacturer's instructions. Two nested primer sets were designed for the 5' and 3' ends of the transgene in the P-Ract1 and nopaline synthase transcriptional terminator, respectively. The identity of cloned PCR products and their relationship to the MluI fragment of PV-ZMGT32 was confirmed by dye-terminator sequencing and alignment with vector sequences with the sequence analysis software (DNASTAR, Inc., Madison, WI). Analysis also included homology searches to investigate the nature of the cloned flanking information. The BLAST algorithm (version 2.0, Althschul et al., 1997) was used to assess homology and look for significant matches to known sequences (e.g., GenBank nonredundant nucleotide database; Benson et al., 2000), at both the nucleotide (BLASTN) and amino acid level (TBLSTX).

PCR reactions used approximately 50 ng of corn genomic DNA, 0.1 µM of the appropriate oligonucleotide primers, 0.2 µM deoxyribonucleotides, 1x REDTaq reaction buffer containing magnesium (1.5 mM), and 1.5 units of REDTaq thermostable DNA polymerase (Sigma, St. Louis, MO) in a 50 µL volume. Oligonucleotide primers used were as follows: A: 5'-TGACGTATCAAAGTACCGACAAAAACATCC-3'; B: 5'-CCTTTGTTTTATTTTGGACTATCCCGACTC-3'; C: 5'-AGATTGAATCCTGTTGCCGGTCTTGC-3'; D: 5'-GCGGTGTCATCTATGTTACTAGATCGGG-3'; E: 5'-CAGCATCAGCGCTCGAAAGTTTCGTCAA-3'; F: 5'-GGCAGGGTGTTGTTGTCCATTTTATGG-3'. The following cycling conditions were used: 1 cycle of 95°C for 2 min; 36 cycles of 94°C for 30s, 60°C for 30 s; 72°C for 1 min; followed by 1 cycle of 72°C for 10 min (using calculated temperature control in an MJ Research PTC-200 DNA Engine thermocycler (MJ Research, Inc., Reno, NV). PCR products were examined by agarose gel electrophoresis.

ELISA Assays and Western Analyses for CP4 EPSPS
ELISAs to quantify CP4 EPSPS protein were done essentially as previously described (Padgette et al., 1996). For forage and leaf samples, an extraction was done at a tissue to buffer ratio of 1:50 and grain at a 1:100 tissue to buffer ratio. For western analysis, extracts were made by homogenizing 0.1 g of frozen leaf tissue in 10 mL of 10 mM KH₂PO₄, 0.1 mM Na₂HPO₄, 1.37 M NaCl, 27 mM KCl, 0.5% (v/v) Tween-20, and 0.1% (v/v) bovine serum albumin, pH 7.4 (PBST+0.1% BSA). Samples were filtered and diluted 1:2 (v/v) in PBST+0.1% BSA before adding an equal amount of 2x Laemmli sample buffer (Laemmli, 1970) and heated to 100°C for 5 min. Proteins were separated on 4 to 20% (w/v) tris-glycine gradient gels (Invitrogen Life Sciences) and transferred to a PVDF membrane (Immobilon-P, 0.45 µM; Millipore, Corp., Billerica, MA). A 1:5000 dilution of primary polyclonal antibodies isolated from CP4 EPSPS goat antiserum was used, followed by a 1:10 000 secondary detection antibody of rabbit anti-goat IgG conjugated to horseradish peroxidase (Sigma). For specific detection of CP4 EPSPS L214P (a form of CP4 EPSPS found in the P-e35S cassette of NK603 that was created by spontaneous mutation during transformation), rabbit antiserum was prepared by immunization with a synthetic peptide of the CP4 EPSPS region at position P214. Western analysis CP4 EPSPS L214P was performed as above, with the exception that the serum was used at a 1:1000 dilution. Immunoreactive bands were visualized on X-ray film with the Enhanced Chemiluminescent Kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) according to manufacturer's instructions. Estimation of the relative contribution of CP4 EPSPS L214P to total CP4 EPSPS accumulation was determined by purifying to 90% homogeneity total CP4 EPSPSs from grain and leaf, and by determining the proportion of CP4 EPSPS L214P immunoreactive to the specific rabbit antibody relative to the total amount of CP4 EPSPSs loaded onto SDS-PAGE before transblotting.

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Development of Expression Elements for Glyphosate Tolerance in Corn
Corn transformations with the CP4 EPSPS transgene began in 1989 and over the next several years, a number of transgenic vectors were created to optimize expression of CP4 EPSPS via promoter, 5'UTR intron and synthetic coding sequence combinations to allow efficient selection during transformation and concomitant glyphosate tolerance in regenerated plants. Many vectors utilized the enhanced Cauliflower mosaic virus 35S promoter (P-e35S) because of its strong and constitutive expression pattern. In some cases, secondary cassettes expressing glyphosate oxidoreductase (GOX, Barry and Kishore, 1995) and N-acyl-phosphonotransferase (phnO, Barry, 1999) were also used in an effort to enhance glyphosate tolerance. Experimentation showed that expression of CP4 EPSPS was sufficient to convey glyphosate tolerance in transgenic corn plants (data not shown).

From these early efforts, corn events expressing CP4 EPSPS were recovered with high vegetative tolerance to glyphosate. However, there was limited recovery of fully male-fertile transgenic events at commercial rates of glyphosate application (0.84 kg a.e. ha^–1). The window of reproductive sensitivity noted in the field and histochemical analyses on greenhouse-grown material indicated that early male reproductive development was impaired in P-e35S/CP4 EPSPS transformants treated with glyphosate. Immunolocalization of CP4 EPSPS in anther sections revealed little to no accumulation in the tapetum that nourishes the developing pollen and the microspore mother cells that are antecedents to pollen, despite presence of CP4 EPSPS in surrounding anther tissue (endothecium, epidermis and connective/vascular tissue) (Fig. 1 , Panel C). The P-e35 expression deficit apparently allowed glyphosate to damage critical cell types during anther development, most notably in the sporophytic tapetum and early stages of gametogenesis leading to male sterility.

View larger version (44K): [in this window] [in a new window]   Fig. 1. Immunolocalization of CP4 EPSPS in developing corn anthers at the microspore mother cell stage of development. Panel A: Toluidine Blue O stain of a transverse section through a developing anther showing one microsporangium. Panel B: Control section treated with pre-immune antiserum as primary antibody. Panel C: Section of a P-e35S/CP4 EPSPS event treated with anti-CP4 EPSPS polyclonal antiserum as primary antibody. Panel D: Section of P-Ract1/CP4 EPSPS event treated with anti-CP4 EPSPS polyclonal antiserum as primary antibody. Positive detection is indicated by development of dark punctate reaction product. M: microspore mother cell. T: tapetum cell layer. Size bar = 20 µm.

Selection of the NK603 event
In 1996, a number of transgenic constructs were created to complement the 35S promoter deficiency in corn and enhance overall glyphosate tolerance. Over 1300 events in total were created in this second transformation series using a variety of transgenic components and configurations. One construct tested was PV-ZMGT32 (Fig. 2) that carried two CP4 EPSPS transgene cassettes, driven by the P-Ract1 and P-e35S promoters, respectively (Table 1). It was created to encompass the regulatory capacity of both promoters and generate glyphosate-tolerant events with full vegetative and reproductive tolerance. PV-ZMGT32 transformation gave rise to a number of highly glyphosate-tolerant transgenic R0 corn events that were advanced to field evaluation.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Plasmid map of PV-ZMGT32. MluI fragment used in transformation of NK603 is indicated by heavy darkline. Vector backbone (hatched segment) was purified away before bombardment. ori (microbial origin of replication); NPTII (neomycin phosphotransferase). Restriction enzyme sites for EcoRV, MluI and MscI are shown.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Schematic diagram of NK603 B73 breeding lineage. Generations referenced in development of the NK603 glyphosate-tolerant corn event are shown. Material used in primary characterizations for regulatory submissions is circled. LH59, and LH82 are commercial inbreds used to make hybrid testing material.

All generations segregated as expected for a single active transgenic integration, except for the BC2F1 generation. The excess of positive plants in the BC2F1 generation may have resulted from gamete selection due to high application rates of glyphosate in the generation before the BC2F1 (i.e., BC1F1). Preferential selection for positive gametes in hemizygous herbicide resistant transgenic plants has been demonstrated following applications of a selective herbicide (Spencer et al., 1998; Conner, 1995). Otherwise, the Chi square segregation analyses are consistent with a single active site of integration for the PV-ZMGT32 transgene into the corn genomic DNA of event NK603. Additionally, the functional stability of the insert has been demonstrated through six generations of crossing and three generations of self-pollination.

CP4 EPSPS Characterization in Transgenic Maize Events
The CP4 EPSPS expressed in the NK603 event was analyzed at a level of nucleotide sequence. Sequencing of the respective CP4 EPSPS coding regions from the P-Ract1 and P-e35S cassettes of the NK603 event revealed two nucleotide changes in the P-e35S CP4 EPSPS coding region. One of these changes lead to a silent codon substitution. The other converted a leucine codon to a proline codon at position 214 (CP4 EPSPS L214P) of the 455 amino acid CP4 EPSPS polypeptide. The P-Ract1 coding region remained unaltered so that two slightly different CP4 EPSPS sequences were produced in the NK603 event. Neither nucleotide change was present in the originating PV-ZMGT32 plasmid but was assayable in archived BC0F1 material and was stably maintained in subsequent generations. MALDI-TOF (Matrix Assisted Laser Desorption and Ionization–Time of Flight) mass spectrometry of purified total CP4 EPSPS from grain also confirmed presence of both protein sequences in NK603 (George et al., manuscript in preparation). The observed change did not affect the active site of the CP4 EPSPS L214P and assays of CP4 EPSPS L214P biochemical activity show it is indistinguishable from CP4 EPSPS (George et al., manuscript in preparation). CP4 EPSPS and CP4 EPSPS L214P also exhibit a common digestibility and immunoreactivity with polyclonal antisera (antiserum raised specifically to the L214P peptide can distinguish the L214P CP4 EPSPS), thus being equivalent in the assessments performed on the NK603 event and prior studies of CP4 EPSPS (Fuchs and Astwood, 1996; Harrison et al., 1996). Corn hybrids containing event NK603 have been shown to be nutritionally equivalent to traditional corn hybrids by direct evaluations of key nutrients (Ridley et al., 2002) and by evaluation in animal feeding studies in broiler chickens (Taylor et al., 2001, 2003), swine (Fisher et al., 2002), cattle (Ipharraguerre et al., 2003), and rodents (Hammond et al., 2002).

ELISA assays (Table 4) and western blot analyses (Fig. 4 and 5) were performed to evaluate accumulation of CP4 EPSPS protein in corn tissues produced by PV-ZMGT32 transformation. As illustrated in the V4 leaf sample, the NK603 event exhibited a midrange CP4 EPSPS accumulation level among four representative events advanced into field evaluations during the summer of 1998. In the NK603 event, these analyses commonly detect CP4 EPSPS and CP4 EPSPS L214P. Steady state levels of transgenic protein in the NK603 event varied by organ and developmental stage with grain accumulating less transgenic protein than leaf or forage material (Table 4). Utilizing the antibody developed for specific detection of CP4 EPSPS L214P, western blotting and subsequent densitometry suggested that CP4 EPSPS L214P accumulation was approximately 30% of the combined total CP4 EPSPS proteins in grain and leaf (Fig. 5). Overall, total accumulation of CP4 EPSPS in event NK603 was lower than that observed in the 40-3-2 glyphosate tolerant soybean event, where 415 to 443 µg/g fwt for leaf and 201 to 288 µg/g fwt for seed was measured (Padgette et al., 1995), although both events meet commercial tolerance specifications. CP4 EPSPS expression also remained stable in the NK603 event over multiple generations sampled (Fig. 5), consistent with glyphosate tolerance observations and Southern analyses of the transgenic insertion.

View larger version (50K): [in this window] [in a new window]   Fig. 4. Western blot estimation of the amount of CP4 EPSPS L214P protein present in the isolated total CP4 EPSPS protein from NK603 grain and leaf tissue. Western blot probed with CP4 EPSPS L214P-specific antibodies. Lanes: 1) molecular weight marker; 2) 4 ng E. coli-expressed CP4 EPSPS; 3) 4 ng E. coli-expressed CP4 EPSPS L214P; 4) 2 ng E. coli-expressed CP4 EPSPS L214P; 5) 1 ng E. coli-expressed CP4 EPSPS L214P; 6) 0.5 ng E. coli-expressed CP4 EPSPS L214P; 7) 4 ng of total CP4 EPSPS purified from grain; 8) 4 ng of total CP4 EPSPS purified from leaves.

View larger version (10K): [in this window] [in a new window]   Fig. 5. Western blot analysis of CP4 EPSPS protein expressed over five generations of NK603. Total protein was extracted from NK603 leaves. Denatured protein was separated by SDS-PAGE, blotted, and visualized by means of a chemiluminescent detection system and CP4 EPSPS antiserum. The corn inbred B73 was used as a recurrent parent in all generational material examined except for hybrid samples which utilized a cross to the LH82 inbred. Lane: 1) molecular weight markers (dye-linked markers visible on blot); 2) 5 ng of E. coli-expressed CP4 EPSPS; 3) 2.5 ng of E. coli-expressed CP4 EPSPS; 4) 1 ng of E. coli-expressed CP4 EPSPS; 5) buffer blank; 6) NK603 B73 BC0F1 7) LH82 x NK603 B73 BC1F1; 8) NK603 B73 BC1F1; 9) NK603 B73 BC5F1; 10) LH82 x NK603 B73 BC2F3; 11) B73 nontransgenic inbred control; 12) LH82 x B73 nontransgenic hybrid control.

Southern analysis was performed on NK603 with probes derived from specific segments of the PV-ZMGT32 plasmid (rice actin 1 promoter, CTP/CP4 EPSPS coding region, e35S promoter, and the nopaline synthase nontranslated region, (Fig. 2). Additional probes (e.g., chloroplast transit peptide and rice actin intron, and vector backbone) were also used in the analysis and were consistent with results presented. All intended transgenic elements were present in the NK603 event and of the correct size relative to the PV-ZMGT32 plasmid (Fig. 6) . Vector backbone sequences (e.g., microbial origin of replication and nptII gene) were not found by Southern blot hybridization using probes spanning the backbone outside the MluI sites (data not shown). Multiple restriction enzyme digestions (additional to the ones shown), short electrophoresis runs before blotting and overexposures of all autoradiograms were completed in addition to the results presented above to confirm that all segments of transgenic DNA, including small subfragments, were accounted for in the analysis. For instance, with this more detailed analysis, a small fragment of P-Ract1 was detectable in the Southern analysis (not visible in Fig. 6, because of its small size, and stringent washing conditions; and <39% G+C content of this 217-bp segment). Sequence determination around the integration site (see below) showed that this fragment was located at the 3' end of the integration. In addition to detecting the anticipated EcoRV restriction enzyme fragments (2.8 and 3.8 kb), all probes localized to a common approximately 8.5-kb MscI fragment. There are no MscI restriction enzyme sites within the PV-ZMGT32 transgene segment, therefore any fragments bearing transgenic segments are generated by digestion of surrounding maize flanking sequence DNA. This provided complementary evidence to phenotypic segregation data that only one functional transgenic locus is present in the NK603 event. Given the unit size of the PV-ZMGT32 transgene segment (approximately 6.7 kb for both CP4 EPSPS cassettes combined), and presence of anticipated EcoRV fragments, only one intact copy is possible at the single locus defined by the approximately 8.5-kb MscI fragment.

View larger version (77K): [in this window] [in a new window]   Fig. 6. Southern analyses of NK603 transgenic elements. Corn genomic DNA was digested with the indicated restriction enzymes, electrophoresed in replicate 0.8% (w/v) agarose gels, transferred to a nylon membrane, hybridized to 32P-labeled segments of PV-ZMGT32, and subjected to autoradiography. Individual lanes: 1) EcoRV digest of 10 µg B73 inbred; 2) EcoRV digest of 10 µg B73 inbred genomic DNA supplemented with 29 pg of PV-ZMGT32; 3) EcoRV digest of 10 µg NK603 B73 BC5S3 genomic DNA; 4) MscI digest of 10 µg of B73 BC5S3 genomic DNA. Panel A, rice Act1 promoter probe; Panel B, CTP/CP4 EPSPS probe; Panel C, e35S promoter; Panel D, NOS transcriptional termination sequence probe. Molecular weight markers are indicated on the side of each autoradiogram (kb, kilobase pairs).

View larger version (34K): [in this window] [in a new window]   Fig. 7. Transgene stability at the NK603 integration site. Corn genomic DNA (10 µg) was digested with EcoRV, electrophoresed in a 0.8% (w/v) agarose gel, transferred to a nylon membrane, hybridized to a 32P-labeled probe from the CTP/CP4 EPSPS portion of PV-ZMGT32, and subjected to autoradiography. Individual lanes: 1, B73 nontransgenic control; 2, LH82 x NK603 B73 BC1; 3, NK603 B73 BC1F1; 4, LH82 x NK603 B73 BC2F3; 5, LH59 x NK603 B73 BC4F4. Molecular weight markers are indicated on the side of the autoradiogram (kb, kilobase pairs).

View larger version (24K): [in this window] [in a new window]   Fig. 8. Schematic diagram of NK603 transgenic locus. The locus includes the 6711-bp MluI fragment from PV-ZMGT32 and cointegrated segments of the P-Ract1 and maize plastid genome. The sense orientation of these cointegrated sequences is indicated below each with an open arrow. Relative positions and orientation of the oligonucleotides used in flanking sequence characterization (A–F) are indicated by arrows.

A second segment of cointegrated DNA with perfect identity to the rpl23 gene cluster of the corn plastid genome (Rapp et al., 1992) is fused downstream of the duplicated portion of P-Ract1. The match spanned a 301-bp portion of the corn plastid open reading frames for rps11 and rpoA (partial segments of each gene corresponding to bases 63–363 of GenBank accession X07810, with open reading frame orientation toward the transgene). As has been seen in a variety of plants such as wheat (Triticum aestivum L.), rice, barley (Hordeum vulgare L., pea (Pisum sativa L.), beet (Beta vulgaris L.), and spinach (Spinacia oleracea L.) (Ayliffe et al., 1998; Rice Chromosome 10 Sequencing Consortium, 2003), numerous portions of DNA of plastid origin lie within the nuclear genomes of higher plants. The sequence homology among the plastid segments can be high and many nuclear equivalents exist. Therefore, it is not possible to determine if the origin of the rps11/rpoA segment at the 3' end of the NK603 integration results from direct plastid genome transfer or incorporation of sequence from elsewhere in the nuclear genome during the process of transformation. As with the P-Ract1 promoter segment, the rps11/rpoA segment is not expected to be a significant contributor to regulation of the transgene or surrounding genomic DNA. First, it lacks the endogenous plastid promoter of the rpl23 gene cluster and second, if cryptic regulatory sequences did exist, they would function poorly in a nuclear context (Cornelissen and Vandewiele, 1989; Scott et al., 1991).

Thus, the entirety of the NK603 transgenic integration is constituted by the MluI fragment from PV-ZMGT32, the 217 bp of duplicated 5' end of the PV-ZMGT32 transformation cassette, and 301 bp of the rps11/rpoA portion of the plastid rpl23 gene cluster. Immediately downstream of the rps11/rpoA segment, 497 bp of true 3' flanking sequence is comprised of novel corn genomic DNA. To verify the 5' and 3' flanking sequences were correctly determined, the site of integration for the NK603 event was cloned from a nontransgenic corn genomic DNA. Using oligonucleotides flanking the transgenic integration (oligonucleotides E and F, Fig. 8), a 237-bp "wild-type" integration allele was amplified by PCR from the nontransgenic B73 inbred and sequenced. The sequence was an exact match to the flanking sequence determined on the respective sides of the integration, with the exception of 3 bp of the target site not represented in either the 5' or 3' cloned flanking genomic sequenced for the NK603 event. Thus, outside of the small deletion of the integration site, the colinearity of the corn genomic sequence surrounding the integration site and the nontransgenic genome was preserved in the NK603 event. The cloned site of integration also did not contain any native plastid sequence indicating that it was introduced during creation of the NK603 event. The understanding of the integration site was further demonstrated by the ability to perform a "zygosity test" with a multiplexed PCR reaction with oligonucleotides D, E, and F (Fig. 9) . In the presence of the NK603 transgenic integration, a 641-bp NK603-specific amplicon was produced, whether a hemizygous or homozygous individual was examined. If the wild-type integration allele was present either in a nontransgenic inbred such as B73, a hemizygous NK603 individual, or a non-NK603 transgenic event, a 237-bp amplicon was evident (a long amplicon spanning the entire transgenic integration is not observed due to short amplification cycles, which bias production of smaller amplicons). All the predicted amplicons were observed when using this multiplexed assay.

View larger version (68K): [in this window] [in a new window]   Fig. 9. NK603 zygosity test. PCR based assay using three oligonucleotides D, E, and F. The schematic diagram (Panel A, not to scale) shows relative placement of oligonucleotides and the expected sizes of amplification products on non-transgenic (Wt) and NK603 templates. The assay was conducted on genomic DNA from a non-transgenic plant (Panel B, lane 3, B73 inbred), a plant hemizygous for the NK603 insertion (lane 4, B73 BC3 composition), a plant homozygous for the NK603 insertion (lane 5, B73 BC2S2 composition), or plants hemizygous for similar but independent transgenic events (Lane 6, event NK560; Lane 7, event NK561; Lane 8, event NK600). Lane 2 is a template-minus control reaction. Lane 1, 100-bp DNA ladder size standard.

	ACKNOWLEDGMENTS

 
We wish to thank the many individuals at Monsanto and our seed company partners who contributed to the development of Roundup Ready® corn and the selection and evaluation of the NK603 event. Among these contributors are individuals in vector development: N. Biest, L. Casagrande, N. Houmard, D. McElroy, and E. Stanton; corn transformation: S. Dozier, D. Genovesi, M, Griffor, B. Haley, T. Kain, A. Lamke, T. Lane, M. Neher, and N. Willetts; seed quality: P. Ball, K. Beazley, M. Ford, R. Krieb, L. Lewis, C. Mielke, J. Soteres; and trait development: J. Arroyo, K. Campbell, N. Probst, J. Rhew, R. Schulze, M. Spaur, and L. Velazquez; plus guidance and counsel: C. CaJacob, T. Carrato; D. Hoerner, C. Lawson, C. Mackey, T. McBride, and S. Padgette.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
¹ Roundup Ready and Roundup herbicide are trademarks of the Monsanto Company.

Received for publication December 16, 2002.

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