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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

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.

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.

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.

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.

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.