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Characterization of Soybean Exhibiting High Expression of a Synthetic Bacillus thuringiensis cry1A Transgene That Confers a High Degree of Resistance to Lepidopteran Pests

^a Monsanto Co., 700 Chesterfield Village Pkwy. West, Chesterfield, MO 63017
^b Monsanto Co., 800 N. Lindbergh Blvd., Creve Coeur, MO 63167

View larger version (26K): [in this window] [in a new window]   Fig. 1. Plasmid map of PV-GMBT01. Not I fragment contains the Bt. expression cassette. ori = microbial origin of replication. Either CP4 EPSPS (5-enolpyruvulshikimate-3-phosphate synthase [EPSPS] sequence isolated from Agrobacterium sp. strain CP4) or NPTII (neomycin phosphotransferase) was the selectable marker used in plant transformation. Spectinomycin or streptomycin resistance was used as a bacterial marker (aadA). Restriction enzyme sites for Eco RV and Not I are shown.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Southern blot analysis of R2 transgeneic and control soybean lines to identify copy number. Each lane represents plasmid DNA or 7ug genomic DNA. DNA was digested with EcoRV and subjected to electrophoresis in a 0.8% agarose gel then transferred to a nylon membrane, hybridized to a 32P-labeled probe with PV-GMBT01, and subjected to autoradiography. Each band represents inserted DNA. The soybean line A3237 was the recurrent parent in all material examined: (1) 100pg PV-GMBT01, (2) 50pg PV-GMT01, (3) blank, (4) 851, (5) 859, (6) 862, (7) 863, (8) 726, (9) 781, (10) 1085, (11) 781 isoline, and (12) A3237 recurrent parent (negative control).

View larger version (116K): [in this window] [in a new window]   Fig. 3. Transgene stability of Bt events. Soy genomic DNA (10 µg) was digested with EcoRV, electrophoresed in a 0.8% agarose gel, transferred to a nylon membrane, hybridized to a 32P-labeled probe with PV-GMBT01, and subjected to autoradiography. Individual lanes: (1) Molecular weight markers, (2) A3237 nontransgenic control, (3) 726 R1, (4) 726 R5, (5) 781 R1, (6) 781 R3, (7) 781 R5, (8) 862 R1, (9) 862 R3, and (10) 862 R5. Molecular weight markers are indicated on the side of the autoradiogram.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Western Blot analysis of R5 transgenic and nontransgenic soybean lines. Total protein was extracted from leaves and digested with trypsin. Denatured protein was separated by sodium dodecyl sulfate–polyacrylamide gel electroporesis, blotted, and visualized using Cry1Ac antiserum and chemiluminescent detection system. The soybean line A3237 was the recurrent parent in all material examined: (1) 862, (2) 726, (3) 781, (4) A3237 soybean genotype (negative control), (5) negative segregent for line 862.

View larger version (80K): [in this window] [in a new window]   Fig. 5. Leaf feeding studies using velvetbean caterpillar. Defoliation of (A) transgenic Bt line 781 compared with (B) M-SOY5826 24 h after supplying foliage from these genotypes to third-instar velvetbean caterpillar larvae.