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Increased Transgene Expression by Breeding and Selection in White Clover

^a The Danforth Center, 975 North Warson Rd, St. Louis, MO 63132
^b The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037
^c Center for Applied Genetic Technologies, University of Georgia, 111 Riverbend Road, Athens, GA 30602-6810

View larger version (43K): [in a new window]   Fig. 1. Transgene expression of T1 plants. Average NPTII concentrations in relation to total soluble protein are shown for T1 plants selected on kanamycin at 200 mg L–1 (samples 13-16), 300 mg L–1 (samples 17-20), and 400 mg L–1 kanamycin (samples 21-24). An untransformed plant (ut) was used as a negative control. Histochemical GUS assay results are denoted below the corresponding sample number and were scored as either positive (+) or negative (–). Vertical bars indicate the standard error.

View larger version (44K): [in a new window]   Fig. 2. Transgene expression analysis over three successive generations. Average NPTII protein concentrations in relation to total soluble protein are shown for 12 T0 plants (samples 1-12); 4 T1 plants selected on 400 mg L–1 kanamycin (samples 21-24); and 28 T2 plants, 11 of which were selected on kanamycin at 300 mg L–1 (samples 25-35), 9 on 400 mg L–1 kanamycin (samples 36-44), and 8 selected on 500 mg L–1 kanamycin (samples 45-52). An untransformed plant (ut) was used as a negative control. Histochemical GUS assay results are denoted below the corresponding sample number and were scored as either positive (+) or negative (–). Vertical bars indicate the standard error.

View larger version (55K): [in a new window]   Fig. 3. Southern blot hybridization of total DNA from T0 plants digested with EcoRI, which cuts only once within the plasmid, outside of any gene cassette, and probed with a 633-bp amplicon of the nptII gene. Plants 5 and 7 appear to be the same.

View larger version (76K): [in a new window]   Fig. 4. Southern blot hybridization of total DNA from T1 plants digested with EcoRI, which cuts only once within the plasmid, outside of any gene cassette, and probed with a 633-bp amplicon of the nptII gene. Plants numbered 21 through 24 survived 400 mg L–1 kanamycin, and were the parents for the T2 plants.

View larger version (45K): [in a new window]   Fig. 5. Southern blot hybridization of total DNA from T2 plants selected on 300 mg L–1 kanamycin, and digested with EcoRI, which cuts only once within the plasmid, outside of any gene cassette, and probed with a 633-bp amplicon of the nptII gene.

View larger version (65K): [in a new window]   Fig. 6. Southern blot hybridization of total DNA from T2 plants selected on 400 mg L–1 kanamycin, and digested with EcoRI, which cuts only once within the plasmid, outside of any gene cassette, and probed with a 633-bp amplicon of the nptII gene.

View larger version (58K): [in a new window]   Fig. 7. Southern blot hybridization of total DNA from T2 plants selected on 500 mg L–1 kanamycin, and digested with EcoRI, which cuts only once within the plasmid, outside of any gene cassette, and probed with a 633-bp amplicon of the nptII gene.