Inactivation of the nopaline synthase gene by double transformation: reactivation by segregation of the induced DNA |
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Authors: | Toru Fujiwara Philip A Lessard Roger N Beachy |
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Institution: | (1) Department of Biology, Washington University, 63130 St. Louis, MO, USA;(2) Present address: Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Yayoi, Bunkyo-ku, 113 Tokyo, Japan;(3) Present address: Division of Plant Biology, Department of Cell Biology MRC-7, The Scripps Research Institute, 10666 No. Torrey Pines Road, 92037 La Jolla, CA, USA |
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Abstract: | Tobacco plants were transformed with derivatives of a binary vector pMON505 and two kanamycin resistant lines that were nopaline positive were selected for second transformation. The plasmids used for the second transformation were derivatives of pMON850 which carries the nopaline synthase gene in addition to a gene for gentamicin resistance. Insertion of each transgene was confirmed by Southern hybridization. Surprisingly, we found that more than 50% of the doubly transformed tobacco plants were nopaline negative. Tobacco plants that were transformed only by the second vector exhibited nopaline accumulation. DNA methylation patterns at the HpaII site in the promoter region of the nopaline synthase gene did not correlate with the nopaline phenotype. In some plant lines, seedlings of the R1 generation which segregated out the second T-DNA insertion recovered the nop+ phenotype. These results indicate that nopaline accumulation was inhibited by the presence of the second T-DNA.Abbreviations T-DNA
transferred DNA
- NPTII
neomycin phosphotransferase II
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uidA
-glucuronidase
- Km
kanamycin
- Gm
gentamicin
- nop+
nopaline positive
- nop–
nopaline negative
- MS
medium, Murashige-Skoog medium |
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