Suppression of transfer of non-T-DNA ‘vector backbone’ sequences by multiple left border repeats in vectors for transformation of higher plants ... View Full Text


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Article Info

DATE

2004-10

AUTHORS

Yoshiki Kuraya, Shozo Ohta, Miyuki Fukuda, Yukoh Hiei, Nobuhiko Murai, Kazuyuki Hamada, Jun Ueki, Hidemasa Imaseki, Toshihiko Komari

ABSTRACT

Vectors for transformation of higher plants mediated by Agrobacterium tumefaciens were modified so that one, two or three additional copies of the left border (LB) sequences were inserted close to the original LB of the T-DNA. A gene for β-glucuronidase (gusA) was placed outside the T-DNA to monitor the transfer to plants of 'vector backbone' sequences. The expression of GUS in immature embryos of rice that had been co-cultivated with A. tumefaciens carrying these constructs was around one tenth of that with A. tumefaciens carrying an unmodified control vector. Between 88 and 127 of independent transformants were regenerated from rice tissues infected with A. tumefaciens carrying each of these vectors. The GUS expressors among the rice transformed with the modified vectors were much less frequent than ones among the control transformants, and rate of reduction in the ratio of transgenic plants that expressed GUS was higher than 93%. Detection of a fragment across the LB region by the polymerase chain reaction and the gusA gene by Southern hybridization correlated well with GUS expression. These results indicate that transfer of the 'vector backbone' from the control vectors resulted mainly from inefficient termination of formation of the transfer intermediate of the T-DNA and additional LB sequences effectively suppressed such transfer. This approach is simpler than the strategy to place a 'lethal gene' outside the T-DNA and will likely help produce 'clean' transformants efficiently. More... »

PAGES

309-320

References to SciGraph publications

  • 2002-09. Transposon-mediated generation of T-DNA- and marker-free rice plants expressing a Bt endotoxin gene in MOLECULAR BREEDING
  • 1996-06. Deviating T-DNA transfer fromAgrobacterium tumefaciens to plants in PLANT MOLECULAR BIOLOGY
  • 1999. Methods of Genetic Transformation: Agrobacterium tumefaciens in MOLECULAR IMPROVEMENT OF CEREAL CROPS
  • 2003-07. Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup in THEORETICAL AND APPLIED GENETICS
  • 2002-12. Transgenic rice with reduced glutelin content by transformation with glutelin A antisense gene in MOLECULAR BREEDING
  • 1992-02. Isolation of plant DNA: A fast, inexpensive, and reliable method in PLANT MOLECULAR BIOLOGY REPORTER
  • 1996-06. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens in NATURE BIOTECHNOLOGY
  • 1995-09. Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA in PLANT MOLECULAR BIOLOGY
  • 2003-05. Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics in THEORETICAL AND APPLIED GENETICS
  • 1983-11. Nucleotide sequence of the T-DNA region from theA grobacterium tumefaciens octopine Ti plasmid pTi15955 in PLANT MOLECULAR BIOLOGY
  • 1992-02. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation in PLANT MOLECULAR BIOLOGY
  • 1990-10. Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542 in PLANT CELL REPORTS
  • 2000-02. Evidence of multiple complex patterns of T-DNA integration into the rice genome in THEORETICAL AND APPLIED GENETICS
  • 2000-10. T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation in MOLECULAR BREEDING
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    http://scigraph.springernature.com/pub.10.1023/b:molb.0000047792.77219.bb

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