In Vitro Cellular & Developmental Biology - Plant

, Volume 36, Issue 6, pp 456–463

The use of the two T-DNA binary system to derive marker-free transgenic soybeans

  • Aiqiu Xing
  • Zhangyuan Zhang
  • Shirley Sato
  • Paul Staswick
  • Tom Clemente
Article

DOI: 10.1007/s11627-000-0082-7

Cite this article as:
Xing, A., Zhang, Z., Sato, S. et al. In Vitro Cell.Dev.Biol.-Plant (2000) 36: 456. doi:10.1007/s11627-000-0082-7

Summary

A binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101. Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate. Transgenic soybeans were grown to maturity in the greenhouse. Fifteen primary transformants (T0) representing 10 independent events were characterized. Seven of the 10 independent T0 events co-expressed GUS. Progeny analysis was conducted by sowing the T1 seeds and monitoring the expression of the GUS gene after 21 d. Individual T1 plants were subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a 100 mgl−1 solution of glufosinate and scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10 independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensitive individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent segregation in progeny is a viable means to obtain soybeans that lack a selectable marker.

Key words

Agrobacterium tumefaciens transformation bar glufosinate Glycine max 

Copyright information

© Society for In Vitro Biology 2000

Authors and Affiliations

  • Aiqiu Xing
    • 1
  • Zhangyuan Zhang
    • 2
  • Shirley Sato
    • 1
  • Paul Staswick
    • 3
  • Tom Clemente
    • 3
  1. 1.Center for BiotechnologyUniversity of Nebraska-LincolnUSA
  2. 2.Department of AgronomyIowa State UniveristyUSA
  3. 3.Department of AgronomyUniversity of Nebraska-LincolnUSA
  4. 4.Department of AgronomyPlant Transformation Core Research Facility, E324 Beadle CenterLincolnUSA

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