Theoretical and Applied Genetics

, Volume 112, Issue 7, pp 1326–1341

Dissociation (Ds) constructs, mapped Ds launch pads and a transiently-expressed transposase system suitable for localized insertional mutagenesis in rice

  • Narayana M. Upadhyaya
  • Qian-Hao Zhu
  • Xue-Rong Zhou
  • Andrew L. Eamens
  • Mohammad S. Hoque
  • Kerrie Ramm
  • Ramannee Shivakkumar
  • Kathryn F. Smith
  • Shu-Ting Pan
  • Suzhi Li
  • Kefan Peng
  • Song J. Kim
  • Elizabeth S. Dennis
Original Paper

DOI: 10.1007/s00122-006-0235-0

Cite this article as:
Upadhyaya, N.M., Zhu, QH., Zhou, XR. et al. Theor Appl Genet (2006) 112: 1326. doi:10.1007/s00122-006-0235-0

Abstract

We have developed a transiently-expressed transposase (TET)-mediated Dissociation (Ds) insertional mutagenesis system for generating stable insertion lines in rice which will allow localized mutagenesis of a chromosomal region. In this system, a Ds containing T-DNA construct was used to produce Ds launch pad lines. Callus tissues, from single-copy Ds/T-DNA lines, were then transiently infected with Agrobacterium harbouring an immobile Ac (iAc) construct, also containing a green fluorescent protein gene (sgfpS65T) as the visual marker. We have regenerated stable Ds insertion lines at a frequency of 9–13% using selection for Ds excision and GFP counter selection against iAc and nearly half of them were unique insertion lines. Double transformants (iAc/Ds) were also obtained and their progeny yielded ~10% stable insertion lines following excision and visual marker screening with 50% redundancy. In general, more than 50% of the Ds reinsertions were within 1 cM of the launch pad. We have produced a large number of single-copy Ds/T-DNA launch pads distributed over the rice chromosomes and have further refined the Ds/T-DNA construct to enrich for “clean” single-copy T-DNA insertions. The availability of single copy “clean” Ds/T-DNA launch pads will facilitate chromosomal region-directed insertion mutagenesis. This system provides an opportunity for distribution of gene tagging tasks among collaborating laboratories on the basis of chromosomal locations.

Supplementary material

122_2006_235_MOESM1_ESM.pdf (47 kb)
Supplementary material

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Narayana M. Upadhyaya
    • 1
  • Qian-Hao Zhu
    • 1
  • Xue-Rong Zhou
    • 1
  • Andrew L. Eamens
    • 1
  • Mohammad S. Hoque
    • 1
  • Kerrie Ramm
    • 1
  • Ramannee Shivakkumar
    • 1
  • Kathryn F. Smith
    • 1
  • Shu-Ting Pan
    • 1
  • Suzhi Li
    • 1
  • Kefan Peng
    • 1
  • Song J. Kim
    • 1
  • Elizabeth S. Dennis
    • 1
  1. 1.CSIRO Plant IndustryCanberraAustralia

Personalised recommendations