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

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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.

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Acknowledgements

The authors wish to thank Celia Miller, Jane Liu for their invaluable assistance. Thanks to Drs. John Watson, Ramesh Bhat, Ming-Bo Wang, Chris Helliwell and Michael Ayliffe for their critical reading. This work was supported by Rural Industries Research and Development Corporation (RIRDC), Australia and New South Wales Agricultural Genomic Center, Australia.

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  1. CSIRO Plant Industry, GPO Box 1600, 2601, Canberra, ACT, Australia

    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

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  1. Narayana M. Upadhyaya
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  2. Qian-Hao Zhu
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Correspondence to Narayana M. Upadhyaya.

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Upadhyaya, N.M., Zhu, QH., Zhou, XR. et al. Dissociation (Ds) constructs, mapped Ds launch pads and a transiently-expressed transposase system suitable for localized insertional mutagenesis in rice. Theor Appl Genet 112, 1326–1341 (2006). https://doi.org/10.1007/s00122-006-0235-0

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