Journal Article

Sequential Monitoring of Transgene Expression Following <i>Agrobacterium</i>-Mediated Transformation of Rice

Hiroaki Saika, Satoko Nonaka, Keishi Osakabe and Seiichi Toki

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 53, issue 11, pages 1974-1983
Published in print November 2012 | ISSN: 0032-0781
Published online October 2012 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcs135
Sequential Monitoring of Transgene Expression Following Agrobacterium-Mediated Transformation of Rice

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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Although Agrobacterium-mediated transformation technology is now used widely in rice, many varieties of indica-type rice are still recalcitrant to Agrobacterium-mediated transformation. It was reported recently that T-DNA integration into the rice genome could be the limiting step in this method. Here, we attempted to establish an efficient sequential monitoring system for stable transformation events by visualizing stable transgene expression using a non-destructive and highly sensitive visible marker. Our results demonstrate that click beetle luciferase (ELuc) is an excellent marker allowing the observation of transformed cells in rice callus, exhibiting a sensitivity >30-fold higher than that of firefly luciferase. Since we have previously shown that green fluorescent protein (GFP) is a useful visual marker with which to follow transient and/or stable expression of transgenes in rice, we constructed an enhancer trap vector using both the gfbsd2 (GFP fused to the N-terminus of blasticidin S deaminase) and eluc genes. In this vector, the eluc gene is under the control of the Cauliflower mosaic virus 35S minimal promoter, while the gfbsd2 gene is under the control of the full-length rice elongation factor gene promoter. Observation of transformed callus under a dissecting microscope demonstrated that the level of ELuc luminescence reflected exclusively stable transgene expression, and that both transient and stable expression could be monitored by the level of GFP fluorescence. Moreover, we show that our system enables sequential quantification of transgene expression via differential measurement of ELuc luminescence and GFP fluorescence.

Keywords: Agrobacterium-mediated transformation; Green fluorescent protein; Luciferase; Rice; Stable transgene expression; T-DNA

Journal Article.  4289 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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