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Comparison of Agrobacterium and particle bombardment using whole plasmid or minimal cassette for production of high-expressing, low-copy transgenic plants

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Abstract

Transgene integration complexity in the recipient genome can be an important determinant of transgene expression and field performance in transgenic crops. We provide the first direct comparison of Agrobacterium-mediated transformation (AMT) and particle bombardment using whole plasmid (WP) and excised minimal cassettes (MC), for transformation efficiency, transgene integration complexity and transgene expression in plants. To enable direct comparison, a selectable marker and a luciferase reporter gene were linked in identical configurations in plasmids suitable for AMT or direct gene transfer into sugarcane. Transformation efficiencies were similar between WP and MC when equal molar DNA quantities were delivered. When the MC concentration was reduced from 66 to 6.6 ng per shot, transformation efficiency dropped fourfold, to a level equivalent with AMT in amenable genotype Q117. The highest proportion of transformants combining low copy number (estimated below two integrated copies by qPCR) with expression of the non-selected reporter gene was obtained using AMT (55 %) or MC at low DNA concentration (30 %). In sugarcane, both of these methods yielded high-expressing, single-copy transgenic plant lines at a workable efficiency for practical plant improvement; but AMT is currently limited to a few amenable genotypes. These methods are best coupled with rapid early screens for desired molecular characteristics of transformants, e.g. PCR screens for low copy number and/or transcription of the gene of practical interest.

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Abbreviations

AMT:

Agrobacterium-mediated transformation

CNI:

Copy number index

LUC:

Luciferase

MC:

Minimal cassette

WP:

Whole plasmid

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Acknowledgments

This work was undertaken through a collaboration between CSR Sugar Limited (Sucrogen) and The University of Queensland with support from the Australian Research Council. The authors acknowledge the excellent technical assistance of Andrea Gray, Kuang-Yu Chen, Mona Singh and Kerrin Henderson.

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Authors and Affiliations

  1. The University of Queensland, Hines Plant Science Building, Mansfield Place, Brisbane, QLD, 4072, Australia

    Mark A. Jackson, David J. Anderson & Robert G. Birch

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  1. Mark A. Jackson
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  2. David J. Anderson
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  3. Robert G. Birch
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Corresponding author

Correspondence to Robert G. Birch.

Additional information

Mark A. Jackson, David J. Anderson contributed equally toward this work.

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11248_2012_9639_MOESM1_ESM.eps

Fig. S1 LUC activity profiles across populations of transgenic plants produced using methods explained in Table 1. LUC activity was measured in extracts from young leaf tissue (EPS 263 kb)

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Jackson, M.A., Anderson, D.J. & Birch, R.G. Comparison of Agrobacterium and particle bombardment using whole plasmid or minimal cassette for production of high-expressing, low-copy transgenic plants. Transgenic Res 22, 143–151 (2013). https://doi.org/10.1007/s11248-012-9639-6

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