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Nature of stress and transgene locus influences transgene expression stability in barley

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Abstract

Stress and the nature of the transgene locus can affect transgene expression stability. These effects were studied in two, stably expressing, T6 populations of barley (Hordeum vulgare): bombardment-mediated, multi-copy lines with ubiquitin-driven bar and uidA or single-copy lines from Ds-mediated gene delivery with ubiquitin-driven bar alone. Imposing the environmental stresses, water and nutrient deprivation and heat shock, did not reproducibly affect transgene expression stability; however, high frequencies of heritable transcriptional gene silencing (TGS) occurred following in vitro culture after six generations of stable expression in the multi-copy subline, T3#30, but not in the other lines studied. T3#30 plants with complete TGS had epigenetic modification patterns exactly like those in an identical sibling subline, T3#31, which had significant reduction in transgene expression in the T3 generation and was completely transcriptionally silenced in the absence of imposed stresses in the T6 generation. Complete TGS in T3#30 plants correlated with methylation in the 5′UTR and intron of the ubi1 promoter complex and condensation of chromatin around the transgenes; DNA methylation likely occurred prior to chromatin condensation. Partial TGS in T3#30 also correlated with methylation of the ubi1 promoter complex, as occurred with complete TGS. T3#30 has a complex transgene structure with inverted repeat transgene fragments and a 3′-LTR from a barley retrotransposon, and therefore the transgene locus itself may affect its tendency to silence after in vitro culture and transgene silencing might result from host defense mechanisms activated by changes in plant developmental programming and/or stresses imposed during in vitro growth.

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Abbreviations

2,4-D:

2,4-dichlorophenoxyacetic acid

BAP:

6-benzylaminopurine

IBA:

indole butyric acid

MS:

Murishige and Skoog

RWC:

relative water content

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Acknowledgements

The authors thank R. Fessenden for technical assistance, Dr. P. Bregitzer for providing T6 seeds of line T3#30; Dr. T. Koprek for generating three Ds-mediated single-copy lines, S. Rangel for generation advance and providing T6 seeds of the three Ds lines, Dr. S. Zhang for helpful discussions, K. Newcomb for care of the plants and B. Alonso for superb graphics assistance on the figures. The Torrey Mesa Research Institute (TMRI) supported LM; the USDA Cooperative Extension Service through the University of California supported PGL.

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  1. Meira Ziv

    Present address: Department of Agricultural Botany, Hebrew University of Jerusalem, 76100, P.O. Box 12, Rehovot, Israel

Authors and Affiliations

  1. Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley , CA , 94720, USA

    Ling Meng, Meira Ziv & Peggy G. Lemaux

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  1. Ling Meng
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Correspondence to Peggy G. Lemaux.

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Meng, L., Ziv, M. & Lemaux, P.G. Nature of stress and transgene locus influences transgene expression stability in barley. Plant Mol Biol 62, 15–28 (2006). https://doi.org/10.1007/s11103-006-9000-7

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  • DOI: https://doi.org/10.1007/s11103-006-9000-7

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