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Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation

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Summary

TransgenicPetunia hybrida clones harbouring the T-DNA gene2 ofAgrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements. In thePetunia line used, floral variegation is due to the presence of the non-autonomous transposable elementdTph1 at theAn1 locus. The gene2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid. Plant cells that express gene2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds. By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene2 was no longer expressed. Southern analysis, using gene2-specific probes, revealed that in one variant the T-DNA was deleted. For 30 other variants no alteration in gene2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene2. Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene2 sequences were methylated. Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene2 expression in some of the variants. These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene2 in this system.

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  1. Laboratorium voor Genetische Virologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640, St-Genesius-Rode, Belgium

    Suzy Renckens, Henri De Greve, Marc Van Montagu & Jean-Pierre Hernalsteens

  2. Laboratorium voor Genetica, Rijksuniversiteit Gent, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium

    Marc Van Montagu

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  1. Suzy Renckens
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  2. Henri De Greve
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Renckens, S., De Greve, H., Van Montagu, M. et al. Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation. Molec. Gen. Genet. 233, 53–64 (1992). https://doi.org/10.1007/BF00587561

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