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Agrobacterium tumefaciens Transformation and Cotransformation Frequencies of Arabidopsis thaliana Root Explants and Tobacco Protoplasts

June 1998 , Volume 11 , Number  6
Pages  449 - 457

Sylvie De Buck , Anni Jacobs , Marc Van Montagu , and Ann Depicker

Laboratorium voor Genetica, Department of Genetics, Flanders Interuniversity Institute for Biotechnology, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

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Accepted 16 February 1998.

In view of the recent finding that different T-DNAs tend to ligate and integrate as repeats at single chromosomal positions, the frequency of transformation and cotransformation was determined during cocultivation of Arabidopsis thaliana root explants and Nicotiana tabacum protoplasts with two Agrobacterium strains. The transformation frequency of unselected A. thaliana shoots was lower than 1% whereas that of cocultivated tobacco protoplasts was approximately 18%. The cotransformation frequencies, defined as the frequencies with which cells transformed with a first T-DNA contained a second unselected T-DNA, were approximately 40% reproducible, irrespective of the selection, the transformation frequency, and the plant system used. Extrapolation of these results suggests that at least two independently transferred T-DNAs were present in 64% of the transformed plant cells. Molecular analysis of cocultivated N. tabacum shoots regenerated on nonselective medium showed that only a few transformants had a silenced (2/46) or truncated (1/46) T-DNA. Therefore, most integrated T-DNAs expressed their selectable or screenable markers in primary transgenic plants. Remarkably, 10 to 30% of the selected A. thaliana shoots or progenies lost the T-DNA marker they were selected on. As these regenerants contained the unselected T-DNA with a high frequency (17%), these selected plants might result from the expression of unstable, transiently expressed T-DNAs. In conclusion, a significant part of the T-DNAs is lost from the transformed cells.

Additional keywords: gene silencing, T-DNA transfer, transgene expression.

© 1998 The American Phytopathological Society