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Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss

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Abstract

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells fromAgrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

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Authors and Affiliations

  1. Plant and Microbial Sciences Department, University of Canterbury, Private Bag 480, Christchurch, New Zealand

    Richard Weld & Jack Heinemann

  2. New Zealand Institute for Crop and Food Research, Private Bag, Christchurch, New Zealand

    Colin Eady

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  1. Richard Weld
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  2. Jack Heinemann
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  3. Colin Eady
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Weld, R., Heinemann, J. & Eady, C. Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss. Plant Mol Biol 45, 377–385 (2001). https://doi.org/10.1023/A:1010798625203

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