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Efficient and sensitive assay for T-DNA-dependent transient gene expression

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Abstract

We describe here a very sensitive and reproducible method to detect the efficiency ofAgrobacterium-mediated T-DNA transfer. This method is based on a quantitative assay of β-glucuronidase activity produced in the plant cell upon transfer of T-DNA carrying a specialuidA gene construct. Analysis of the transfer efficiency of a transfer-proficient bacterium compared with that of the same bacterium diluted at different ratios with a transfer-defective bacterium shows a high sensitivity of the β-glucuronidase activity in the plant. Five orders of magnitude in T-DNA transfer efficiency can be covered when the activity is measured combining the fluorimetric MUG assay (for high activity) and the histochemical X-Gluc assay (very sensitive for low activity).

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Abbreviations

T-DNA:

transferred DNA

MUG:

4-methyl-umbelliferyl-glucuronide

Ti-plasmid:

tumor-inducing plasmid

X-Gluc:

5-bromo-4-chloro-3-indolyl-glucuronide

uidA :

gene encoding β-glucuronidase

GUS:

β-glucuronidase. Other abbreviations are defined underMaterials, Solutions

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Authors and Affiliations

  1. Friedrich Miescher-Institut, P.O. Box 2543, CH4002, Basel, Switzerland

    L. Rossi, J. Escudero, B. Hohn & B. Tinland

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  1. L. Rossi
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  2. J. Escudero
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  3. B. Hohn
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  4. B. Tinland
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Rossi, L., Escudero, J., Hohn, B. et al. Efficient and sensitive assay for T-DNA-dependent transient gene expression. Plant Mol Biol Rep 11, 220–229 (1993). https://doi.org/10.1007/BF02669849

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