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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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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DOI: https://doi.org/10.1007/BF02669849