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Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium
Published: Jul 20, 2011 DOI: 10.21769/BioProtoc.95 Views: 65313
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Abstract
Transient expression in tobacco plant (Nicotiana benthamiana) is used to determine the subcellular location of a protein of interest when tagged with a reporter such as green fluorescent protein (GFP), or to mass produce proteins without making transgenic plants. The root tumor bacteria, Agrobacteria, are used as medium to introduce the target gene expression cassette into benthamiana mesophyll cells.
Keywords: Transient expression
Tobacco
Agrobacterium
Fluorescence
Leaves
Materials and Reagents
- Agrobacterium strain hosting a plant expression construct (usually driven by Cauliflower mosaic virus 35S promoter)
- Healthy Nicotiana benthamiana (N. benthamiana) plants 2-4 weeks
- MES
- MgCl2 stock
- Antibiotics
- Acetosyringone
- LB media with appropriate antibiotics (see Recipes)
- Acetosyringone stock (see Recipes)
- MES-K (see Recipes)
- Resuspension solution (see Recipes)
- Acetosyringone datasheet (Sigma-Aldrich) (see Recipes)
Equipment
- Centrifuge for 50 ml tubes
- Spectrometer
- Syringe
- UV lamp (optional)
- Fluorescence microscope (optional)
- Confocal laser scanning microscope (optional)
Procedure
Category
Plant Science > Plant transformation > Agrobacterium
Biochemistry > Protein > Expression
Molecular Biology > Protein > Expression
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