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Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium   

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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

  1. Agrobacterium strain hosting a plant expression construct (usually driven by Cauliflower mosaic virus 35S promoter)
  2. Healthy Nicotiana benthamiana (N. benthamiana) plants 2-4 weeks
  3. MES
  4. MgCl2 stock
  5. Antibiotics
  6. Acetosyringone
  7. LB media with appropriate antibiotics (see Recipes)
  8. Acetosyringone stock (see Recipes)
  9. MES-K (see Recipes)
  10. Resuspension solution (see Recipes)
  11. Acetosyringone datasheet (Sigma-Aldrich) (see Recipes)


  1. Centrifuge for 50 ml tubes
  2. Spectrometer
  3. Syringe
  4. UV lamp (optional)
  5. Fluorescence microscope (optional)
  6. Confocal laser scanning microscope (optional)


  1. Inoculate one single colony of Agrobacterium in 5 ml LB with appropriate antibiotics. Grow overnight at 28-30 °C.
    Note: I usually use 100 μg/ml gentamicin (maintain the virulence of Agrobacterium strain GV3101) and 50 μg/ml spectinomycin (selective marker for shuttle vector) for most of the shuttle vectors.
  2. Use 1 ml of the overnight culture to inoculate 25 ml LB (with same antibiotics, plus 20 μM acetosyringone added after autoclaving and immediately before use) and grow overnight.
  3. Measure the A600 of overnight culture. 
  4. Precipitate the bacteria (5,000 x g, 15 min), resuspend the pellet in Resuspension Solution. The final A600 should be adjusted to 0.4. 
  5. Leave on the bench (room temperature) for 2-3 h (or overnight) before infiltration.
  6. Perform the infiltration with 5 ml syringe. Simple press the syringe (no needle) on the underside of the leaf (Note: Avoid cotyledons), and exert a counter-pressure with finger on the other side. Successful infiltration is often observed as a spreading “wetting” area in the leaf.
  7. (Optional) Check the GFP fluorescence by a portable long-wavelength UV lamp 2-5 days after infiltration. This only applies to strong expression of GFP signal (as green from red background).
  8. Observe the fluorescence labeled protein under a fluorescent microscope or confocal laser scanning microscope. Or harvest leaves for protein purification.


  1. LB media with appropriate antibiotics
    Usually two antibiotics used: one to maintain Agrobacteria virulence, one for the shuttle vector
  2. Acetosyringone stock
    100 mM in ethanol, stored at -20 °C
  3. MES-K (0.5 M) (pH 5.6)
    First make 0.5 M MES, adjust pH with KOH to 5.6
  4. Resuspension solution
    10 mM MgCl2
    10 mM MES-K (pH 5.6)
    Autocloave 15 min
    100 μM acetosyringone (note: Added after autoclaving and immediately before using)
  5. Acetosyringone datasheet
    Synonyms     3’, 5’-Dimethoxy-4’-hydroxyacetophenone
    Synonyms     Acetosyringone
    4’-Hydroxy-3’, 5’-dimethoxyacetophenone
    Molecular Formula              C10H12O4
    Molecular Weight                196.20
    CAS Number                       2478-38-8
    Beilstein Registry Number   1966119
    EG/EC Number                   2196105
    MDL number                      MFCD00008748


  1. Li, X., Chanroj, S., Wu, Z., Romanowsky, S. M., Harper, J. F. and Sze, H. (2008). A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process. Plant Physiol 147(4): 1675-1689.
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Li, X. (2011). Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium. Bio-101: e95. DOI: 10.21769/BioProtoc.95.

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Rashmi R
Jamia Millia Islamia
Is there any specific reason behind using GV3101 strain? Can LBA4404 be used for infiltration?
9/13/2019 2:38:24 AM Reply
Noroza Umer
Boyce Thompson Institute
is this method also works efficiently for N.tobaccum ?
5/18/2018 9:47:27 AM Reply
Sarah Fernandes
University of Cape Town

Please could you advise me as to how to prepare slides for N benthamiana for fluorescent microscopy. Is it necessary to use a microtome or can one peel the epidermis away? My sample preparation will be made a bit more challenging as I will be dehydrating the plants for about a week.

Many thanks
8/15/2017 12:12:31 PM Reply
Marek Szecowka
University of Cambridge

It depends on what you are trying to see. For observation of fluorescent proteins usually you can use small piece of a leaf and suspend it in watter. You don't have to remove an epidermis.

9/14/2017 6:20:35 AM

kaushik ghose
kaushik ghose

I have used this protocol for BiFC and it worked perfectly. I agreed with Marek. Just a piece of leaf suspended in water is good enough. Make sure you water the plants well before infiltration. I normally kept the plants in dark between 3-4 days before monitoring the fluorescence.

9/16/2019 5:17:47 PM

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