Simplified Protocol to Demonstrate Gene Expression in Nicotiana benthamiana Using an Agrobacterium-Mediated Transient Assay

Materials and reagents

1. N. benthamiana seeds (available from numerous plant science research labs)

2. pC1300S-XB3: Agrobacterium tumefaciens strain EHA105 (Intact Genomics, catalog number: 1084-06) harboring the construct pC1300S-XB3 [1]

3. EV: A. tumefaciens strain EHA105 harboring pC1300S (empty vector)

4. P19: A. tumefaciens carrying the p19 construct. The P19 protein from tomato bushy stunt virus is an efficient suppressor of posttranscriptional gene silencing, which enables higher expression of the gene of interest [6]

5. Luria-Bertani broth (LB Broth) (Fisher Scientific, catalog number: 10855001)

6. Kanamycin sulfate (Fisher Scientific, catalog number: BP906-5)

7. Rifampicin (PhytoTechnology Lab, catalog number: R501)

8. 100 mM acetosyringone (PhytoTechnology Lab, catalog number: A1104)

9. Filtered water

10. Soilless potting mix (Growing Mix, PRO-LINE HFC/B HydraFiber, from Jolly Gardener)

1. Kanamycin sulfate stock solution (50 mg/mL) (see Recipes)

2. Rifampicin stock solution (50 mg/mL) (see Recipes)

3. Infiltration buffer [1] (see Recipes)

Recipes

1. Kanamycin sulfate stock solution (50 mg/mL)

   Dissolve 0.5 g of kanamycin sulfate (powder) in 10 mL of distilled water at room temperature. Filter through a 0.2 μm filter. Aliquot in 1 mL fractions and store in a -20 °C freezer.




2. Rifampicin stock solution (50 mg/mL)

   Dissolve 0.5 g of rifampicin (powder) in 10 mL of 100% methanol at room temperature. Aliquot in 1 mL fractions and store in a -20 °C freezer.




3. Infiltration buffer [1]

   Dissolve 1.95 g of MES [2-(N-Morpholino)ethanesulfonic acid] and 1.96 g of MgCl₂ in 1,000 mL of sterile water at room temperature. Adjust pH to 5.6. Aliquot in 1 mL fractions and store in a -20 °C freezer.

Laboratory supplies

1. 9-cm square disposable pots with drainage holes (any manufacturer)

2. 15 mL culture tubes (Carolina Biological Supply, catalog number: 215090)

3. 50 mL conical screw cap tubes (Carolina Biological Supply, catalog number: 215095)

4. Syringes (1 cc) (Carolina Biological Supply, catalog number: 697765)

5. 10 mm cork borer (Carolina Biological Supply, catalog number: 712202)

Equipment

1. Forceps for transplanting seedlings (Carolina Biological Supply, catalog number: 624790)

2. Incubator shaker (New Brunswick Scientific, model: C24KC)

3. Conductivity meter with electrode (Eutech, model: Cond 6+ ECCON603PLUS)

4. Deep freezer with -20 °C (any manufacturer)

5. Bunsen burner (Carolina Biological Supply, catalog number: 706706)

6. Laminar flow hood (any manufacturer)

Procedure