Preparation of Agrobacterium Culture for Gene Delivery into Plant Cells

Abstract

Source: Shi, J., et al., Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens. J. Vis. Exp. (2020)

This video demonstrates the procedure for preparing an Agrobacterium tumefaciens culture by inducing virulence gene expression with acetosyringone, followed by antibiotic removal through washing and resuspension, to generate a transformation-ready suspension for gene delivery into plant tissues.

Protocol

1. Preparation of Agrobacterium culture for infiltration

1. Pick a positive colony from the Luria-Bertani (LB) plate and inoculate the cells into a glass tube containing 5 mL of LB medium supplemented with 50 µg/mL kanamycin and 50 µg/mL rifampicin. Grow the cells at 30 °C with shaking at 200 rpm for 24−48 h.
2. Transfer 100 µL of culture into 5 mL of LB medium supplemented with the same antibiotics, 10 mM 2-(N-morpholino) ethanesulfonic acid (MES; pH 5.6), and 20 µM acetosyringone (AS). Grow bacteria at 30 °C with shaking at 200 rpm for 16−20 h.
3. Centrifuge cells at 4,000 x g for 10 min. Pour off the supernatant and resuspend the pellet in 2 mL of 10 mM MgCl₂ buffer.
4. Repeat step 1.3 to ensure the complete removal of antibiotics.
5. Determine the density of the Agrobacterium culture by measuring the optical density at 600 nm (OD₆₀₀). Adjust to an OD₆₀₀ of 1.5−2.0 with 10 mM MgCl₂ buffer.
6. Add 10 mM MES (pH 5.6) and 150 µM AS to the final suspension cultures and incubate the cells at RT for at least 3 h without shaking.
   NOTE: Do not leave the final suspension cultures overnight.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Morpholinoethanesulfonic Acid (MES)	Biofroxx	1086GR500	Buffer
Acetosyringone (AS)	Sigma-Aldrich	D134406-5G	Induction of Agrobacterium
Agar	Sigma-Aldrich	A1296-1KG	LB medium
Bacto Tryptone	BD Biosciences	211705	LB medium
Bacto Yeast Extraction	BD Biosciences	212750	LB medium
Kanamycin Sulfate	Sigma-Aldrich	K1914	Antibiotics
Rifampicin	MP Biomedicals	219549005	Antibiotics
Sodium Chloride	Amresco	0241-10KG	LB medium