Preparation of Agrobacterium Culture for Gene Delivery into Plant Cells

Begin with an Agrobacterium tumefaciens culture grown in medium supplemented with two antibiotics to prevent contamination and ensure plasmid retention.

The bacteria contain a plant expression vector carrying a gene of interest and an antibiotic-resistance gene.

They also contain a helper plasmid with a region of virulence genes required for delivering the gene of interest into plant cells.

Transfer a portion into a tube containing a buffered medium supplemented with acetosyringone, a plant-derived signal molecule, and the antibiotics. Incubate with shaking.

Acetosyringone diffuses into the cell, triggers a signaling pathway, and activates expression of virulence genes required for the transfer of the gene of interest.

Transfer the culture, centrifuge to collect the bacteria, and discard the supernatant.

Add a salt buffer and wash the cells repeatedly to remove the added antibiotics.

Resuspend the bacteria in the buffer with acetosyringone and incubate without shaking to maintain virulence gene expression.

Use the culture immediately for efficient transformation.

Use a tip to pick a positive colony from the LB plate and inoculate the cells into a glass tube containing 5 milliliters of LB medium supplemented with 50 micrograms per milliliter Kanamycin and 50 micrograms per milliliter of Rifampicin. Grow the cells at 30 degrees Celsius with shaking at 200 RPM for 24 to 48 hours.

Transfer 100 microliters of the culture into 5 milliliters of LB medium supplemented with the same antibiotics, 10 millimolar MES at pH 5.6, and 20 micromolar AS. Grow the bacteria at 30 degrees Celsius with shaking at 200 RPM for 16 to 20 hours. Centrifuge the cells at 4,000 times g for 10 minutes. Discard the supernatant and resuspend the pellet in 2 milliliters of 10 millimolar magnesium chloride buffer.

Repeat the wash to ensure the complete removal of antibiotics. Determine the density of the Agrobacterium culture by measuring the optical density at 600 nanometers. Adjust the cell culture with 10 millimolar magnesium chloride buffer to an OD₆₀₀ of 1.5 to 2.0.

Add 10 millimolar MES at pH 5.6 and 150 micromolar AS to the final suspension culture and incubate the cells at room temperature for at least 3 hours without shaking.