Benefits and applications

Agrobacterium is a soil phytopathogen that infects plant wound and causes Crown Gall disease
by horizontal gene transfer(Transferred T-DNA plasmid) through bacterial type IV secretory
system. This manifests as a tumor like outgrowth. The ability of plasmid transfer by
Agrobacterium is harnessed as a tool for transformation of plants for the purpose of genetic
modification. Over the years agrobacterium mediated gene transfer has become a well
accepted method that yields high results.
Agrobacterium-mediated gene transfer is a strong tool for producing secondary metabolites and
recombinant proteins (namely, molecular farming), phytoremediation and pharmaceuticals, crop

improvement, targeted gene knockdown, and the development of biotic and abiotic resistant
transgenic plants.

Maintenance

Short-term storage

For short-term storage of Agrobacterium, cells can be kept on agar media and stored at 4°C for
several weeks.

Long-term storage

The ideal way to store Agrobacterium strains, ensuring that genetic changes due to laboratory
selection are limited, is to keep them frozen at −80°C in 30% glycerol

Growth conditions

A. tumefaciens grows optimally at 28°C. The doubling time can range from 2.5–4 h
depending on the media, culture format and level of aeration.
LB is a common laboratory medium used to support growth of A. tumefaciens. This
minimal medium provides a non-stressful environment with all essential nutrients and
limits the development of auxotrophic mutants. LB medium is the standard growth
medium and will allow for a doubling time ranging from 2.5h 4h (dependent on specific
carbon/nitrogen sources and antibiotics) and will approach stationary phase at an
OD600 of 0.9.
Induction Broth (IB medium) is thought to mimic aspects of the vir-inducing conditions of
a plant wound. In this medium, Ti plasmid bearing cells grow markedly slower with a
lower yield (due to the high cost of vir gene expression and phosphorus limitation) and
will reach stationary phase at an OD600 of approximately 0.2–0.4. In shaken liquid
cultures, clumps of cells can be easily visualized in the medium. It has been observed
that even in very light cultures that appear clear, there are high numbers of cells likely
attributable to this clumping and perhaps smaller cell size.

Agrobacterium Competent Cell Preparation

From a frozen glycerol stock grow a 20 ml culture of Agrobacterium overnight at room
temperature
The following morning dilute the culture by adding 0.5 μl of culture to 2 ml of H2O making a
4000 fold dilution. From the 4000 fold dilution take 0.5 μl and add to 2 ml H2O making a 16,000
fold dilution, from the 16,000 fold dilution take 0.5 μl and add to 2 ml H2O making a 64,000 fold
dilution
Add 5 μl of the 64,000 fold dilution to an LB plate and thoroughly spread the culture on the
plate, incubate at room temp or 28°C for 1 - 3 days
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Once you have single colonies on a plate, pick a colony and start a 20 ml overnight culture in
LB at 27°C .
Inoculate two 500 ml flasks (use 1 liter flasks) of LB with 9 ml of the overnight culture, do not
use antibiotics here.
Grow cells to an OD600 nm of 0.5 - 1
Precool Centrifuge to 4°C
When cells are ready to harvest chill flasks on ice for 15 - 30 minutes
Centrifuge at 4000 g in bottles for 15 minutes at 4°C
Remove as much of the supernatant as possible and resuspended the pellet in each bottle
in 500 ml of ice cold water, use graduations on side of centrifuge bottle to measure volume
Centrifuge at 4000 g for 15 min
Remove supernatant and resuspended the pellet in each bottle in 250 ml of ice cold water
Centrifuge at 4000 g for 15 min
Remove supernatant and resuspended the pellet in each bottle in 10 ml ice cold 10%
glycerol
Combine contents of bottle into 1 50 ml Falcon tube
Centrifuge at 4000 g for 15 min
Remove supernatant and resuspend the pellet in 2 - 3 ml ice cold 10% glycerol. 18. Store in
1.5 ml microfuge tubes in 50 μl aliquots, freeze in LN2, store in -80°C, cells are good for at last 6
months

Transformation of Agrobacterium with vector

Thaw 50 μl electrocompetent cells of hypervirulent Agrobacterium tumefaciens strain
AGL1 on ice.
Add 1 μl (∼100 ng) binary vector DNA to the competent cells, gently mix by flicking, and
place back on ice.
Transfer the cells and DNA mixture on ice. Gently tap to ensure the contents are at the
bottom of the cuvette. Maintain ice.
Electroporate using a Bio-Rad GenePulser, with the settings 2.50 kV, 25 μFD, 400 Ω,
and time constant between 8.0 and 9.5 ms.
Immediately add 100 μl liquid LB medium to the cuvette to allow cells to recover.
Transfer the cells to 500 μl LB medium in a sterile Falcon tube. Wrap the lid of the
Falcon tube with Parafilm to secure.
Incubate the cells at room temperature, lay the Falcon tube horizontally on a shaker,
and gently shake at 100 rpm for 4-6 hr.
Spread 20 and 40 μl of the electroporation culture, respectively, onto two plates solid LB
medium supplemented with appropriate antibiotics.
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Incubate the plate upturned at 28°C for ∼48 hr.
The cells are pelleted down 3100 rpm for 10 min at 24°C and resuspended in inoculation
media.

** Transformation protocols depending on plant variety and explant may vary although initial
steps remain constant.

Agrobacterium tumefaciens