3.2 Preparation
of Agrobacterium
Cultures
for Agroinfiltration
3.2.1 Agrobacterium
Culture Growth
1. Inoculate 2.5 mL of LB containing the appropriate antibiotics
(25 μg/mL gentamicin and 100 μg/mL kanamycin when
using T-DNA vectors with the kanamycin-resistance gene)
with Agrobacterium transformed with the fluorescent marker
of interest, from a glycerol stock.
2. Incubate the 2.5 mL culture overnight at 28
C in a shaking
incubator with good aeration (pre-culture) (see Notes 4 and
5).
3. Inoculate 50 mL of LB containing the appropriate antibiotics
with 0.5 mL of the 2.5 mL pre-culture (1/100 dilution) (see
Note 6).
4. Incubate the 50 mL culture overnight at 28
C, in a shaking
incubator with good aeration.
3.2.2 Agrobacterium
Suspension
for Agroinfiltration
1. Grow the overnight Agrobacterium culture until it reaches an
OD600 of 2.2 (see Note 7).
2. Centrifuge 30 mL out of the 50 mL Agrobacterium overnight
culture (see Note 8) for 15 min at 6000 Â g (RCF) at room
temperature.
3. Remove the medium and keep the bacterial pellet. Resuspend
the bacterial pellet with 2 mL of liquid MS medium (see Notes
9 and 10).
4. Measure the OD 600 of the bacterial suspension. For this, take
50 μL of bacterial pellet suspension and add 950 μL of distilled
water to get a 1:20 dilution. Homogenize the dilution by
inversion before measuring (see Notes 11 and 12).
5. Prepare the infiltration buffer (see Note 13): liquid MS at room
temperature with 0.005% Silwet L-77 (see Note 14) and
200 μM acetosyringone (see Notes 15 and 16).
6. Dilute the Agrobacterium suspension with the infiltration
buffer to have an OD 600 of 2 (see Notes 17, 18, and 19). In
this method, a highly concentrated bacterial suspension is used
to improve the transfection efficiency.
7. Keep Agrobacterium in the infiltration buffer for 30–45 min at
room temperature to let the acetosyringone be assimilated by
the Agrobacterium and activate its virulence genes.
3.3 Agroinfiltration
1. Pour the Agrobacterium in the infiltration buffer onto the
plates with the Arabidopsis seedlings. All the seedlings should
be covered by the Agrobacterium solution (see Note 20).
2. Place the plates with the infiltration buffer into the vacuum
desiccator. To obtain reproducible results, apply vacuum
(400 mbar) with the help of a manometer (Fig. 1) for 1 min.
After, increase the pressure slowly and apply the vacuum again
for another minute (see Notes 21–24).
150
Ce ´ sar Bernat-Silvestre et al.
of Agrobacterium
Cultures
for Agroinfiltration
3.2.1 Agrobacterium
Culture Growth
1. Inoculate 2.5 mL of LB containing the appropriate antibiotics
(25 μg/mL gentamicin and 100 μg/mL kanamycin when
using T-DNA vectors with the kanamycin-resistance gene)
with Agrobacterium transformed with the fluorescent marker
of interest, from a glycerol stock.
2. Incubate the 2.5 mL culture overnight at 28
C in a shaking
incubator with good aeration (pre-culture) (see Notes 4 and
5).
3. Inoculate 50 mL of LB containing the appropriate antibiotics
with 0.5 mL of the 2.5 mL pre-culture (1/100 dilution) (see
Note 6).
4. Incubate the 50 mL culture overnight at 28
C, in a shaking
incubator with good aeration.
3.2.2 Agrobacterium
Suspension
for Agroinfiltration
1. Grow the overnight Agrobacterium culture until it reaches an
OD600 of 2.2 (see Note 7).
2. Centrifuge 30 mL out of the 50 mL Agrobacterium overnight
culture (see Note 8) for 15 min at 6000 Â g (RCF) at room
temperature.
3. Remove the medium and keep the bacterial pellet. Resuspend
the bacterial pellet with 2 mL of liquid MS medium (see Notes
9 and 10).
4. Measure the OD 600 of the bacterial suspension. For this, take
50 μL of bacterial pellet suspension and add 950 μL of distilled
water to get a 1:20 dilution. Homogenize the dilution by
inversion before measuring (see Notes 11 and 12).
5. Prepare the infiltration buffer (see Note 13): liquid MS at room
temperature with 0.005% Silwet L-77 (see Note 14) and
200 μM acetosyringone (see Notes 15 and 16).
6. Dilute the Agrobacterium suspension with the infiltration
buffer to have an OD 600 of 2 (see Notes 17, 18, and 19). In
this method, a highly concentrated bacterial suspension is used
to improve the transfection efficiency.
7. Keep Agrobacterium in the infiltration buffer for 30–45 min at
room temperature to let the acetosyringone be assimilated by
the Agrobacterium and activate its virulence genes.
3.3 Agroinfiltration
1. Pour the Agrobacterium in the infiltration buffer onto the
plates with the Arabidopsis seedlings. All the seedlings should
be covered by the Agrobacterium solution (see Note 20).
2. Place the plates with the infiltration buffer into the vacuum
desiccator. To obtain reproducible results, apply vacuum
(400 mbar) with the help of a manometer (Fig. 1) for 1 min.
After, increase the pressure slowly and apply the vacuum again
for another minute (see Notes 21–24).
150
Ce ´ sar Bernat-Silvestre et al.