Biology Reference
In-Depth Information
15.2
MATERIALS
15.2.1
Sterile media for sterile plant growth
Add 2.2 g MS powder, 500
l Gamborg's micronutrients, and 0.5 g 2-(
N
-morpho-
lino)ethanesulfonic acid (MES) to 1 l distilled water. Adjust the pH to 5.7 with po-
tassium hydroxide and then add agar that has been tested for plant cell culture. For
many experiments, 0.8% agar (w/v) is suitable. Less rigid (0.6%) plates are recom-
mended when selecting T1 seedlings to ease the removal of the seedling roots em-
bedded in the agar. Sterilize the media by autoclaving for at least 15 min and then
cool the media to 50
C in water bath. Supplements (50
m
g/ml kanamycin, 25
g/ml
m
m
hygromycin B, and 500
g/ml carbenicillin) can be added to the media after it has
cooled. Pour the media into sterile Petri plates and allow to solidify.
m
15.2.2
A. tumefaciens suspension for plant transformation
Grow a 500-ml
A. tumefaciens
bacterial culture in LB supplemented with the appro-
priate antibiotic for selection determined by the T
i
plasmid. The culture should be
grown for 48 h at 28-30
C. The common
A. tumefaciens
strain, GV3101, is resistant
to both rifamycin (10
g/ml). Pellet the bacteria by
centrifugation for 20 min at 4000 rpm at room temperature (the resulting pellet will
be pinkish in color). Resuspend the bacterial pellet in 500 ml of virulence buffer (rec-
ipe below). If performing a cotransformation with two different transgenes, resus-
pend each pellet in 250 ml of buffer and then combine the resuspended pellets.
To induce virulence, shake the cultures at 28-30
C at 250 rpm for 2 h. Pellet the
cultures by centrifugation as described above and then resuspend them in 500 ml
of 5% sucrose (sugar from the grocery store is adequate) and 0.002% Silwet L-77.
15.2.2.1
Virulence buffer
85 ml of 20
g/ml) and gentamycin (25-50
m
m
AT salts (recipe below)
17 ml of 50% Glucose (filter sterilized)
34 ml of 1 M MES buffer, pH 5.6
850
l of 100 mM Phosphate
m
850
l of 200 mM Acetosyringone
Add distilled water to 1.7 l.
m
20
AT salts
80 g Ammonium sulfate
6.4 g Magnesium sulfate heptahydrate
0.4 g Calcium chloride dihydrate
0.48 g Manganese sulfate monohydrate
Add distilled water to 2 l and then autoclave.
200 mM Acetosyringone
780 mg Acetosyringone
20 ml of DMSO
Sterilize using a 0.2
20
C in 900
m filter and then store at
l aliquots.
m
m
