Transgenic Plants

Direct Agrobacterium transformation of GV3101(pMP90RK) with pLH vectors

Although the transformation frequency by this method is low compared to the triparental mating method, the technique is reliable and very rapid. This transformation procedure also eliminates much of the plasmid rearrangement that often occurs during triparental mating.

A. Preparation of competent cells

Grow an Agrobacterium strain with the appropriate antibiotics in 5 ml LB medium overnight at 28°C.
Add 2 ml of the overnight culture to 50 ml LB medium and incubate at 28°C until the culture grows to an OD600 of 0,5 to 1,0
Chill the culture on ice. Centrifuge the cell suspension at 3000 g for 5 min at 4°C.
Discard the supernatant solution. Resuspend the cells in 1 ml of 20 mM CaCl2 solution (ice-cold). Dispense 0,1 ml aliquots into 2 ml Eppendorf tubes.
Freeze the cells in liquid nitrogen and store at -80°C or use directly for transformation.

B. Agrobacterium transformation with binaries

Thaw the competentcells carefully on ice
Add 0,1-1 µg of DNA to the cells and mix.
Freeze the cells at -80°C
Thaw the cells by incubating in a 37°C water bath for 5 min.
Place the cells on ice for 30 min.
Spread the cells on prewarmed LB plates (28°C) containing all the appropriate antibiotics
After 2 days incubation at 28°C about 20-100 colonies will appear.

ElectroMAX™ A. tumefaciens LBA4404 Cells

https://www.lifetechnologies.com/order/catalog/product/18313015 18313-015

5 x 40 µL $325.00

with pBI121 vector

II.2.1.7 Preparation of competent A. tumefaciens cells for electroporation

A single colony of A. tumefaciens grown on a YEB-agar containing 100μg/ml rifampicin (Rif) and 25 μg/ml kanamycin (Km) (YEB-Rif-Km) was inoculated in 5 ml
of YEB-Rif-Km medium in a 100 ml Erlenmeyer flask and incubated at 28°C for two days with shaking (250 rpm). One ml of the culture was transferred into 100 ml of
YEB-Rif-Km medium and cultivated at 28°C for 15-20 h with shaking (250 rpm) until the OD 660 reached 1-1.5. The cells were chilled on ice for 15 min and spun down by
centrifugation (4,000g/4°C/5 min). The culture medium was decanted and the cells were washed three times with 10 ml of dH 2 O by centrifugation and resuspended in 500 μl of sterile 10% (v/v) glycerol. 45 μl-aliquots of the suspension were dispensed into prechilled eppendorf tubes, frozen immediately in liquid nitrogen and stored at -80°C.

II.2.1.8 Transformation of Agrobacterium tumefaciens by electroporation
0.2-1.0 μg of plasmid DNA (II.2.3.1) in sterile dH 2 O was added to a thawed aliquot of A. tumefaciens electrocompetent cells (II.2.1.7) and placed on ice for 3 min. The
cell/DNA mixture was transferred into a prechilled electroporation cuvette (0.2 cm) and assembled into a safety chamber. After application of the pulse (25 μF, 2.5 kV, 200 Ω),
the cells were diluted in 1 ml of YEB medium in a 4.0-ml tube and incubated at 28°C with shaking (250 rpm) for 1 h. Finally, 50-100 μl of the cells were plated on YEB-agar
containing 100μg/ml rifampicin (rif), 25μg/ml kanamycin (Km) and 100μg/ml carbenicillin (carb) (YEB-Rif-Km-Carb) and incubated at 28°C for 2-3 days. As a
negative control, dH 2 O was used to transform competent Agrobacterium cells.

II.2.2 Transformation of plants and suspension cultures
II.2.2.1 Growth and maintenance of Nicotiana tabacum
Tobacco plants were grown in a greenhouse in ED73 standard soil (Patzer, Sinntal-
Jossa) with 0-30% (v/v) sand under supplementary illumination of 10 000 Lux (plus the
sun light), 70-90% humidity and 16h photoperiod) at 24°C (or higher depending on the

outside temperature). To prevent pollination from other plants, flowers were covered
with plastic bags with micro pores. Mature, dried seeds were stored in paper bags at RT.
II.2.2.2 Growth and maintenance of Nicotiana tabacum cv. BY-2 cells
N. tabacum cv. BY-2 suspension cells and calli were grown in MSMO medium. Liquid
medium was adjusted to pH 5.2, while medium solidified with 0.8% (w/v) agar was
adjusted to pH 5.8. Both suspension cells and calli were cultivated at 26°C in the dark.
Suspension cells were sub-cultured weekly using a 2% (v/v) inoculum while pinpoint-
sized clumps of callus cells were transferred to fresh plates monthly.
MSMO medium
MSMO salts
thiamin/HCl
2,4-D
KH 2 PO 4
sucrose
4.4 g/L
0.4 mg/L
0.2 mg/L
0.2 g/L
30 g/L
pH 5.2
II.2.2.3 Preparation of recombinant Agrobacterium. tumefaciens
A single colony of a positive clone was checked by Agrobacterium-control PCR
(II.2.3.6.1) and inoculated in 5 ml of YEB medium and cultivated at 28°C for 2-3 days
with shaking at 250 rpm. One ml was transferred into 100 ml of YEB-Rif-Km-Carb
medium (II.2.1.2) and cultivated o/n at 28°C with shaking at 250 rpm. The following
day the cells were pelleted by centrifugation at 5,000 g for 10 min at 15°C and
transferred into 250 ml of induction medium in a 500-ml flask and cultivated o/n at
28°C with shaking at 250 rpm. Bacterial cells were centrifuged (4,000g/15-25°C/15
min) and resuspended in 50 ml of MMA solution and kept for 2 h at RT. The cell
suspension was diluted to an OD 600nm of 2.4 and 100 ml of the diluted cell suspension
was used for vacuum infiltration of plant leaves (II.2.2.5).

Induction medium
YEB medium (pH 5.6)
MES
10 mM
pH 5.6
2 mM MgSO 4 , 25μg/ml kanamycin, 100μg/ml rifampicin, 100μg/ml carbenicillin, and
20μM acetosyringone were added after autoclaving and cooling.
MMA Buffer
MS salts
MES
Sucrose
0.43% (w/v)
10 mM
2% (w/v)
pH 5.6
200μM acetosyringone was added after autoclaving and cooling.
II.2.2.4 Transformation of Nicotiana tabacum cv. BY-2
N. tabacum cv. BY-2 cells were transformed by co-cultivation with recombinant
A. tumefaciens. Three ml of a 3 days old BY-2 culture in the log phase of growth were
mixed in an empty petri dish (5cm) with 150μl of an o/n culture of recombinant A.
tumefaciens. After incubation for 3 days in the dark at 24°C, the cells were transferred
to centrifuge tubes and sedimented (3min/100g/RT). The supernatant was removed and
the pelleted cells washed with 10ml MS medium (II.2.2.6). After three cycles of
sedimentation and washing in MS, the cells were resuspended in an equal volume of
MS medium and 200μl aliquots were plated on MS agar supplemented with 75μg/ml
kanamycin and 250μg/ml cefotaxime. The plates were incubated in the dark at 24°C.
Pinpoint-sized clumps of kanamycin-resistant cells were visible after 20-25 days and
were transferred to fresh plates containing the same medium and cultivation continued
until sufficient cell material for screening by immunoblot (II.4.8) was available.
II.2.2.5 Vacuum infiltration of intact plant leaves
For transient expression of recombinant proteins in different N. tabacum cultivars and
edible plants like zucchini and bean; young leaves (4 leaves for each construct) were
placed in 100 ml of recombinant A. tumefaciens (II.2.2.3) suspension in a Weck glass
and a continuous vacuum (60-80 mbar) was applied for 15-20 min. The vacuum was

MS medium
MS-salts
Myo-Inosite (SERVA)
Sucrose
Thiamin-HCl
A. bidest
Material and Methods
0.43% (w/v)
0.1% (w/v)
2% (w/v)
0.4 mg/l
add to 1000 ml
The pH was adjusted to 5.8 with 1 N NaOH (for preparation of solid medium, 0.8%
(w/v) agar were added), autoclaved and 500 μl of vitamin solution I were added upon
cooling to 55°C.
MS-II medium
MS medium supp. with
BAP (in DMSO)
Kanamycin
Claforan
Betabactyl 1 mg/l
100 mg/l
200-500 mg/l
200-250 mg/l
MS-III medium
MS medium supp. with
Kanamycin
Claforan
Betabactyl 100 mg/l
200-500 mg/l
200-250 mg/l
Vitamin solution I
Glycine
Nicotinic acid
Pyridoxine
Filter sterilized and stored at 4°C.
0.4% (w/v)
0.1% (w/v)
0.1% (w/v)
II.2.2.7 Bombardment of wheat embryos
Immature wheat (Triticum aestivum L. cv. Bobwhite) embryos were used as explants
for the delivery of the HSA-KDEL gene integrated in pal76 vector (II.1.5.8). DNA for
particle bombardment was prepared by mixing plasmids containing the HSA-KDEL
gene with the selectable marker in equimolar ratios not exceeding 15 μg of total DNA
and 2.5 mg of gold particles (0.7 μm). Coating of DNA and bombardment of wheat
embryos in a BioRad He2000 apparatus at 900psi Helium pressure was done by Dr. Eva
Stöger (RWTH Aachen, Institute for Biology VII). Following selection of wheat on
PPT (2.5 mg/l), PPT resistant plant material was recovered.