Plant Cell Lines in Cell Morphogenesis Research - Plant Cell Morphogenesis: Methods and Protocols - page 212

3.2 Cultivation
of Calli

Perform all steps under sterile conditions in laminar fl ow-box.
1. Transfer a piece (about 5 mm in diameter, i.e., ca. 50-100 mg
fresh weight) of the callus tissue (upper, light-yellow part)
onto the solid modifi ed MS (BY-2 and LE lines) or V4 (VBI-0
line) media ( see Note 7 ). Work with a spatula sterilized by
washing in 70 % ethanol followed by fl ame sterilization.
2. Cultivate the calli at room temperature in darkness.
3. Subculture onto the fresh medium every 4-5 weeks (Fig. 1d ).

1. Day 0: inoculate 2 ml of BY-2, 4 ml of LE or 16 ml of VBI-0
cell suspension (stationary phase) into 100 ml of modifi ed MS
medium in 250 ml Erlenmeyer fl ask under sterile conditions.
2. One day preceding the transformation procedure, inoculate
overnight culture (about 16 h before use) of A. tumefaciens
strain (i.e., in YEP medium) carrying binary vector with your
gene construct.
3. Harvest the cells by fi ltration on the third day (BY-2, LE) or
sixth day (VBI-0) of SBI and resuspend them in the same vol-
ume of the fresh modifi ed MS medium.
4. Add acetosyringone stock solution to plant cell culture, to get
the fi nal concentration 1

3.3 Transformation
of Cell Cultures

l/ml.
5. Before proceeding further, it is necessary to prepare plant sus-
pension cells for easier gene transfer by pipetting thoroughly
with regular uncut tip ( see Note 8 ). Use 5 ml or 10 ml pipette
with standard tips and aspirate and dispense the full pipette
volume about 20 times (BY-2) or 60 times (VBI-0 or LE).
6. Cocultivation: for each sample, mix 4 ml of plant cell suspen-
sion with 20-100

μ

l of Agrobacterium overnight culture ( see
Note 9 ) in 90 mm sterile Petri dishes. For each gene con-
struct, prepare at least four parallel plates. Seal the Petri dishes
with parafi lm and incubate the mixture for 3 days at 27 °C in
darkness, without shaking ( see Note 10 ).
7. Washing: after 3 days, transfer the mixture of the plant cells
and bacteria (by pipetting with cut off tips) into a sterile cell
fi ltration device with 20

μ

m mesh fi lter, and let the medium
fl ow through under atmospheric pressure. If the suspension
cells in all parallel Petri plates are viable, they can be mixed and
fi ltered at once. Add 2.5 ml cefotaxime stock solution into
500 ml of sterile sucrose solution to get 500

μ

g/ml fi nal con-
centration and stir. Close the valves on the fi ltration unit and
pour 1/3 of the sucrose/cefotaxime solution to the cells. Let
incubate shortly (ca. 10 min) and then let the liquid fl ow
through. Repeat this step twice more ( see Note 11 ).
8. After the last washing step, close the valves on the fi ltration
device. Add 150

μ

μ l of cefotaxime stock solution into 30 ml of