Plant Cell Lines in Cell Morphogenesis Research - Plant Cell Morphogenesis: Methods and Protocols - page 213

liquid modifi ed MS (BY-2, LE) or V4 (VBI-0) medium to get
fi nal concentration 500 g/ml. Pour this mixture to the cells

in the fi ltration unit and, by opening the valves, let the liquid
drain partly. Close the valves to leave approximately 2-3 ml of
suspension on the fi ltration device.
9. Transfer this suspension (by pipetting with cut off tips) onto
60 mm Petri plates with solid modifi ed MS (BY-2, LE) or V4
(VBI-0) medium supplied with appropriate antibiotics
(100

μ

g/ml hygromycin). Carefully
spread cells over the surface of the agar plate. This is the crucial
step of the whole procedure: cells have to be spread in a layer
that is dense enough to allow regeneration (about 2 mm high
layer of cells equally spread on the whole plate; see Note 12 ).

μ g/ml kanamycin or 20 μ

10. Place the Petri dishes into one big sterile Petri dish (Fig. 1e ),
do not seal with parafi lm and incubate in darkness at 27 °C.
First regenerating transformed calli (antibiotic resistant) can
be observed after 3-4 weeks of incubation in case of BY-2 cells
and 6-8 weeks in case of VBI-0 and LE cells.
11. Transfer the regenerated calli onto fresh solid medium sup-
plied with antibiotics and incubate in darkness at 27 °C.
Alternatively, prepare cell suspensions by transferring the
regenerated small calli (several mm in diameter) into 2-3 ml of
liquid medium supplied with selection antibiotics and shake in
darkness at 27 °C (tobacco) or 24 °C (Arabidopsis).

3.4 Assessment
of the Growth Curve
of Suspension-
Cultured Cells

Work under sterile conditions in laminar fl ow-box.

1. Inoculate 8 ml of BY-2 or LE cell suspension into 240 ml of
modifi ed MS medium (or 40 ml of VBI-0 cell suspension into
250 ml of V4 medium) in 500 ml Erlenmeyer fl ask, mix thor-
oughly by shaking the fl ask in hand. Remove 1 ml of the
freshly inoculated cell suspension with a cut off tip into a test
tube (Fig. 1c ). Use this sample as the fi rst one for the cell den-
sity evaluation ( see Subheading 3.6 ) and/or cell viability test
( see Subheading 3.5 ).
2. Prepare seven aliquots of BY-2 or LE cell suspensions by pipet-
ting smaller amounts (e.g., 10 ml) consecutively in 100 ml
Erlenmeyer fl asks. It is important to keep the stock cell culture
homogeneous by shaking the 500 ml stock fl ask well by hand
before every transfer. Final volume of cell suspension aliquots
in each 100 ml Erlenmeyer fl ask is 30 ml. Close the fl asks with
aluminium foil ( see Note 13 ). For VBI-0 cell culture, prepare
two 100 ml aliquots in 250 ml Erlenmeyer fl asks. Consecutively
remove smaller cell culture aliquots (e.g., 50 ml) with a gradu-
ated cylinder to reach the fi nal volume (100 ml). Shake the
500 ml stock fl ask well by hand before every transfer. Cultivate