Plant Cell Lines in Cell Morphogenesis Research - Plant Cell Morphogenesis: Methods and Protocols - page 216

6. Count the number of cells in individual cell fi les or aggregates,
compare the frequency of fi les/aggregates composed of the
similar cell number (e.g., 2-4, 5-8, 9-16) as one of parameters
illustrating cell division rate ( see Note 21 ).
7. Transfer the data to a spreadsheet (e.g., OpenOffi ce/
LibreOffi ce Calc and Microsoft Excel). Plot the measured data
in a graph (Fig. 2b ).

4 Notes

1. Medium can be autoclaved directly in Erlenmeyer fl asks or in
Duran laboratory glass bottles.
2. Use double aluminium foil of 30

m thickness.
3. Before adding antibiotics to the autoclaved medium, let the
bottle cool so you can hold it in a bare hand. Work
aseptically.
4. Use of DL-phosphinothricin (PPT) is not possible in cell
suspensions.
5. The aluminium foil and the glass bottle neck are better to be
sterilized additionally by gas burner fl ame during the process.
6. In general, the growth activity of both callus and suspension
cultures is determined by two key factors: nutrition (includ-
ing, sensu lato, also necessary growth regulators) and proper
aeration. The use of inocula of subcritical size/fresh weight in
case of callus cultures, or subcritical cell density in case of sus-
pension cultures, pronouncedly impairs the viability and mul-
tiplication ability of cells as well as their phenotype (“dilution
effect”). Therefore, the initial cell density of the fresh BY-2,
VBI-0, or LE subculture should not be lower than ca. 1-5 × 10 5
cells per ml. On the contrary, too high inoculum density unde-
sirably shortens the exponential phase of the subculture inter-
val owing to the cell competition for oxygen supply. As a rule,
the fi nal cell density in the suspension of BY-2, VBI-0, or LE
cells reaches max. 10-60 × 10 5 cells per ml. Consequently, in
case of the use of the abovementioned inoculum density and
with respect to equal reproduction ability of the most of cell
populations of BY-2, VBI-0, or LE lines, the cultures pass
maximally 6-7 subsequent cell divisions during standard sub-
culture interval.
7. There should be about 12 pieces of calli on the 90 mm Petri
plate to maintain the optimal hormone and nutrient
conditions.
8. By producing small lesions at the surface of the plant cells in
this step, we help the successful transformation by
Agrobacterium.

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