Plant Cell Lines in Cell Morphogenesis Research - Plant Cell Morphogenesis: Methods and Protocols - page 217

9. Approaching the plant cultured cells transformation for the
fi rst time, start with at least three Agrobacterium volumes.
Suitable volumes are 20, 40, and 60

μ l for BY-2 cell culture or

l for VBI-0 and LE cells. Do not forget to
have one plate without Agrobacterium as a control.
10. You may check the condition of cell culture using inverted
microscope. If the transformation successfully progresses,
attachment of bacteria to the plant cell walls can be seen, and
the majority of plant cells remain viable. Medium appears
milky because of Agrobacterium growth.
11. Because of the fi lamentous or cluster-like character of the
VBI-0 and LE cell suspensions, it is highly recommended to
incubate plant cells for 15 min in the washing sucrose solution
to remove Agrobacterium completely.
12. Avoid medium overfl ow on the plates.
13. Preparation of seven aliquots of BY-2 or LE cells is optimal to
avoid suspension density changes due to samples removal.
14. Trypan (Evans) blue “dye exclusion” test is based on the
inability of injured or dead cells to exclude the dye actively
from their cytoplasm and vacuoles. However, the reliability of
the test is not absolute; in a very few cases, also cells with pre-
served internal structure and even dividing cells can be stained
and thus mimic the positive (i.e., nonviable) ones.
15. The fl uorescein diacetate (FDA) test [ 20 ] is based on the
activity of cell esterases which catalyze the release of free fl uo-
rescein from FDA and its accumulation in vacuoles of viable
cells. Inactive esterases or damaged cell membranes prevent
fl uorescein accumulation inside cells so that dead cells exhibit
no fl uorescence.
16. To evaluate cell density in samples containing higher fraction
of multicellular spherical aggregates that are hardly observable
using standard light microscopy, one can use methods of fl uo-
rescent staining of cell nuclei, e.g., using the Hoechst dye. By
means of proper software, it is possible to count individual
nuclei with reasonable reliability. In such a way, cell number is
determined indirectly.
17. Before counting, mix the cell culture with Pasteur pipette to
evenly distribute cells in the counted samples.
18. As the most reliable method for determination of actual cell
viability in suspension cultures, the combination of standard
light microscopy and proper cell staining is recommended. For
routine work we prefer to combine Nomarski DIC (differential
interference contrast) microscopy either with trypan (Evans)
blue or FDA technique. For experienced scientists, who are able
to recognize damaged cells (e.g., destruction of the network of
cytoplasmic bands), light microscopy alone is suffi cient.

60, 80, and 100 μ