Protocols for Micropropagation of Woody Trees and Fruits

2.6. Flow Cytometry Analysis
MICROPROPAGATION OF VACCINIUM VITIS-IDAEA L. 463
Flow cytometry analysis was perfomed to determine potential undesirable variations
in genome size induced during in vitro regeneration process. Three clones of V.
vitis-idaea cv. ‘Red Pearl’ derived by adventitious regeneration and one meristemderived
clone were analysed. Flow cytometry analyses were performed on Ploidy
analyzer PA II, with mercury arc lamp using UV excitation. The samples were
prepared from young leaves of in vitro plants of Vaccinium vitis-idaea L. by two
step procedure using reagent kit Partec CyStain UV precise P (Partec GmbH,
Münster, Germany) containing Extraction Buffer and Staining Buffer with DAPI:
– Approximately 20 mg of young leaf tissue were chopped with a sharp razor
blade in a plastic Petri dish containing 400 µl Extraction Buffer for 30–60 s.
After incubation in Extraction Buffer for 1–2 min at the room temperature
the sample was filtered through a 50 µm filter.
– Staining Solution 1.6 ml was added to the test tube and sample was
incubated for 30–60 s. Samples were analysed in flow cytometer in the blue
fluorescence channel.
The histograms obtained after nuclear DNA analysis contained a single dominant
peak corresponding to nuclei in G1 phase of the cell cycle with 2C DNA content.
Very few nuclei were found having 4C DNA content. The distribution of nuclear
DNA content obtained after the analysis of nuclei isolated from meristem-derived
clone was similar to those of adventitious regeneration-derived clones (Figure 3A–D).
It can be stated that all tested clones are characterized by significant genetic stability
and polyploid cells occurred in tissues individually without a significant tendency
towards polyploidization. These finding suggest that no changes in ploidy levels
occurred during regeneration process (Gajdošová et al., 2006).
Considering the results of flow cytometry analyses, it seems highly probable
that the stability of ploidy level under in vitro conditions is a more general phenomenon
in Vaccinium spp. plants.
3. CONCLUSION
In conclusion we can state, that an efficient cloning protocol was developed for V.
vitis-idaea L. cultivars, which enables large-scale propagation of high quality, true
to type plants for need of practice. Our results, as well as results of other authors,
prevailingly confirmed that in vitro regeneration ability is highly genotype depending,
therefore small protocol modifications may be necessary in different cultivars.
Acknowledgements. The work was supported by Slovak Grant Agency VEGA, project no.
2/5128/25 and COST 863 Action.