Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF SELECTED VACCINIUM SPP. 453
number (1–3) in all ten genotypes. The number of synthesized DNA fragments was
in average 8 per genotype while the size of synthesized fragments reached 250–1550
bp. A high DNA polymorphism level and low distance index values determined by
DNA microsatellite polymorphism have pointed out a stable in vitro cultures of
Vaccinium spp., however independently from the plant origin. Two primers out of
five have clustered as the closest genotypes number 8 and 9 (‘Blueray’ and ‘Darrow’)
originated from meristematic tissue. Three out of five primers have clustered genotypes
3 and 4 (‘Berkeley’) and two have clustered genotypes 6 and 7 (‘Bluecrop’).
In both cases a leaf tissue was the primary explant. It seems that a primary explant
origin might play a role in stability of in vitro culture.
Dissimilarity (EDAC) 1
85
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Figure 5. Dendrogram of Vaccinium genotypes analyzed by ISSR using five primers. (1) EDAC
– Euclidian Distance Averages of Clusters, (2 ) Genotypes, 1–4 Berkeley, 5–7 ‘Bluecrop’, 8
‘Blueray’, 9 ‘Darrow’, 10 ‘Linnea’.
2.7. Flow Cytometry Analysis
1 4 5 6 7 2 3 8 10 9 Genotypes 2
Flow cytometry analysis was perfomed with the aim to determine undesirable
variations in genome size induced during in vitro regeneration process. Three clones
of V. corymbosum cv. Berkeley derived by adventitious regeneration and one
meristem-derived 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 corymbosum 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: