Protocols for Micropropagation of Woody Trees and Fruits

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2.1. Explant Preparation
M.G. OSTROLUCKÁ ET AL.
2. EXPERIMENTAL PROTOCOL
In vitro regeneration was tested in such Quercus spp., as Q. robur L., Q. virgiliana
Ten., Q. cerris L., Q. rubra L. resulting in the optimal experimental protocol assessment.
2.1.1. Growing Conditions of Mother Plants
Q. robur L., Q. virgiliana Ten. and Q. cerris L. are the autochthonous species,
naturally widespread in Slovakia. Q. rubra is an introduced species, which also
grows well in conditions of Slovakia. The acorns and stem cuttings with dormant
buds, which were used as source of explants, were collected from selected mature
trees of the natural populations in Slovakian territory or from arboretum.
1. Q. robur L. acorns were collected from locality Gabčikovo, situated in
southern Slovakia.
2. Q. virgiliana Ten. acorns were collected from trees of natural population in
Čankov, situated in southern Slovakia. Locality is Corneto-Quercetum forestation
on neogeneous eruptive rocks.
3. Q. rubra L. and Quercus cerris L. acorns were obtained from collection of
woody plants in Arboretum Mlyňany, dendrological park situated in southern
Slovakia at the foothills of the Western Carpathian Mountains at an altitude
160–206 m above sea level.
Seedlings, used as a source of dormant buds, were produced outside from acorns
obtained after open pollination. The acorns were collected from different localities
in Slovakia.
2.1.2. Explant Excision and Sterilisation
Embryo cultures. The mature seeds were collected during October and November.
containing the embryonic axes were surface desinfected in 70% ethanol for 10 min
followed by 15–20 min treatment in 0.1% solution of mercuric chloride and finally
axes were aseptically isolated from the surrounding cotyledons with preservation of
cotyledonary nodes and plumule. Isolated explants were treated in 100 mg.l –1 Seeds were washed under top water for 5 min. After pericarp removal the acorns
washed with sterile distilled water under aseptic conditions for 3 × 15 min. Embryonic
solution
of ascorbic acid to prevent oxidation for 30 min. After removing the radicular pole,
the embryos were placed upright on the culture medium.
Cultures of dormant buds. The stem cuttings with dormant buds were collected from
2 month to 1.5 year-old seedlings, as well as from selected mature trees during
February to mid March. Apical and nodal segments with dormant buds were used
for initial culture establishment. The material was sterilised by washing under top
water for 1 h, treating for 5–10 min in 70% ethanol and 10–15 min in 0.1% solution