Protocols for Micropropagation of Woody Trees and Fruits

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A. GAJDOŠOVÁ ET AL.
Disadvantages of in vitro propagation in Vaccinium vitis-idaea L. is higher cost of
in vitro plants in comparison with classically propagated plants, mainly in cultivars
with natural rich shooting (‘Koralle’, ‘Linnea’). However, this technique could be
effectively used for a production of cultivars characteristic by weak shooting (‘Ida’,
‘Sanna’) (Gustavsson & Trajkovski, 1999 Ondrušková et al., 2006).
The present paper is focused on description of efficient in vitro regeneration and
propagation protocol for Vaccinium vitis-idaea L.
2.1. Explant Preparation
2. EXPERIMENTAL PROTOCOL
2.1.1. Growing Conditions of Mother Plants
The mature mother plants of Vaccinium vitis-idaea L. were cultivated in sandy-clay
soil on the plantation of Research Station Krivá na Orave, Slovak Republic. Since
1994, several cultivars of this species were tested for their suitability to grow under
local climatic conditions. The Research Station is located at 700 m above sea level,
with an average annual temperature 6°C and annual precipitation 800–900 mm, the
highest during June and July. Each plant was planted into acid peat-made bed, with
the volume of 10 l peat per plant, and pH 3.8.
2.1.2. Explant Excision and Sterilisation
As an initial plant material stem cuttings with dormant buds were collected from the
selected cultivars of mature mother plants during the month of February and
beginning of March. Cuttings with apical and axillary buds were washed under
running water for 1 h followed by 70% ethanol treatment for 2 min and 0.1%
mercuric chloride with 3 drops of Tween for 6 min, and finally washed with sterile
distilled water for 3 × 15 min.
2.2. Culture Medium and Conditions of Cultivation
For primary explant cultivation Anderson’s Rhododendron medium – AN (Anderson,
1980) was prepared (Table 1) from packaged powder macro and micro mineral salt
mixture including vitamins (Duchefa, The Netherlands). For 1 liter medium preparation,
2 g powder was completely dissolved in double distilled water. Heat stable
supplements, such as 30 g sucrose, 8 g Phyto agar, desired plant hormones, etc. were
added and medium raised to the final volume by adding double distilled water.
Medium pH was adjusted with HCl and KOH and heated until the solution was
clear. Medium was autoclaved at 1 kg –1 cm –2 (15 psi), 121°C, for 20 min. Heat labile
constituent zeatin was filter sterilized and added to the medium after autoclaving.
All used plant growth regulators and Phyto agar were supplied by Duchefa, The
Netherlands. Cultures were maintained in the growth chamber at 23 ± 2°C under 16-h
photoperiod with light intensity 50 µmol m –2 s –1 provided by white fluorescent lamps.