Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF SELECTED VACCINIUM SPP. 449
2.3.3 Influence of Medium pH on Micropropagation of Vaccinium spp.
It is known that by changing pH levels nutrient availability and plant metabolism are
affected. Vaccinium spp. production is limited to soils with naturally low pH or soils
treated with acidifying amendments (Finn et al., 1991). For understanding of pH
tolerance, in vitro screening with modification of pH values in AN medium with 0.5
mg l –1 zeatin within a tested pH range 3.0–6.0 was performed in selected cultivars of
V. corymbosum during shoot formation from apical and axillary buds (Ostrolucká
et al., 2004).
With increasing pH values of the medium in the range 3.0–5.0, also the number of
shoots per explant increased in the range 2.33–7.34 in ‘Duke’ and 4.02–11.03 in
‘Brigitta’ as a mean number of shoots after 3rd subculture. At pH 5.5 considerable
decreasing in the shoot number was observed. Statistically significant differences in
the shoot proliferation were found among all tested pH variants with optimum
medium pH 5.0.
2.3.4 Shoot Proliferation
For long-term proliferation of in vitro regenerated axillary or adventitious shoots
AN medium supplemented with 0.5 mg l –1 zeatin was successfully used. On this
medium formation of vigorous multiple shoot cultures was observed with different
intensity of shoot proliferation in tested cultivars. Increasing of shoot proliferation
intensity can be achieved by segmentation of regenerated, elongated microshoots on
one-node segments and their further cultivation on medium with 0.5 mg l –1 zeatin
(Figure 1C).
2.4. Rooting and Hardening
Satisfactory rooting of isolated microshoots is achieved by following steps:
1. ex vitro rooting by short (2–3 min) dipping of 15–20 mm long microshoots
–1
into 0.8 mg l IBA solution followed by direct planting in containers
50 mm in diameter filled with peat substrate
–1
2. in vitro rooting on AN medium supplemented with 0.8 mg l IBA and
–1
0.8 g l activated charcoal.
With both approaches the percentage of rooting reached 80–90–95% in dependence
on cultivar. The rooted plantlets transplanted into peat substrate were subsequently
acclimatized. Hardening of plantlets was performed under greenhouse conditions
without any special light treatment, with regular irrigation, in containers at the
beginning (2 weeks) covered by lid, later opened. Plantlets in length 100–150 mm
(after 2 months) were replanted into bigger containers (120 mm in diameter) and
transferred to Research Station Krivá, where they were placed under open-air
conditions. Transfer of regenerants from in vitro to ex vitro conditions and their
acclimatization was successful, as almost 80–90% of transferred plants survived
(Figure 2A,B).