Protocols for Micropropagation of Woody Trees and Fruits

PROTOCOL FOR MICROPROPAGATION OF QUERCUS SPP.
2.3.2. Shoot Regeneration from Dormant Apical and Axillary Buds
Results confirmed that successful plant regeneration and rapid propagation in tested
Quercus species is readily achieved through cultures of dormant apical buds and
axillary buds from both explant types (buds from seedlings and from mature trees).
The best shoot regeneration was observed on the WPM medium supplemented with
0.5–1.0 mg.l –1 BAP and 0.1–0.5 mg.l –1 NAA in dependence on the species. The
multiple vigorous shoot cultures were formed on the above mentioned combinations
of plant growth regulators (Figure 1D, E). Differences in intensity of shoot proli-
feration were species dependent ranging from 3.13 to 6.34 shoots/explant, the lowest
being in Q. robur and the highest in Q. rubra (Ostrolucká & Bežo, 1994).
2.3.3. Shoot Proliferation
For long-term proliferation of in vitro regenerated shoots WPM medium supplemented
with 0.5 mg.l –1 BAP and 0.01 mg.l –1 NAA was successfully used. On this
medium formation of multiple shoot cultures with good elongation growth was
observed, along with different intensity of shoot proliferation in tested species.
Increasing of shoot proliferation intensity can be achieved by segmentation of regenerated,
elongated microshoots on one-node segments and their further cultivation on
the same medium (Figure 1F).
2.3.4. Rooting and Hardening
Spontaneous rooting of microshoots or multiple shoots derived from apical and
In many cases, spontaneous rooting was also observed in embryonic axis cultures.
Microshoots exhibited a good rooting ability on WPM medium with low concentration
of auxins IBA 0.3 mg.l –1 and NAA 0.1 mg.l –1 supplemented with activated
charcoal 3 g.l –1 axillary buds was recorded on media containing low BAP concentration (Figure 1G).
. The percentage of rooting reached 85–90% depending on
species. The addition of activated charcoal to the medium is important for the
reduction of light intensity at the base of the microshoots and also for the phenolics
absorption (Table 1). After 3–5 weeks of cultivation the root formation occurred on
the mentioned medium (Figure 1H).
The rooted plantlets were transplanted into the mixture of peat and perlite (3:1)
(Figure 1I) and immediately covered under a plastic tunnel for keeping high air
humidity. After 2–3 weeks was air humidity gradually reduced to normal values.
The rooted plants were successfully acclimatised under greenhouse conditions without
any special light treatment, with regular irrigation, in plastic containers. Plants after
2–3 month of acclimatisation in the greenhouse were subsequently transferred to
the soil into the bigger containers and placed outside. Winterisation and survival of
plants was successful. After 2 years the oak plants reached the height of 32–45 cm
(Figure 1J).
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