Protocols for Micropropagation of Woody Trees and Fruits

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M. BERUTO AND P. CURIR
6. Initial explants start to develop in shoot cluster after 8 week of cultures. A
reaction depending on genotype can be observed and the aptitude to multiplication
can be enhanced after a further subculture on the WPMP2 medium
(Figure 6).
Figure 5. Micropropagation of tree peony: steps in the procedure of inoculation. a) collection
of axillary buds from mother plant; b) Removing of bud scales before sterilization; c) Inoculation
onto WPMP1 medium; d) Browing medium observed after 1 day of culture; at this time, the
explants are transferred to the same fresh medium, without any further cutting.
2.2. Stage 2: Multiplication Phase
Accordingly to our protocol, initial explants start to develop in shoot clusters of
axillary shoots and no adventitious shoots are observed. In our experiments, most
cultivars (about 70%) already showed their aptitude to propagate at the end of the
initiation stage, and this trend was reinforced during the subsequent subculture; only
one cultivar presented a delayed response, showing multiplication after 16 weeks of
culture. Two third of the tested cultivars reached 100% of propagating explants after
16 weeks in culture (Figure 6).
Plant growth regulators and calcium level in medium have been recognized as
important factors controlling the in vitro multiplication of tree peony. In order to
achieve higher multiplication rates, Bouza et al. (1994a) used the mineral salts of
MS with 6 mM Ca 2+ (instead of 3 mM). Other researchers confirmed signs of Cadeficiency
when MS and WPM media were used for in vitro culture tree peonies
(Černá et al., 2001; Wang & van Staden, 2001).