Protocols for Micropropagation of Woody Trees and Fruits

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M. BERUTO AND P. CURIR
produces its own sustaining roots. The choice of the propagation method is related to
the degree of branching of plants which can present a different number of divisible
bushes. Taking into consideration that these conventional propagation methods lead
to a high cost of the plants due to technical and time-consuming difficulties (Aoki &
Inoue, 1992), the peony culture could benefit greatly from micropropagation. A
reliable micropropagation system might overcome these difficulties and it could also
be used for the multiplication of virus-free stock material or new cultivars.
In vitro culture of peony for industrial applications has for long time been faced
by the difficulties such as serious explant contamination, browning and establishment
of an ideal medium. Table 1 summarizes the relatively scarce information
available on tissue culture of P. suffruticosa. The in vitro culture of P. suffruticosa
has been applied principally for plant breeding (haploid embryogenesis, Zenkteler
et al., 1975) and for the production of secondary metabolites (Yamamoto, 1988).
The propagation system has also been provided at disposal for the horticultural
market. Different explant sources have been tested with variable degree of success
(Meyer, 1976 a, and b; Li et al., 1984; Buchheim & Meyer, 1992; Bouza et al., 1994a)
but the results are sometimes limited to a particular cultivar. Callus could be
induced from zygotic embryos (Demoise & Partanen, 1969; Wang & van Staden,
2001), flower buds (Meyer, 1976), stems (Gildow & Mitchell, 1977) and roots
(Meyer, 1976b; Gildow & Mitchell, 1977) but neither organogenesis nor embryogenesis
was achieved. Results on plantlet regeneration were referred when axillary
buds (Li et al., 1984; Bouza et al., 1994a; Beruto et al., 2004), leaves and petioles
(Li et al., 1984), filaments and petals (Beruto et al., 2004) and stems (Harris &
Mantell, 1991) were cultured. By culturing axillary buds, Li et al. (1984) estimated
that rather high multiplication rates could be achieved, but this parameter was
strictly dependent on cultivars (generally speaking, the dwarf types are more difficult
to be in vitro propagated) and sometimes on shoots derived from callus. The
objective of this paper is to describe a detailed protocol for in vitro propagation of
tree peony, P. suffruticosa, through axillary budding.
2. EXPERIMENTAL PROTOCOL
Procedures described here have been developed for different genotypes of P.
suffruticosa and, consequently, these techniques should be, in principle, applicable to
other tree peony cultivars. Taking into consideration that a prerequisite to apply a
micropropagation protocol is that no somaclonal variations occur and that changes on
ploidy number was found in callus cultures of P. suffruticosa (Buchheim & Meyer,
1992), in this study we have considered the in vitro propagation through axillary bud
stimulation which, generally, has proven to be the most applied and reliable system for
true-to-type in vitro propagation. Moreover, our choice is supported by the observation
that for many cultivars shoot development from adventitious regeneration is still an
important constraint to the application of a propagation protocol for the tree peony
(Wang and van Staden, 2001; Beruto et al., 2004). The protocol developed in our
laboratory was applied to seventeen Chinese cultivars, two Japanese cultivars (‘Orange’
and ‘Red’) and an old traditional cultivar from our region (‘Old Pink’) (Figure 1).