Protocols for Micropropagation of Woody Trees and Fruits

IN VITRO CULTURE OF TREE PEONY 493
pronounced with 2 to 3 principal roots per plant (mean length being 8.5 ± 0.7 cm)
and also development of secondary roots may be observed (2 to 4 secondary roots
per plant with 2–3 cm mean length). The second step is carried out under greenhouse
conditions where the plantlets start to receive the standard culture practices regarding
nutrition, irrigation and pest control. This system allowed us to produce quality
plants with a survival rate of 80 ± 10% (Beruto et al., 2004).
Figure 13. Rooting of tree peony shoots following a two-step procedure (see text).
2.4.1. Steps in the Procedure
Step 1
1. Fill glass vessels (500 mL) with a mixture (200 mL) of milled peat (Rekiva,
cd 1A9 and perlite (# 3) (1:1) and sterilize at 120°C, 101 kPa for 40 min.
2. Add 30–40 mL of sterilized deionized water to the sterilized mixture in
order to lightly moisten the substrate.
3. Take the rooted plantlets from stage 3, pay attention not to break the roots
and wash the root system to get rid off the agar pieces.
4. Place the rooted shoots (about 3 cm in height) on the mixture, close the
vessel with the corresponding polycarbonate lids and polyethylene film and
incubate at the usual culture conditions as described above for the micropropagation
process.
5. The culture period of the first step of acclimatization is 4 weeks; along this
period excessive humidity accumulation in the vessel should be avoided
by removing the polyethylene film and progressively increasing the gasexchange
in the culture.
6. At the end of the first step, select the rooted plantlets (Figure 14A,B) ready
to be transferred to greenhouse conditions (step 2).