Protocols for Micropropagation of Woody Trees and Fruits

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that is a modified MS medium with glutamine, 9.0 µM 2,4-D and 4.4 µM BA. For
biolistic transformation the MS preculture medium with 5.7 µM IAA and 0.05 µM
thidiazuron (TDZ) has been appropriate (Valjakka et al., 2000).
Induction medium. Medium appropriate to induce adventitious shoots is WPM with
1.1 µM 2,4-D and 2.3 µM TDZ for material regenerated after Agrobacterium mediated
gene transfer (Keinonen, 1999) and for material regenerated after biolistic transformation
MS with 0.1 µM TDZ (Valjakka et al., 2000).
Multiplication medium. Potential multiplication medium for individual shoots was
MS or WPM with 2.85 µM IAA and 2.22 µM BA in the case of shoots derived from
biolistic transformation (Valjakka et al., 2000) and WPM with 2.2 or 4.4 µM BA when
transformation was done via Agrobacterium-mediated gene transfer (Keinonen, 1999).
Rooting medium. Rooting medium used for shoots regenerated after Agrobacteriummediated
transformation was WPM with 2.9 µM IAA (Keinonen, 1999) and for shoots
from biolistic transformation WPM without growth regulators (Valjakka et al., 2000).
2.5. Method
The regeneration method can be divided into four main steps: explant excision and
sterilisation, establishment and proliferation of shoot cultures, rooting, and hardening.
2.5.1. Explant Excision and Sterilisation
1. Collect the dormant silver birch shoot tips, shoot or leaf pieces. Prepare
them immediately or let the twigs with dormant buds to vernalise at room
temperature (RT) in water containers until the buds start to swell. For genetic
transformation experiments use leaf pieces or nodal stem segments derived
from in vitro shoot cultures (Keinonen-Mettälä et al., 1998; Valjakka et al.,
2000).
2. Disinfect the shoot tips or leaves in 70% ethanol for 60, 90 or 120 s (Ryynänen
& Ryynänen, 1986; Lemmetyinen et al., 1998). In some cases the explants
have needed more severe disinfection. In leaf explants, the ethanol treatment
is followed by the immersion of the material in 3% sodium hypochlorite for
2 min (Simola, 1985). For shoot buds or tips 10% sodium hypochlorite (Jones
et al., 1996) or 7% calcium hypochlorite (Welander, 1988) with 0.1% Tween-
20 for 15 min. For severely infected material also 0.2% HgCl2 with Tween-20
for 7 min followed by several rinses with sterile water.
2.5.2. Initiation and Multiplication of Shoot Cultures
1. Remove the outer layers of the apical or axillary bud scales, to expose the
shoot apex, and place them on shoot initiation medium. Especially the buds
from adult material will secrete high amounts of phenolic compounds into
the culture medium quite rapidly and they need to be either replaced in the