Protocols for Micropropagation of Woody Trees and Fruits

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includes the same macronutrients, micronutrients and vitamins as N7 medium described
by Simola (1985) and it is used for callus induction as presented below.
Multiplication medium. Multiplication medium applicable for silver birch is WPM
with the plant growth regulators 2.2 µM BA and 2.85 µM IAA or 4.4 µM BA
together with 0.03 µM NAA or only 4.4 µM BA, 2.0% sucrose and 0.6–1.0% agar.
MS medium with 2.2 µM BA and 2.85 µM IAA is also appropriate (Valjakka et al.,
2000). In Betula pendula var carelica bud formation was further induced on MSmedium
with 4.5 µM BA and 1.07 µM NAA and shoot development occurred on
MS-medium containing half strength of macronutrients, all micronutrients and
vitamins, 2.22 µM BA and 2.85 µM indole-3-acetic acid (IAA), 1.5% sucrose and
1.0% agar (Ryynänen & Ryynänen, 1986).
Rooting medium. For silver birch appropriate rooting media are WPM without any
growth regulators, containing 1.0% sucrose and 1.0% agar (e.g. Ryynänen, 1999) or
WPM with one fifth concentration of the macroelements and indole-3-butyric acid
(IBA) at 0.5 µM (Jones et al., 1996), MS without any growth regulators, containing
half strength of macro nutrients, all micronutrients and 1.5% sucrose (e.g. Viherä-
Aarnio & Ryynänen, 1994).
2.3. Sterile Culture Media with Leaf or Twig Pieces as Explant Material
use so called N7-medium (Simola, 1985) which contains the macroelements according
to Chu et al. (1975) and microelements and vitamins according to Murashige &
Skoog (1962). In addition, the N7 callus induction medium included 2.3 or 4.6 µM
kinetin, 9 or 22.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 50 mg l –1 Callus induction medium (Table 2). As a callus induction medium it is possible to
ferric-
EDTA, 0.56 mM myoinositol, 0.1% casein hydrolysate, 2.0% sucrose and 0.6% agar.
Callus cultivation medium. Callus cultivation medium (Simola, 1985) included 2.3
µM kinetin, 9 µM 2,4-D, 2.0% sucrose and 0.6% agar. Callus cultivation has also been
successful on MS medium including 0.14 mM IAA and 2.3 µM kinetin (Huhtinen &
Yahyaoglu, 1973).
Shoot differentiation medium. Shoot differentiation medium (Simola, 1985) included
5 or 10 mg l –1 zeatin or zeatin riboside, 0.5 or 1.1 µM NAA.
Rooting medium. Rooting medium (Simola, 1985) does not necessarily include growth
regulators. On the other hand Iliev & Tomita (2003) reported that the highest rooting
percentage was achieved when half-strength MS was used together with 2.7 µM
NAA and 2.5 µM IBA. Rooting has sometimes also been achieved on half-strength
MS medium with 2.9 µM IAA (Lemmetyinen et al., 1998). Our own results indicate
that silver birch shoots can be rooted also ex vitro without any plant growth regulator
treatments. However, ex vitro rooting could be improved by submerging the shoots
in 2.5 µM IBA for 30 min and then rinsed in water before planting in soil.