Protocols for Micropropagation of Woody Trees and Fruits

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D. EWALD
ability of the plant material used as well as other factors might have been responsible
for the negative results. Therefore, based on experiences in micropropagation of
conifers, a method was worked out in detail to multiply selected yew clones.
2.1. Explant Preparation
2. EXPERIMENTAL PROTOCOL
2.1.1. Growing Conditions of Mother Plants
Cuttings of adult selected donor trees harvested in September were rooted using
rooting paste containing 2 g l –1 indole-3-butyric acid (IBA) under high pressure fog
in a greenhouse (Figure 1A). These rooted plants were used as donor plants for establishing
tissue cultures. Closed buds or shoot tips were harvested as explants after pretreatment
with a fungicide (0.2% Euparen by Bayer, 50% dichlorfluanide) for 24 h.
2.1.2. Disinfection of Plant Material
The surface of the explants was kept dry to allow an effective disinfection. Therefore,
no washing was carried out before disinfection and the plants were not watered one
day before use. Disinfection was carried out by washing the explants in mercuric
chloride solution (0.25%) with one drop of a detergent (e.g. TWEEN 80) for 10–15
min. Afterwards, the explants were rinsed three times with sterile water and placed
on nutrient medium in 100 ml-Erlenmeyer flasks.
2.2. Culture Media
Nutrient media used for micropropagation are listed in Table 1. The concentration
of basic components (dilution or increase) from well-known plant nutrient media is
shown there, whereas growth regulators, which were added, are mentioned in the
text separately. In different experiments Woody Plant Medium (WPM, according to
Lloyd & McCown, 1981) was found to support the growth and vitality of Taxus
explants best. Therefore, nutrient media for propagation as well as for elongation
was based on this basal medium. The basal medium for rooting was a modified LS
medium (L9, based on LS according to Linsmaier & Skoog, 1965).
Table 1. Basal nutrient media compositions used for larch micropropagation (Macroelements,
microelements given as dilution or increase of original medium).
Medium Macroelements Microelements Carbon source g l –1
pH
W 1 1 20 sucrose 5.7
Wdouble 2 2 20 sucrose 5.7
L9 1/3 1 5 sucrose 5.7