Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF SALIX CAPREA L. 215
Nursery plants. Rooted cuttings from adult donor trees were grown in plastic pots in
the nursery during spring and summer under irrigation. One year after rooting, the
new shoots were used for tissue culture establishment.
Greenhouse plants. The most favourable preconditions for the establishment of plant
material in vitro were given after growing potted willow plants (e.g. from cutting
propagation attempts) for a period of 2–6 months in the greenhouse. In these conditions
the shoots were elongated quickly and the surface of leaves and stems was
relatively clean.
2.1.2. Explant Excision and Sterilization
Explants from sprouting twigs in the greenhouse. Four to six weeks after transferring
the twigs to the greenhouse, 2–2.5 cm long apical shoot tips and nodal segments
with leaves were excised from the newly growing shoots and were used as explants.
The area of the biggest leaves was reduced to 1/3 their full size and the explants
were shaken in 0.2% Euparen solution for 2 min. After drying with a paper towel,
the disinfection procedure was immersion for 9 min in 0.25% mercuric chloride with
two drops of the detergent Tween80, followed by rinsing three to four times in
sterile deionised water. Before transfer to culture vessels, the base of the explants
was cut again for better nutrient uptake (final length of the explants: 1.5–2 cm).
Nursery plants. From the middle of August to the beginning of September, healthy
apical shoot tips and nodal segments, each 2–2.5 cm long, were taken from containergrown
plants in the nursery when the weather was warm and dry. Big leaves were cut to
1/3 their size, afterwards the explants were rinsed for 2 min in 0.2% Euparen. After
drying between paper towel sheets, the disinfection with 0.25% mercuric chloride
and a few drops of Tween80 was extended to 20 min because of the high contamination
risk from the explants grown outdoors.
Greenhouse plants. When mother plants were growing for several months in the
greenhouse before establishment in vitro, shaking of the explants in a fungicide could
be omitted, but all other steps of surface disinfection had to be done as described
above. Since the lignification level of the shoot tips and nodal segments was different,
mercuric chloride for surface disinfection had to be applied for 8 min with explants
taken in April, but 10 min with explants taken in October followed by the rinsing in
sterile deionised water.
2.2. Culture Medium
2.2.1. Media Composition
Two different basic nutrient media were modified for the successful establishment of
Salix caprea clones (see Table 1): MCM according to Bornman (1983) and WPM by
Lloyd and McCown (1981). The propagation media MCMAK, WPMAK and WPM1/2AK
were prepared without any plant growth-regulator, but with 0.1% activated charcoal
(Darco G60 by SERVA). The medium WPM1/250 contained 50 mg l –1 indole-3-butyric