Protocols for Micropropagation of Woody Trees and Fruits

216 G. NAUJOKS
acid (IBA) for root induction. SERVA agar, 10 g l –1 (gel strength ~800), was used as
gelling agent.
2.2.2. Medium Preparation
For preparing WPM based media variants, powdered media supplied for example by
DUCHEFA Biochemie B.V. were used. MCMAK had to be produced with stock solutions
of the different components. The high amount of auxin (50 mg l –1 IBA) was
dissolved separately with a few ml of undenaturated ethanol (70%) before it was
added to the total quantity. After adjusting the pH, the media were heated till all
compounds were completely dissolved. While stirring carefully, the medium was
distributed with a dispenser pump to suitable culture vessels. Twenty five ml medium
were filled into 100 ml Erlenmeyer flasks and 10 ml medium into small glass tubes,
95 mm long with 28 mm diameter. After closing with aluminium foil the medium
was autoclaved at 121°C (pressure: 105 kPa) for 20 min.
2.3. Shoot Regeneration and Maintenance
For culture initiation after surface disinfection, the shoot tips and nodal segments
were put into the glass tubes with medium MCMAK or WPMAK. The cultures were
kept at 20–22°C with a light intensity of 1600 to 1700 lux (27.5 to 28 µmol m –2 s –1 )
supplied by warm-white fluorescent tubes (e.g. Phillips TLD 58W/93, OSRAM
L58W/31-830 Warmton Lumilux warm white) and a 16-hours photoperiod. If enough
explants are available for each clone, both nutrient media variants should be tested,
because different sallow genotypes may have different media preferences for establishment
of viable cultures.
During the first month, explants from donor trees with the best tissue culture
ability showed spontaneous rooting. Subsequently shoots started elongating and the
shoot tip could be cut if it was about 1.5–2 cm long (Figure 1A). At least one or two
axillary buds were left at the remaining rooted part of the plantlet. Within the next
weeks, new shoots began sprouting from these buds. After transfer to fresh nutrient
medium, the shoot tips rooted again spontaneously and could also be used as mother
plants for microcuttings which could be harvested periodically every 3–4 weeks.
Rooted plants had to be transferred to fresh medium not later than after 3 months if
they were set aside as further stock plants.
Explants from some recalcitrant donor trees were not able to form roots spontaneously.
In these cases, shoot tips had to be put on a half concentrated Woody Plant
Medium (WPM1/250) supplemented with 50 mg l –1 IBA for 1 week for root induction
and afterwards on the growth-regulator-free medium (WPM1/2AK) for root development.
During the following 3 weeks, after the appearance of roots shoot elongation
started. Further propagation in vitro could be done as described above via repeated
cutting of the newly formed shoots and application of a pulse treatment with a high
amount of auxin. After several microcutting cycles executed in this manner, it became
obvious that the difficult-to-root sallow clones were capable of forming roots spontaneously.