Protocols for Micropropagation of Woody Trees and Fruits

2.3. Shoot Regeneration and Maintenance
MICROPROPAGATION OF YEW
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2.3.1. Bud Formation and Propagation
Testing of very efficient cytokinins, like 6-benzylaminopurine (BAP), to stimulate
bud and shoot development in combination with nutrient media supplemented with
activated charcoal, as it was reported in the literature, led to a rapid necrosis of the
material. Tests of different basic media showed that Woody Plant Medium (W) was
the most efficient for the growth and vitality of explants. Experiences with other
conifers regarding the use of cytokinins (larch, Norway spruce) were also applied to
yew. Woody Plant medium with 1.5 mg l –1 zeatin (medium called Wz) was used for
bud induction. Sometimes, zeatin was replaced by 2iP (N 6 -(� 2 -isopentenyl) adenine
– 2.84 mg l –1 ). The explants were kept under continuous red light conditions (30 µE
m –2 s –1 , 650 nm peak emission) at a temperature of 23°C. Although medium Wz led
to a reduced elongation of shoots, the formation of axillary buds was forced (Figure 1B).
If the concentration of basic medium was doubled (Wdouble) and used with zeatin
in identical concentrations as Wz (Wzdouble) a remarkable improvement in shoot
elongation, number of lateral buds and needle length was achieved. Nevertheless, a
continuous use of Wzdouble reduced the propagation rate. A callus phase – comparable
to adventitious bud propagation in other conifers (e.g. Norway spruce) – was
not observed. Resulting from these experiments, the nutrient medium Wz was used
for continuous propagation of clusters of axillary buds because of its efficiency.
Propagation rates of 1.2–1.8 per month were achieved. The addition of 200 mg l –1
spermidine boosted these positive results. Using exclusively one medium to get propagation
as well as elongation, e.g. as it was elaborated for poplar in our laboratory,
was not possible. The conclusion was to establish and to maintain a propagation
culture and to transfer buds to an elongation medium to get rootable shoots. Problems
during propagation which derived from the occurrence of endophytic bacteria were
solved by the addition of 500 mg l –1 ticarcilline.
2.3.2. Bud Elongation
Elongation of lateral buds. Tests concerning the serial propagation of bud-bearing
stem segments by dissection of elongating shoots comparable to the system used for
larch, failed because it was not possible to force the development of axillary buds in
stem segments. On the other hand, elongation of shoot tips in this experiment was
achieved best using double concentrated W nutrient medium (Wdouble) and a combination
of zeatin and kinetin (0.66 + 0.64 mg l –1 ) with addition of PVP and arginine
(100 mg l –1 each). Elongation of lateral buds was not stimulated on this medium, but
was stimulated most when a subculture free of phytohormones was included, followed
by a subculture with low concentration of thidiazuron (TDZ, 0.01 mg l –1 ).