Protocols for Micropropagation of Woody Trees and Fruits

26
S.S. KORBAN AND I.-W. SUL
1. In vitro establishment: Wolter and Skoog (WS) (1966) basal medium (4.4
g/l WS salts, 20 g/l sucrose, and 7 g/l agar) and Staba vitamins.
2. In vitro shoot proliferation: WS basal medium + Staba vitamins + kinetin
(4.7 µM) + 6-benzyladenine (BA; 4.4 µM) + zeatin (15 µM).
3. Shoot elongation and rooting: ½ WS salts + Staba vitamins + activated
charcoal.
Table 1. List of media components for micropropagation of Sequoia sempervirens.
Medium
component
Culture
establishment
(per liter)
Shoot proliferation
(per liter)
Shoot elongation
and rooting a
(per liter)
WS salts 4.4 g 4.4 g 2.2 g
Staba vitamins 10 ml 10 ml 10 ml
Myoinositol 10 mg
10 mg 10 mg
6-benzyladenine 1 mg
kinetin 1 mg
zeatin 3.3 mg
Sucrose 20 g 20 g 20 g
Agar (Difcobacto)
7 g 7 g 7 g
Charcoal 2 g
pH 5.6 5.6 5.6
a
Spontaneous rooting is observed on this medium; however, it may be necessary to transfer
cultures to a fresh similar medium, but containing an auxin such as indolebutyric acid (IBA)
at 0.5 mg/l to increase the frequency of rooted shoots.
2.2.2. Regeneration via Shoot Organogenesis
The overall protocol of micropropagtion of S. sempervirens is maintained by
continuous in vitro shoot proliferation and subsequent ex vitro rooted shoot production.
The protocol used in our laboratory can be divided into three stages as
follows: 1) establishment of explants; 2) shoot proliferation; and 3) shoot elongation
and spontaneous rooting (Figure 1).
1. Establishment of explants: Greenhouse- or field-grown shoots are cut into
10 cm stem segments, and transferred to a WS medium without PGR for
4 weeks. Any contaminated shoots are discarded, and clean stem segments,
about 1–2 cm in length, are maintained for shoot proliferation.
2.
Shoot proliferation: sterilized shoot segments containing 2–3 axillary buds
are cultured horizontally on WS medium containing zeatin (15 µM) for 4
weeks. Depending on the genotype, it is expected that variations in shoot
proliferation rate will be observed. Although a range of 5 to 15 µM zeatin
promotes shoot proliferation, 15 µM zeatin showed the best frequency of
shoot proliferation for our own tested genotypes. Therefore, it is important
to determine the optimum zeatin concentration for the genotype used.