Protocols for Micropropagation of Woody Trees and Fruits

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2.4. Establishment of Shoot Cultures
D.T. NHUT ET AL.
Use young stems 1.5–2 cm long and/or shoots 2–2.5 cm long to establish the shoot
cultures.
2.4.1. Shoot Growth and Development
Most of the T. wallichiana needles from stem-derived explants were green and alive
whereas more than half of needles of the shoot-derived explants became yellowish,
turned brown and died after 8 weeks of culture. All shoot-tip cultures produced only
one elongated shoot per explant in all the culture media tested, whereas stem
explants were characterized by axillary bud elongation (Figures 3 and 4).
Percentage of explants showing axillary bud growth (%)
50
45
40
35
30
25
20
15
10
5
0
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11
Medium
Figure 3. Percentage of T. wallichiana explants on different basal MS tissue culture media
without activated charcoal showing axillary bud elongation.
.l –1
mented with 1 mg.l –1 IBA) (Figures 3 and 4).
At high concentration, auxin inhibits the development of the primordial or
axillary buds and induces the formation of callus. Shoots elongated better on media
containing lower concentrations of PGRs (Chang et al., 2001). Almost all explants
placed on culture medium supplemented with 3 mg.l –1 and 5 mg.l –1 auxin showed lower
growth rate, produced phenolics, and formed calli.
To promote shoot elongation and reduce the release of phenolic compounds,
add activated charcoal into the culture media. The percentages of explants showing
axillary bud growth on culture medium supplemented with 2 g.l –1 For obtaining axillary buds, culture shoots on C7 medium (supplemented with
1 mg IBA). For obtaining elongated shoots, place shoots on C4 medium (suppleactivated
charcoal
can reach to over 80% with an average shoot length of about 3.32 cm, and nearly
90% with an average shoot length of about 3.7 cm on C10 medium and C11 medium,
respectively.