Protocols for Micropropagation of Woody Trees and Fruits

IN VITRO PROPAGATION OF NUTMEG
3. They were then cut into several single nodes about 1 cm in length or 2-node
explants.
4. Nodal explants were cultured upright with the lower end inserted into the
culture medium.
Stem segments
1. Shoot was cut into segments.
2. Stem segments were then sterilized with 0.1% HgCl2 for 5 min followed by
several rinses with sterile distilled water and cultured.
Leaves
1. Tender, immature leaves were cut transversely after discarding 2–3 mm of
the margin, into pieces of about 1.5 cm width with midrib.
2. Segments were then sterilized with 0.1% HgCl2 for 3 min followed by
several rinses with sterile distilled water.
3. Segments were placed mostly with the adaxial surface in contact with the
culture medium.
4. Petioles was cultured with the lower end inserted into the culture medium.
In some cases the petiole was placed horizontally on the surface of the
medium.
2.3.2. Culture Medium and Growth Conditions
The media used were based on those devised by Murashige and Skoog (1962).
Various growth regulators were added singly or in combination at different levels to
the media together with sucrose (Table 3). After adjustment of the pH to 5.7, the media
were solidified with agar or gelrite and sterilized in an autoclave for 15 min at 121 ±
2ºC. The cultures were incubated in a temperature-controlled room at 25 ± 2ºC in
continuous light (25 µmol m –2 s –1 ) from cool white fluorescent lamps.
Table 3. Several concentrations of plant growth regulators were tested in the culture media
used for culturing explants from juvenile leafy shoots.
Sl. No. Plant growth regulator
concentration (mg/ L )
C1 C2 C3 C4
1 BAP 5.0 — — —
2 Kinetin — 1.0 3.5 —
3 2,4 - D — — 5.0 2.0
4 NAA — 5.0 — —
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2.3.3. Regeneration
Formation of axillary shoot from nodal explants. Axillary bud break was obtained
after 3 weeks in medium C1. Single axillary shoots were formed in nodal explants
(Figure 3A) in medium C1.