Protocols for Micropropagation of Woody Trees and Fruits

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2.2. Culture Medium
R.I. IYER
There was severe browning and necrosis, even in darkness, owing to the heavy
leaching of phenolics in the culture medium when zygotic embryos were used
as explants. The problem was overcome by addition of activated charcoal to the
medium which absorbs all the phenolic substances resulting in survival, growth and
massive proliferation of the cultured tissues. Somatic embryos from intact and
fragmented pieces of zygotic embryos could be obtained (Iyer et al., 2000)
regardless the use of the basal media and plant growth regulators after addition of
activated charcoal in the medium. Table 1 describes the mineral salt composition of
the basal media A (Anderson, 1978) and media B1– B7 (Murashige & Skoog, 1962 )
used for various steps of plant regeneration.
2.2.1. Medium Preparation
1.
appropriate proportion.
2.
3. The pH of the nutrient media was adjusted to 5.7. After addition of
activated charcoal (0.25%), the media were solidified with agar or gelrite
and sterilized in an autoclave for 15 min at 121 ± 2 C.
4. Cultures were incubated in a temperature-controlled room at 25 ± 2 C in
continuous light (25 µmol m –2 s –1 Medium preparation involved the following steps:
Stock solutions with the mineral salts and vitamins were mixed in the
Various growth regulators were added singly or in combinations at different
levels to the media together with sucrose and other growth supplements
(Table 2).
°
°
) from cool white fluorescent lamps.
2.2.2. Regeneration and Maintenance
Direct formation of green somatic embryos (12–14 per explant) without any intervening
callus stage (Figure 1A) was obtained from intact medium-sized (9–10 mm
long) zygotic embryos in the culture media supplemented with activated charcoal
(Iyer et al., 2000) in 3–5 weeks. Direct somatic embryogenesis was also obtained
from zygotic embryos of large (13–14 mm long) size (Figure 1B) as well as from
broken zygotic embryos. There was no response from embryos of very small size (3–4
mm long). The embryogenic competence could be maintained by repeated subculture
in the same or different media for about 10 months and establishment of long term
embryogenic cultures that could serve as stable sources of the various secondary
metabolites of nutmeg could be achieved. High frequency somatic embryogenesis
(Figure 1C) could be obtained from fragmented zygotic embryos. Light was absolutely
essential for induction of somatic embryogenesis.
2.2.3. Establishment and Maintenance of Embryogenic Cultures
1.
activated charcoal (Table 2).
2. The cultures are maintained in continuous light (25 µmol m –2 s –1 The intact and fragmented zygotic embryos are placed on media with
) from cool
white fluorescent lamps at 25 ± 2° C.
3. Formation of green somatic embryos takes place after 3–5 weeks.