Protocols for Micropropagation of Woody Trees and Fruits

2.3. Tissue Culture Media
MICROPROPAGATION OF CRESCENTIA CUJETE L. 429
Figure 1. Crescentia cujete L. mature tree.
C. cujete is propagated on media containing MS (Murashige & Skoog, 1962) salts,
B5 (Gamborg et al., 1968) vitamins and various growth regulators (Table 1). Media
are prepared by dissolving all chemical components in a flask containing distilled
water placed on a stirring/hot plate and the pH of the solution adjusted to 5.7. For
solidification, 2.2 g l –1 of gelling agent (Gellan gum, Sigma Chemical Co., St. Louis,
MO) is added to the medium and dissolved by heating the medium on the
stirring/hot plate. The medium is dispensed into tissue culture vessels using a
dispenser and then autoclaved at 121 C for 20 min (1.19 kg cm –2 °
).
2.4. In vitro Seedling Cultures from Seeds
1. Place the surface sterilized seeds in Petri dishes (90 mm) containing 25 ml
MS medium devoid of growth regulators.
2.
3. Culture the re-sterilized seeds individually in Petri-dishes each containing
25 ml of MSO medium (Table 1) and examine the cultures regularly to
ensure that they are free of contamination.
4. Incubate the cultures in a culture growth room at 25°C ± 2ºC with a 16 h
photoperiod (25–40 µmol m –2 s –1 If bacterial or fungal contamination is observed, sterilize the seeds from
contaminated cultures again by dipping the intact or germinated seeds in
70% alcohol for 30 s, then immerse in 10% bleach for 3–5 min, and rinse 5
times with sterilized distilled water.
) provided by cool white fluorescent lamps
(Figure 2A).
5. Seeds germinate after 2 weeks of culture on MS medium, and are transferred
to fresh medium every month.