Protocols for Micropropagation of Woody Trees and Fruits

2.5. Initiation of Aseptic Stock Cultures
MICROPROPAGATION OF CRESCENTIA CUJETE L. 431
1. Individual seedlings are used to establish axenic cultures.
2. Seedlings are maintained on a medium containing MS salts, B5 vitamins,
3% sucrose, and 1.0 µM kinetin (Figure 2A).
3. Once aseptic cultures are established, de novo regeneration is induced on
petiole explants from the axenic cultures.
4. Transfer petiole sections to C. cujete shooting (CS) medium containing 10
µM thidiazuron (TDZ) in combination with 2.5 µM 2, 4-dicholorphenoxyacetic
acid (Figure 3), and subculture every 3 weeks.
5. Grow cultures for about 6 weeks in a culture growth room adjusted to 25°C
± 2 C with a 16 h photoperiod (25-40 µmol m –2 s –1 °
) provided by cool white
fluorescent lamps (Figure 2B–D).
Figure 2. Micropropagation of Crescentia cujete L. a) In vitro germinated seedlings (bar = 5 cm).
b) Callusing of the petiole explants after 28 days (bar = 5 mm). c) De novo shoot organogenesis
on petiole explants on a medium supplemented with 5 µM TDZ (bar = 5 mm). d) Protrusion of
shoot and root primordial (bar = 5 mm). e) Plantlets grown on solid MS medium with 1.0 µM
kinetin (Bar = 3 cm). f) Plantlets grown in liquid MS medium with 1.0 µM kinetin (bar = 3 cm).
g) Plantlets grown in temporary immersion system with liquid MS medium with 1.0 µM kinetin
(bar = 3cm). h) Mature plant grown in the greenhouse 1 month after transplanting (bar = 5 cm).