Protocols for Micropropagation of Woody Trees and Fruits

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C. LIU ET AL.
C. cujete L. is commonly grown throughout Costa Rica. However, the Calabash
tree is not native to North America and is grown only in a relatively small number of
nurseries in the United States and Canada. C. cujete is cultivated in Costa Rica
in yards and fences. The shell of Crescentia fruit are used as containers, cups
and spoons. The cups, referred to as “jícaras”, were specially used for drinking
chocolate. The indigenous people also used the fruits to make artwork. The name
“jícara” arose from the Nahualt language “xicalli”, a word used by the Native
Americans to refer to cups for drinking chocolate (León & Poveda, 2000).
Mass propagation of elite trees for commercial use as well as for the extraction
of chemically consistent medicinal preparations requires that the plants be grown in
an environment with consistent light, nutrition and temperature as well as in
isolation from abiotic and biotic contaminants (Murch et al., 2001; Saxena, 2001).
This chapter provides details of various steps involved in the micropropagation of
C. cujete including:
1. establishment of in vitro seedling cultures of C. cujete,
2. development of protocols for proliferation and maintenance of axenic
cultures in perpetuity,
3. protocols for de novo regeneration, and
4. determination of the optimal configuration of bioreactors for large-scale
production.
2.1. Plant Materials
2. PROPAGATION PROTOCOLS
Mature fruits of C. cujete can be used in micropropagation experiments. Plant material
employed in our experiments was collected from mature trees (Figure 1) in March
2001 from Mercedes Sur, Puriscal, in the Pacifico Central Conservation Area, Costa
Rica.
2.2. Explant Preparation from Donor Trees for in vitro Cultures
1. Collect mature fruits from donor trees.
2. Place the fruit explants in a 1 L beaker and wash thoroughly with running
tap water.
3. Clean the fruits with a rigorous washing them using water and soap followed
by surface flaming using 90% alcohol.
4. Sterilize seeds from opened fruits in a 3% sodium hypochlorite solution
for about 15 min with occasional swirling of the seeds to ensure uniform
exposure to the sterilant.
5. Remove the unhealthy seeds with a pair of forceps.
6. Wash the seeds 4–5 times with sterile distilled water.