Protocols for Micropropagation of Woody Trees and Fruits

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The development of in vitro culture systems for C. fissilis is therefore the basis
to ensure the introduction of genes for insect resistance, clonal propagation of
selected resistant clones, germplasm conservation, synthetic seed technology and
secondary metabolite production. The few studies available on in vitro culture of the
genus Cedrela are on micropropagation by shoot tip culture, in vitro conservation
and organogenesis by hypocotyl segment culture of C. odorata (Maruyama et al.,
1989, 1997 a,b; Cerdas et al., 1998). Micropropagation procedures have been
established for Cedrela odorata L. and Swietenia macrophylla (Lee & Rao, 1988;
Maruyama et al., 1989; Cerdas et al., 1998) and a somatic embryogenesis system
was developed for Swietenia macrophylla (Maruyama & Ishii, 1999). An efficient
micropropagation protocol was developed for C. fissilis by Nunes et al. (2002) using
nodal segments from juvenile origin for axillary shoot proliferation. Shoot prolixferation
was significantly affected by medium salt content, explant origin and 6benzyladenine
concentration. Rooting was achieved on half strength Murashige and
Skoog medium either with or without growth regulators. Regenerated plants were
successfully acclimatized on sterilized sand and for further plant development the
sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets under
ex vitro conditions was 100% after 3 months.
Storage of calcium-alginate encapsulated shoot tips of the Amazonian species
Cedrela odorata L., Guazuma crinita Mart. (Sterculiaceae) and Jacaranda mimosaefolia
D. Don. (Bignoniaceae) at above freezing temperatures (Maruyama et al., 1997a)
has been reported. Cryopreservation, using a vitrification procedure has also been
undertaken for Guazuma crinita Mart. bud clusters (Maruyama et al., 1997b). The
development of germplasm conservation programs for Cedrela spp. is of increasing
importance and these should include biotechnological techniques (Nunes et al.,
2003) including tissue culture (artificial seeds comprising alginate-encapsulated
vegetative propagules) and cryopreservation.
Rapid screening at the callus level is required to select the most promising
culture conditions for the establishment of ideal plant cell suspension cultures to
produce secondary metabolites. The solid phase micro extraction technique (SPME)
was successfully used to extract volatile compounds released by small samples of
embryogenic cultures and somatic embryos of sweet oranges (Alonzo et al., 2001). A
sensitive headspace solid phase micro extraction method was developed to analyze
the volatiles produced by callus culture of C. fissilis.
The protocols described herein are efficient and reproducible for shoot initiation,
rooting, acclimatization, artificial seeds using alginate encapsulated shoot tips and
nodal segments of C. fissilis. The chapter also deals with protocols on callus culture
initiation from juvenile seedling stock explants to study organogenesis and secondary
metabolism and a rapid and sensitive method to analyze the volatiles produced by
small samples of calli.