Protocols for Micropropagation of Woody Trees and Fruits

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2.4.2. In Intact Seedlings
1. Surface sterilize seeds in commercial bleach (2.5% active chlorine) with
2–3 drops of Tween 20 for 75 min.
2. Rinse three times for 10 min in sterile distilled water and culture on MS
medium supplemented with 2% (w/v) sucrose, 0.2% (w/v) Phytagel and 0,
10.74, 21.48 and 42.96 µM NAA.
3. Incubate the cultures in the dark.
4. After 60 days evaluate the cultures for morphogenic events.
Direct organogenesis (rhizogenesis) is observed in the distal portion of cotyledons of
seedlings (20–30%) grown at either at 10.74 or at 21.48 µM NAA (Figure 2C). The
competence of cotyledons to produce roots without callus formation is a clear
indication that they may also have the ability to originate shoots directly when
properly manipulated with cytokinins. These are important achievements to foster
studies on the development of consistent direct shoot/root regeneration for genetic
transformation.
3. CONCLUSION
This chapter highlights C. fissilis tissue culture and a reproducible micropropagation
protocol based shoot multiplication. The results showed that BA is indispensable for
the sprouting and multiplication of axillary buds of cotyledonary node cuttings. They
can then be used as starting plant material for further rapid multiplication of selected
genotypes and in alginate-encapsulation of in vitro derived vegetative propagules for
synthetic seed production. We established a reliable micropropagation and callus
culture protocols from juvenile seedlings of C. fissilis. This technology may provide
a valuable tool in a tree improvement program, although the study on micropropagation
from adult trees will be essential.
In vitro germplasm conservation of C. fissilis requires 6–8 subcultures per year
in fresh culture media. We have achieved in reducing maintenance cost, risk of
contamination by preserving ca. 44% viability of encapsulated shoot tips. However,
more studies are necessary to improve the efficiency of the system. Another advantage
of the described protocol is that it does not require liquid nitrogen and reduce the
cost of storage. Therefore storage of alginate-encapsulated germplasm at room temperature
is a practical alternative for the conservation of tropical forest trees.
The callus culture protocols will foster the studies on secondary metabolites and
genetic transformation systems. Lago et al. (2004) identified volatile oils from
leaves and stem barks of C. fissilis by direct analysis with GC-MS performed by
hydrodistillation in a Clevenger-type apparatus. This is a laborious method and
requires relatively large amount of fresh plant material. It is unsuitable to perform
rapid screening for volatile production of newly initiated cell cultures. Therefore, the
headspace SPME technique would be an ideal tool to probe calli for the production
of volatiles of C. fissilis. The method was validated preliminary on characterization
of callus grown in different culture conditions and it is suitable for screening of
in vitro cultures for volatile oil production.