Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF CEDRELA FISSILIS 227
2.2.4. Post-storage Recovery
1. After 3, 6 and 9 months of storage transfer the alginate-encapsulated
explants to recovery medium consisting of MS medium supplemented with
2% (w/v) sucrose, 0.2% (w/v) Phytagel and 2.5 µM BA.
2. Evaluate shoot regeneration after 14 days incubation in the recovery
medium.
3. Transfer the microshoots to MS medium containing 2% (w/v) sucrose,
0.2% (w/v) Phytagel and 2.5 µM IBA (microshoots from cotyledonary
nodes) and 5.0 µM IBA (microshoots from encapsulated shoot tips).
4. After 45 days the microplants are ready for acclimatization.
Nunes et al. (2003) observed high regeneration rates (90–100%) for propagules
exposed to all treatments. They survive storage at 25°C for 3 months, however, after
6 months storage, survival of encapsulated shoot tips on 0.4% (w/v) agar decreases
to 40–44%. This value is significantly greater than those obtained for cotyledonary
nodes, at all agar concentrations tested, and when compared to shoot tips on 1%
(w/v) agar. After 9 months storage the viability of the explants declines drastically
(6–8%). Plants regenerated from encapsulated cotyledonary nodes and apical shoot
tips did not show morphological variation after 15 days in the recovery medium
(Figure 1E, F). Maximum rooting (58%) occurs in plantlets derived from encapsulated
cotyledonary nodes, in 2.5 µM IBA, and from shoot tips, in 5.0 µM IBA. Vegetative
propagules for synthetic seed production and germplasm storage at above freezing
temperatures have been reported in Cedrela odorata, Guazuma crinita and Jacaranda
mimosaefolia (Maruyama et al., 1997a).
2.3. Callus Induction
The culture media for different protocols of callus induction are shown in Table 2.
2.3.1. Callus Induction from Cotyledonary Nodes and Shoot Tips
1. Inoculate either cotyledonary node cuttings or epicotyl node cuttings (0.8–1.0
cm long) obtained from 30-day-old aseptically grown seedlings on MS
medium supplemented with 2% (w/v) sucrose, 0.2% (w/v) Phytagel and
combinations of 2.5 µM BA and 5µM α-naphthalene acetic acid (NAA).
Place explants horizontally into the medium.
2. Incubate cultures at 25°C in either light (under 16-h photoperiod and
photon flux of 20–25 µmol⋅ m –2 ⋅s –1 at culture level, supplied by fluorescent
light tubes) or in the dark.
3. After 8 weeks evaluate the callus fresh and dry mass.
For both cotyledonary and epicotyl node explants, high callus fresh weight (ca. 1.3
g, for cotyledonary nodes and 0.7–0.9 g, for epicotyl nodes) is obtained on the
culture medium containing 2.5 µM BA and 2.5 or 5 µM NAA (Nunes et al., 2002).
When BA and NAA are used separately, the fresh weight of calli is decreased.
Differences in dry weight are detected when cotyledonary node-derived calli grow in