Protocols for Micropropagation of Woody Trees and Fruits

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E.C. NUNES ET AL.
5. Use a Hewlett Packard 5890 II capillary gas chromatograph equipped with
a mass spectrometer and a split-splitless injector to carry out the HS-
SPME-GC experiments.
6. Use a HP-5 fused silica capillary column of 30 m × 0.25 mm ID and a
phase thickness of 0.25 µm for GC separations of the pre-concentrated
compounds.
7. Set the temperature program for the analyses as follows: hold the initial
temperature of 35 C for 3 min and then increase to 280 at 10°C min –1 . Set
the injector and detector temperatures at 250 and 280°C, respectively. The
run time is 28.6 min. Use helium as a carrier gas at a flow rate of 1.0 mL
min –1 °
.
8. Inject the samples in the splitless mode. Operate the spectrometer in electron
impact mode (EI) with 70 eV detection volts and scan range of 35–450 m/z.
Although the CG-MS profiles do not correspond to the actual composition of the
sample due to the nature of the equilibrium in the SPME method, this method is
suitable for extracting and pre-concentrating the volatile compounds. It helps in
comparing chromatographic profiles of different samples extracted under similar
experimental conditions. Comparison of retention indexes and mass spectra allows
the identification of 13 compounds (eight monoterpenes, three sesquiterpenes and
two alkanes), of the 21 volatiles detected by the headspace SPME technique in
callus grown in the light (Table 3). Ten compounds were identified in callus cultured
in the dark. Tridecane, germacrene D and α-alaskene were detected only in callus
produced in the light. Only germacrene D was previously reported as a constituent of
volatile oils from leaves and stem bark of C. fissilis.
2.3.3. In Vitro Low Temperature Storage of Epicotyl Nodes and Shoot Tips
for Callus Production
1. Inoculate either epicotyl node cuttings or shoot tips (0.8–1.0 cm long)
obtained from 30-day-old aseptically grown seedlings on MS medium
supplemented with 2% (w/v) sucrose and 0.2% (w/v) Phytagel.
2. Incubate the cultures at 5°C in the dark.
3. After storage transfer the explants to MS medium supplemented with 2%
(w/v) sucrose, 0.2% (w/v) Phytagel, 2.5 µM BA and 5.0 µM NAA.
4. Incubate the cultures at 25°C in the light.
5. After 8 weeks collect callus to evaluate the fresh and dry mass and for the
secondary metabolism studies.
The explants withstand storage at 5°C in the dark up to 8 weeks. No differences in
callus dry weight are detected when the explants are stored at low temperature when
compared with the explants stored at 25°C. However, callus from stored epicotyl
nodes shows higher dry weight (ca. 56 mg) than the shoot tips (ca. 38 mg). Low
temperature storage of vegetative propagules is very simple and effective method for
short-term germplasm storage of callus for secondary metabolism studies.