Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF CEDRELA FISSILIS 229
Table 2. Composition of culture media used for callus induction and direct organogenesis of
Cedrela fissilis. Basic MS medium containing 20 g⋅l –1 sucrose, 2 g⋅l –1 phytagel, and plant
growth regulators in appropriate concentrations was used for different objectives. The
photosynthetic photon flux at culture level was 20–25 µmol m –2 s –1 .
Objective Explant *PGR (µM) Culture
condition
Callus induction Cotyledonary
and epicotyl
nodes
BA (2.5) + NAA (2.5–5) light
Epicotyl nodes BA (2.5) + NAA (2.5–5) light
NAA (2.7) light or dark
2,4-D (1.15–4.6) light
2,4-D (4.6) dark
Root segments 2,4-D (2.3–4.6) dark
Cotyledon basal 2,4-D (2.3–4.6)
end segments
dark
Cotyledon
middle portion
segments
BA (2.5–15) + NAA (10–15) light
Direct organogenesis Intact cotyledon BA (0.6) light
(shoots)
TDZ (0.3) light
TDZ (0.6) dark
Direct organogenesis Intact cotyledon — light or dark
(roots) Seedlings NAA (10.74–21.48) dark
*PGR = Plant Growth Regulator
2.3.2. Headspace Solid Phase Micro Extraction (SPME) and Mass Spectrometer
Analysis Procedure for Rapid Screening of Calli for Production of Volatiles
1. SPME device and fiber of polydimethylsiloxane (PDMS) (100 µm) were
obtained from Supelco (Bellefonte, PA, USA). Condition the fiber thermally
(300°C) prior to their first absorption in the hot port of the gas
chromatograph instrument according to the supplier’s instructions.
2. Collect a portion of 0.5 g (wet weight) of fresh callus produced after 8
weeks culture on MS basal medium supplemented with 2% sucrose, 2.5 µM
BA, 5 µM NAA and 0.2 % (w/v) Phytagel under either light or dark and
smash it gently.
3. Add a portion of 10-mL tissue sample in a 16-mL vial containing a
magnetic spin bar (PTFE) and seal the vial immediately with a septum to
prevent sample evaporation.
4. Pierce the septum vial with the protecting needle and expose the PDMS
fiber to the sample headspace. After 1 h of extraction of the organic compounds
from the headspace onto the fiber, with the maximum agitation of
the sample, withdraw the fiber into the needle, remove from the vial and
immediately insert into the heated GC-MS injection port.