Protocols for Micropropagation of Woody Trees and Fruits

2.1. Establishment of Shoot Cultures
MICROPROPAGATION OF CEDRELA FISSILIS 223
2. EXPERIMENTAL PROTOCOL
The culture media for different steps of in vitro propagation are shown in Table 1.
2.1.1. Seed Surface Sterilization and Germination
1. Surface sterilize seeds of C. fissilis in commercial bleach (2.5% active
chlorine) added 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
basal medium (MS, Sigma Co., USA) (Murashige & Skoog, 1962) supplemented
with 2% (w/v) sucrose and 0.2% (w/v) Phytagel (Phytagel, Sigma
Co., USA). Adjust the pH to 5.8 before adding Phytagel and dispense the
medium into 25 × 150 mm glass culture tubes (10 ml/tube) before
autoclaving at 121°C for 18 min.
3. Seal the vessels with transparent polypropylene film (76 × 76 mm) and
place the cultures 25°C, 70% RH under a 16-h photoperiod and photosynthetic
photon flux of 20–25 µmol m –2 s –1 supplied by fluorescent light
tubes. Use these culture conditions in all the protocols mentioned below
unless otherwise stated.
After 4 weeks of in vitro growth the seedlings are about 8.2 ± 1.0 cm long, root
length of 8.6 ± 1.2 cm, and 0.505 ± 0.1 g fresh weight. They have one cotyledonary
node, two epicotyl nodes, one apical bud and 3.1 ± 0.5 leaves.
2.1.2. Shoot Initiation and Multiplication
1. Remove the cotyledonary (0.8–1.0 cm long comprising two axillary buds)
and epicotyl node cuttings (comprising two axillary buds inserted at the
base of the first pair of leaves above the cotyledons) from 30-day-old
seedlings aseptically grown in the light.
2. Place the explants horizontally and on MS medium supplemented with 2%
(w/v) sucrose, 0.2 % (w/v) Phytagel and 2.5 µM 6-benzyladenine (BA).
3. Incubate the cultures at 25°C, 70% RH under a 16-h photoperiod and
photosynthetic photon flux of 20–25 µmol m –2 s –1 supplied by fluorescent
light tubes.
Shoot initiation starts by releasing axillary buds from apical dominance on the
culture medium containing 2.5 µM BA. The highest number of nodes per explant
could then be excised for further multiplication and production of new microplants.
Originally there are only two axillary buds per explant. Calli proliferate and adventitious
buds are produced at the base of the axillary buds (Figure 1A). The multiplication
rate increases to 6–7-fold after 60–90 days in the second subculture cycle.
The subculture of each of the single node cuttings produces a new plant (Figure 1B).
After 60–90 days shoot cultures from cotyledonary nodes produce ca. 6–7 nodes per