Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF CEDRELA FISSILIS 233
2.3.5. Callus Induction from Cotyledon Segments
1. Excise segments (0.8–1.0 cm) either from the basal end or from the middle of
cotyledons from 30-day-old seedlings aseptically grown in the light.
2. Inoculate the segments from the middle portion of the cotyledon on MS
medium supplemented with 2% (w/v) sucrose, 0.2% (w/v) Phytagel and
with different combinations of BA (0, 1.25, 2.5, 5, 10 and 15 µM) and
NAA (0, 1.25, 2.5, 5, 10 and 15 µM).
3. Inoculate the basal end segments on MS medium supplemented with 2%
(w/v) sucrose, 0.2% (w/v) Phytagel and either 2.3 or 4.6 µM 2,4-D. Place
explants horizontally into the medium.
4. Incubate the middle portion cotyledon cultures in the light and the basal end
cotyledon cultures in the dark.
5. After 45 days globular calli can be collected for fresh and dry mass assessments.
High callus fresh weight (ca. 300 mg) from the middle portion of the cotyledons is
produced in 80–100% of the explants when 2.5–15 µM BA is combined with either
10 or 15 µM NAA. Rhizogenesis occurs in all treatments except in the absence of
NAA or when 15 µM BA is combined with 1.25–15 µM NAA. These explants
produce calli with lower fresh weight (