Protocols for Micropropagation of Woody Trees and Fruits

2.4. Axillary Shoot Proliferation
MICROPROPAGATION OF ALBIZIA ODORATISSIMA
2.4.1. Nodes from Two-year-old Saplings
With the help of sterile forceps and a sterile surgical blade (No. 10), trim the nodal
explants (with two axillary buds) by removing the tissues at the basal end of the
explants which turned brown during the process of sterilization. This would substantially
help improve the responsiveness of the explants as the deteriorated dead
cells were removed and thereby exposing the fresh live tissues to the culture medium.
Inoculate nodal segments vertically on MS-2 medium in such a way that the axillary
bud is in contact with the medium. Buds sprout and turn green in 10–15 days and
callus develops at the basal cut end of the nodes. After 8 weeks 2–3 shoots with the
average height of 1.5 cm develop from nodes (Figure 4A).
2.4.2. Multiplication in Subculture
Take elongated shoots with 2 or 3 internodes. Cut the shoot into small segments so
that each segment includes nodal region with single axillary bud. Inoculate on MS-3
medium. After about 3–4 weeks each segment gives rise to 2 to 3 shoots which can
be further subcultured (Figure 4B).
2.4.3. In Vitro Germinated Seedling Explants
Inoculate cotyledonary nodes and leaf nodes on MS-4 medium and MS-5 medium,
respectively. Place the explants vertically with the basal end slightly inserted, 1–2
mm deep, into the medium taking care that the axillary buds are in contact with the
medium. Incubate cultures at 25 ± 2°C in the light (16-h photoperiod with a photosynthetic
photon flux (PPF) density of 60 µmol m –2 s –1 ). Within 20–25 days about
6–7 shoots with the average height of 2–2.5 cm develop from cotyledonary nodes
(Figure 4C) whereas about 3–4 shoots with the mean height of 1-1.5 cm develop from
leaf nodes (Figure 4D). These shoots can either be subcultured for further
multiplication or kept for rooting.
2.5. Adventitious Shoot Organogenesis from Hypocotyls
Inoculate hypocotyl segments vertically with the basal cut end inserted slightly 0.5
cm deep into the MS-6 medium. After incubation for about a week, callus develops
at the basal cut end of hypocotyls. However, shoot buds start emerging after 3 weeks
of incubation at both the cut ends. After about 3–4 weeks each segment gives rise to
14–15 shoots with the average height of 1.5–2 cm (Figure 4E).
2.6. Cytological Changes during Adventitious Shoot Organogenesis
The in vitro organization and morphogenesis of multicellular plants depends upon
the integration and mutual interaction of various organs, tissues and cells. There is a
need for much more intensive anatomical examination by both light and electron
microscopy to trace the emergence of organs within the initially relatively uniform cell
mass of callus or suspension culture aggregates. This part of the work was carried
out to analyze the ontogenesis of adventitious buds from hypocotyls which would
contribute to the understanding of the mechanisms controlling morphogenesis.
207