Protocols for Micropropagation of Woody Trees and Fruits

2.5. Regeneration
MICROPROPAGATION OF ELITE NEEM TREE 351
2.5.1. Node
Culture nodal explants either on Nitsch’s medium for bud break (Figure 1a) or on
shoot regeneration medium (NB1) for 6 weeks where multiple (Figure 1c) or pseudomultiple
shoots (Figure 1d) will regenerate. Transfer these explants to multiple shoot
induction medium (NB2) for another 6–8 weeks. The nodal explants will develop
numerous accessory shoots on this medium (Figure 1e).
Multiple shoots will differentiate in nodal regions in two ways, i.e. (i) several
shoots will be induced in the nodal region in a cluster (Figure 2a) where it will be
difficult to count the exact number of shoots, and (ii) in some explants, a shoot will
emerge through bud break, later other shoot buds will develop near its base (Figure 2b).
The percentage of explants forming multiple shoots and the number of shoots per
explant, will be higher on medium containing adenine sulphate (Ad.S., NB2 medium).
2.5.2. Microcuttings
Cut the axenically raised shoots into 1 - 1.5 cm long pieces (Figure 2c) and use
them as microcuttings. Inoculate them on multiple shoot induction medium where
they multiply profusely (Figures 2d,e). Culture the in vitro raised smaller shoots on
shoot elongation medium (NB3) before inducing roots (Figures 2h,i).
2.5.3. Apical Meristem
Excise the apical meristem (Figure 2f), specially if the nodal explants are infested
with inborn bacterial infection which is a very common problem in tree tissue cultures,
and rear them on meristem culture medium (NB4). Within 10–15 days. shoot buds
will be visible (Figure 2f) which will grow into healthy shoots (Figure 2g).
2.5.4. Elongation of Shoots
Subculture in vitro raised small shoots on gibberellic acid supplemented medium
(NB3) for elongation (Figures 2h,i). Within 20–25 days of culture, explants will start
elongating and after 8 weeks explants will have several healthy elongated shoots.
2.6. Rhizogenesis
Excise 2–3 cm long in vitro raised or elongated shoots (on elongation medium) and
culture on rooting medium.
2.6.1. Rooting of Excised Shoots
Culture in vitro raised shoots on either (i) ½ strength Nitsch’s (Figure 3a) or (ii) ½
strength Knop’s medium supplemented with 0.02 mg l –1 activated charcoal (Figure
3b). Roots will be induced at the base of shoots, within 15–20 days of inoculation.