MSc

The study under the title "Micropropagation and evaluation of
azadirachtin production in the plantlets of neem (Azadirachta indica A. Juss.)",
was carried out at the Tissue Culture Laboratory of College of Forestry and
Biochemistry laboratory, College of Horticulture, Vellanikkara during the period
2000-2002. The objective of the programme was to standardize the
micropropagation protocol and also to evaluate the secondary metabolite
production potential of in vitro produced plantlets and callus in neem (Azadirachta
indica A. Juss.).
Culture contamination mainly due to fungus was prominent in the rainy
season. To get contamination free cultures, dipping of explants in a fungicidal
mixture of 0.1 per cent each of Bavistin (Carbendazim) and Indofil M-45
(Mancozeb) for 30 min and their sterilization with mercuric chloride (0.10%) for
15 min was found effective in controlling the contamination. Larger sized explants
(2.00 cm long) with significantly low culture contamination was found to be better
than 1.00 cm long explants.
Murashige and Skoog (MS) medium was found to be better than WPM
for culture establishment and growth individual supplementation of Kn to MS
medium was found more effective than BA. MS medium supplemented with 1.5
mg r' BA + 0.5 mg r' NAA was found to be the best media for shoot proliferation.
Maximuni in vitro rooting (93.33%) of micro shoots was obtained on ~
MS + 1.5 mg r' IAA. Vermiculite and vermiculite + sand (1:1) were found to be
the best media for hardening of in vitro raised plantlets.
The auxins evaluated for stimulating callus production were, IAA, IBA
and 2,4-D among them IAA was the most potent in callusing followed by 2,4-D.
The combination of 1.5 mg r' 2,4-D + 1.5 mg r' IAA and 1.5 mg r' 2,4-D + 1.5
mg r' IBA produced maximum callus.
Azadirachtin content was estimated by using TLC and colorimetry
techniques. In the case of TLC for eluting azadirachtin into a single condensed

spot, the running solvent system comprising of methanol: water (30:70) was found
to be the best. One per cent vanillin in concentrated sulphuric acid was used as a
spray reagent to detect the azadirachtin on TLC plates. Amount of azadirachtin
varied depending on the plant part used which was estimated at various growth
stages and different concentration of growth regulators supplemented to the
medium. It ranged from 0.11 to 6.81 ug g" in in vitro plants sample and 9.42 to
12.45 ug g-I in leaves of in vivo plants. Both these methods can be followed for the
preliminary estimation of azadirachtin.