Protocols for Micropropagation of Woody Trees and Fruits

354 B.K. BISWAS AND S.C. GUPTA
tree. Unfortunately, one will have to wait for a few years to check it because the
in vitro raised plants should first reach physiological maturity so that they can
flower and set seeds, since seeds possess the highest azadirachtin content in a plant.
Therefore, the only viable alternative is to assess genetic fidelity of the offsprings
raised from the elite neem tree through tissue culture. Though this will not guarantee
the same azadirachtin levels in the seeds produced by the offsprings but will certainly
detect if any change takes in the genomic DNA.
2.7.1. Total DNA Extraction
Extract DNA from the leaves of selected elite tree and its in vitro raised offsprings.
Extract total genomic DNA following Cetyl Trimethyl Ammonium Bromide (CTAB)
method of Murray and Thompson (1980).
Procedure
1. Grind 5 g leaves to fine powder in liquid nitrogen using a mortar and pestle.
2. Transfer the powdered tissue into a 50 ml sterile screw capped centrifuge
tube.
3. Add 20 ml CTAB extraction buffer to the tube, mix thoroughly and then
incubate the homogenate at 60°C for 1 h.
4. Add equal volume of chloroform:isoamyl alcohol (24:1) to the homogenate
and mix gently by inverting it for 10 min.
5. Centrifuge at 15,000 rpm for 10 min at 25°C.
6. Then transfer aqueous phase to 50 ml sterile glass tube.
7. Add 0.7 volume chilled isopropanol to the aqueous phase, cover with parafilm
and mix gently for about 2 min.
8. Observe the precipitation of DNA into the tube and then transfer the DNA
to 1.5 ml Eppendorf tube.
9. Add 0.75 ml 70% ethanol and keep at room temperature for 1 day.
10. Dry DNA either in vacuum drier for 30–45 min or keep the tube open for
several hours on the working bench.
11. Dissolve DNA in 1.5 ml TE buffer and store at –20°C.
2.7.2. RAPD Analysis
RAPD Analysis of Micropropagated Offsprings. To test the genomic fidelity of micropropagated
neem plants through random amplified polymorphic DNA (RAPD), take
the genomic DNA of mother plant (Figure 4a). and of micropropagated plants (Figure
4b). Presently, 13 micropropagated plants along with their parent tree have been
analysed. Isolate the DNA as described earlier. Take a set of random decamer primers
(viz. OPA, OPC and OPK, from Operon Techonologies, Alameda, California, USA) to
generate comparative RAPD profiles in AP-PCR mode.