PROTOCOL:
1. Collect immature fruits of neem from the experimental plant and thoroughly wash in 1% savlon solution for 5min, followed by rinsing with SDW.
2. Bring cleaned immature fruits inside the laminar-air-flow. After rinsing in 90% ethanol for 30sec and two changes in SDW, surface sterilize the fruits with 0.1% HgCl2 solution for 8min.
3. After washing with SDW three times, using sterile forceps, inoculate the immature fruits at early dicot stage, horizontally on either of the following media in Petriplates or test-tubes -
MS + TDZ (5µM) / MS + Kinetin (5µM) / MS+BAP (5µM)
MS + BAP (5µM) + IAA (0.1µM) / MS +BAP (2µM) + NAA (5µM) + CH (500 mgl-1)
MS +BAP (5µM) + 2iP (5µM)/ +BAP (5µM) + NAA (2µM) + CH (500 mgl-1)
4. Seal the Petriplates with parafilm or test-tube with cotton plug.
5. Label each Petriplate/test-tube with date and media combination used.
6. Incubate the cultures at 25 ± 2°C temperature and 50-60% relative humidity under a 16/8 hour (light/dark) photoperiod with diffuse light (1000-2000 lux).
7. Observe the cultures at regular intervals for callus proliferation from endosperm in split open immature seeds.
8. Remove the green embryo and subculture the calli into BA containing regeneration medium.
9. Observe the cultures for adventitious shoot proliferation from the calli, and remove the contaminated culture, if any. Record the time of shoot proliferation, number of shoots per culture and the size of each shoot in single culture. If required use the Stereo zoom microscope to see shoot development and take photographs for the record.