Myristica fragrans

Here’s a detailed tissue culture protocol for Myristica fragrans (nutmeg), a valuable yet challenging species for micropropagation due to its high phenolic content and long juvenile phase:

???? Tissue Culture Protocol for Myristica fragrans (Nutmeg)

1. Explant Source:

  • Best explants: Juvenile shoot tips, nodal segments, immature zygotic embryos
  • Explants from seedlings or young trees are preferred for better regeneration

2. Surface Sterilization:

  • Rinse with running tap water for 20–30 min
  • Soak in 70% ethanol for 30 seconds
  • Treat with:
  • 0.1% HgCl₂ for 5–7 minutes or
  • 1.0% sodium hypochlorite + 1 drop Tween-20 for 10–15 minutes
  • Rinse 3–5 times with sterile distilled water

3. Culture Medium – Initiation Phase:

  • Basal Medium: MS (Murashige and Skoog) or WPM (Woody Plant Medium)
  • Supplements:
  • BAP (0.5–2.0 mg/L) – for shoot induction
  • NAA (0.1 mg/L) – to support initial callus/shoot base formation
  • Ascorbic acid (100 mg/L) or citric acid – to reduce browning
  • Activated charcoal (0.1–0.2%) if browning persists

4. Shoot Multiplication:

  • Subculture to MS + BAP (1.5–2.0 mg/L) every 4 weeks
  • Kinetin (0.5–1.0 mg/L) may be added to enhance multiple shoot development
  • Maintain cultures under 25±2°C with 16 h light/8 h dark photoperiod

5. Rooting:

  • Use half-strength MS or WPM medium
  • Auxins:
  • IBA (1.0–2.0 mg/L) or
  • NAA (0.5–1.0 mg/L)
  • Activated charcoal (0.2–0.5%) supports root development
  • Rooting typically takes 3–5 weeks

6. Acclimatization:

  • Transfer well-rooted plantlets to sterile soil mix (cocopeat + sand or peat + vermiculite)
  • Use humidity chambers or mist setups for hardening
  • Gradual transition to ambient conditions over 4–6 weeks

✅ Special Notes:

  • M. fragrans exudes excess phenolic compounds, causing browning. This can be minimized with:
  • Frequent subculturing
  • Use of antioxidants (ascorbic acid/citric acid)
  • Activated charcoal in the media
  • In vitro plantlets may require support from mycorrhizal inoculation in the acclimatization phase for better survival.

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