Tissue Culture Protocol

COPI | Propagation | Seed | Tissue Culture | Sexual | DNA | Nursery Management

Micropropagation of Aquilaria malaccensis is a tissue culture–based method used to mass-produce genetically uniform, disease-free, and high-quality planting materials. This approach is especially valuable for conservation and commercial agarwood plantation programs.

1. Explant Selection

  • Source: Young, healthy shoots from elite mother trees (ideally <1 year old).
  • Preferred explants: Shoot tips, nodal segments, and axillary buds.

2. Surface Sterilization

  • Rinse in running water (15–30 min) to remove debris.
  • Dip in 70% ethanol (30 seconds).
  • Treat with 0.1% mercuric chloride (HgCl₂) or 1% sodium hypochlorite (NaOCl) for 5–10 minutes.
  • Rinse thoroughly (3–5x) with sterile distilled water.

3. Culture Initiation

  • Medium: Murashige and Skoog (MS) basal medium
  • Supplements:
    • 3% sucrose
    • 0.7% agar
    • Growth regulators (for shoot induction):
      • BAP (6-Benzylaminopurine): 1–2 mg/L
      • NAA (Naphthaleneacetic acid): 0.1–0.5 mg/L
  • Conditions:
    • 25 ± 2°C
    • 16-hour photoperiod
    • Light intensity: ~2000–3000 lux

4. Shoot Multiplication

  • Subculture every 3–4 weeks onto fresh MS medium.
  • Use BAP (2–3 mg/L) and KIN (Kinetin, 0.5–1.0 mg/L) to enhance shoot proliferation.
  • Multiplication rate: 4–6 shoots per cycle, depending on genotype.

5. Rooting

  • Transfer elongated shoots (~3–5 cm) to rooting medium:
    • Half-strength MS medium
    • IBA (Indole-3-butyric acid) or NAA at 0.5–2 mg/L
  • Rooting duration: 2–4 weeks
  • Success rate: 60–90%, depending on genotype and auxin concentration.

6. Acclimatization

  • Transfer rooted plantlets to sterilized cocopeat, peat moss, or vermiculite in nursery trays.
  • Gradually reduce humidity using a humidity dome or intermittent mist system.
  • Hardened plants are moved to soil-polybag systems or agroforestry nursery beds.

Applications

  • Mass production of elite lines with known sex (if pre-screened).
  • Clonal propagation of resin-producing or disease-resistant phenotypes.
  • Integration into CBFMA or farm forestry schemes.
  • Supports CITES compliance through traceable, nursery-origin stock.

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