Canarium luzonicum

Here is a comprehensive tissue culture protocol for Canarium luzonicum (locally known as Pili or Elemi tree), an important Philippine endemic valued for its fragrant resin (Manila Elemi) and edible nuts. Tissue culture of Canarium species is still considered challenging due to high phenolic exudation and low in vitro response, but protocols have been developed using shoot tip, nodal explants, and embryo culture.

???? Tissue Culture Protocol for Canarium luzonicum (Manila Elemi)

1. Explant Selection

  • Preferred explants: Juvenile nodal segments, shoot tips, or zygotic embryos
  • Source: Seedlings grown under sterile or semi-sterile conditions

2. Surface Sterilization

  • Wash in running tap water for 20–30 minutes
  • Dip in 70% ethanol for 30 seconds
  • Sterilize in:
  • 1% NaOCl + 1–2 drops Tween-20 for 10–15 minutes
  • Alternatively: 0.1% HgCl₂ for 5–8 minutes (with caution)
  • Rinse 3–5 times with sterile distilled water

3. Culture Medium

A. Initial Culture / Shoot Induction

  • Basal Medium: MS (Murashige & Skoog) or WPM (Woody Plant Medium)
  • Supplements:
  • BAP (1.0–2.0 mg/L) – promotes axillary bud break
  • NAA (0.1–0.5 mg/L) – assists in callus support
  • Activated charcoal (0.2%) – binds phenolics
  • Ascorbic acid (100 mg/L) or citric acid – reduces oxidation
  • Sucrose (30 g/L)
  • Agar (7 g/L)
  • Incubate at 25 ± 2°C with 16-hour photoperiod

4. Shoot Multiplication

  • Subculture to MS medium with:
  • BAP (1.5–2.0 mg/L) + Kinetin (0.5–1.0 mg/L)
  • Optional: GA₃ (0.1 mg/L) to elongate compact shoots
  • Subculture every 4 weeks

5. Rooting of Shoots

  • Use half-strength MS or WPM
  • Add:
  • IBA (1.0–2.0 mg/L) or NAA (0.5–1.0 mg/L)
  • Activated charcoal (0.2%) may improve rooting
  • Incubate in low light or dark for first 7 days to enhance rooting
  • Roots typically develop in 3–5 weeks

6. Acclimatization

  • Transfer to pots or trays with sterile peat moss:cocopeat:sand (1:1:1)
  • Maintain high humidity with misting or plastic domes
  • Harden for 3–4 weeks in nursery shade before full sun exposure

✅ Key Considerations

  • Phenolic exudation is a major challenge—activated charcoal and antioxidants are essential.
  • Juvenile explants respond better than mature tissues.
  • Wounding (cut ends) should be minimal and processed quickly.
  • Explants may benefit from pre-treatments with antioxidant solution (100 mg/L ascorbic acid soak for 15 minutes).

Would you like:

  • A visual flowchart summarizing the protocol?
  • A combined PDF of tissue culture protocols for Manila Elemi, Magnolia, Myristica, and Citrus species?
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