Tissue Culture (Micropropagation) Protocol for Aquilaria malaccensis, designed for elite mother tree cloning and Agaris™ Elite production. This is suitable for research, commercial nursery SOPs, or training manuals.
- Explant Selection & Preparation
Source Material:
- Young, actively growing shoots from elite mother trees
- Apical or nodal segments preferred (≈1–2 cm)
Surface Sterilization:
- Rinse explants under running water (15–30 min)
- Wash with mild detergent + rinse thoroughly
- Treat with 70% ethanol (30 sec–1 min)
- Immerse in 0.1–0.5% sodium hypochlorite solution (10–15 min)
- Rinse 3–5 times with sterile distilled water
Notes:
- Work under laminar airflow
- Minimize explant damage to reduce contamination
2. Culture Medium Preparation
Basal Medium:
- Murashige & Skoog (MS) medium, full strength
Additives:
- Sucrose 3% (w/v)
- Plant growth regulators (depending on stage):
- Shoot induction: BAP 1–3 mg/L, NAA 0.1 mg/L
- Shoot multiplication: BAP 0.5–1.5 mg/L
- Rooting: IBA 0.5–1 mg/L
Gelling Agent:
- Agar 0.7–0.8%
pH Adjustment:
- Adjust to 5.7–5.8 before autoclaving
3️⃣ Culture Conditions
| Parameter | Optimal Condition |
|---|---|
| Temperature | 25 ± 2°C |
| Light | 16 h photoperiod, 40–50 μmol m⁻² s⁻¹ |
| Relative Humidity | 50–60% |
| Subculture Interval | Every 3–4 weeks |
4️⃣ Micropropagation Stages
Stage 1: Establishment
- Place sterilized explants on shoot induction medium
- Monitor for contamination and initial growth (2–3 weeks)
Stage 2: Shoot Multiplication
- Transfer growing shoots to multiplication medium
- Encourage lateral shoot proliferation
- Repeat subculture 2–3 times until sufficient shoots are produced
Stage 3: Rooting
- Transfer 3–4 cm shoots to rooting medium (IBA supplemented)
- Root induction in 3–5 weeks
Stage 4: Acclimatization
- Gently remove plantlets from culture medium
- Wash agar residues from roots
- Plant in sterilized potting mix (cocopeat + sand or peat)
- Maintain high humidity (mist chamber / plastic covering)
- Gradually reduce humidity over 2–3 weeks
- Transfer to nursery under partial shade before field deployment
5️⃣ Quality & Genetic Verification
- Morphological Assessment: uniform growth, leaf health
- Molecular Verification: DNA barcoding (ITS/matK) to confirm identity
- Batch Documentation: Track mother tree, culture batch, propagation date
Optional: Integrate QR code for Agaris™ Elite molecular certification
6️⃣ Notes & Best Practices
- Minimize contamination: work carefully under sterile conditions
- Use healthy mother trees to ensure elite traits
- Rotate media batches to avoid hormonal imbalance
- Maintain records for each subculture to track growth performance
- Consider scaling in temporary immersion bioreactors for mass production
7️⃣ Timeline Overview
| Stage | Duration |
|---|---|
| Explant preparation & sterilization | 1–2 days |
| Shoot induction | 2–3 weeks |
| Shoot multiplication | 2–3 subcultures (6–9 weeks) |
| Rooting | 3–5 weeks |
| Acclimatization & nursery | 3–4 weeks |
| Total time from explant to field-ready plantlet | ~3–4 months |
If you like, I can next create a visual infographic of this Tissue Culture Protocol, similar in style to your Seed Germination infographic, showing stages, media, growth progression, and Agaris™ Elite branding integration.
Do you want me to make that infographic?