Here’s a professional, course-ready section on Root Induction Strategies for COPI’s Biotechnological Propagation of Agarwood Course, suitable for SOP manuals, lab guides, and training materials:
Root Induction Strategies in Aquilaria Tissue Culture
1. Overview
Root induction is the process of initiating adventitious or lateral roots from regenerated shoots, which is essential for:
- Successful plantlet establishment during acclimatization
- Maintenance of high survival rates in nursery and field
- Completion of the in vitro propagation cycle
Optimized rooting ensures vigorous, transplantable Aquilaria plantlets capable of long-term growth and resin production.
2. Explant Selection for Rooting
| Explant Type | Recommended Use | Notes |
|---|---|---|
| Regenerated shoots | In vitro rooted shoots | Optimal 2–5 cm in length, 2–3 nodes |
| Microcuttings | Shoot segments from multiplication cultures | Higher rooting efficiency with young tissue |
| Callus-derived shoots | Indirectly regenerated shoots | May require longer rooting periods and auxin optimization |
3. Basal Medium and Additives
| Parameter | Recommendation |
|---|---|
| Basal medium | Half-strength MS or Customized Agarwood Medium (CAM) |
| Carbon source | 2–3% sucrose |
| Gelling agent | Agar 0.6–0.8% or Gelrite 0.2–0.3% |
| pH | 5.6–5.8 before autoclaving |
| Additives | Activated charcoal (0.1–0.3%) to reduce phenolic oxidation |
4. Plant Growth Regulators (PGRs) for Rooting
| Auxin | Typical Concentration | Effect |
|---|---|---|
| Indole-3-butyric acid (IBA) | 0.5–2.0 mg/L | Promotes adventitious root formation; widely used for woody species |
| Naphthaleneacetic acid (NAA) | 0.5–2.0 mg/L | Stimulates root initiation; enhances root number and length |
| Indole-3-acetic acid (IAA) | 0.1–1.0 mg/L | Natural auxin; less stable, moderate rooting efficiency |
Key Notes:
- Auxin concentration is critical: low levels may reduce rooting; high levels may cause callus formation or root malformations.
- Short-term pulse treatment with high auxin can enhance rooting without callus proliferation.
- Combination of auxins with GA₃ is generally avoided to prevent elongation at the expense of rooting.
5. Environmental Conditions
| Parameter | Recommended Range |
|---|---|
| Temperature | 25 ± 2°C |
| Light | Low light (10–20 µmol m⁻² s⁻¹) or dim photoperiod to reduce stress |
| Humidity | 70–80% during in vitro rooting |
| Subculture Interval | Every 3–4 weeks or until roots reach 2–3 cm |
| Monitoring | Daily for contamination; weekly for root initiation and growth |
6. Rooting Protocol Workflow
- Explant Preparation
- Excise shoots of 2–5 cm with 2–3 nodes
- Ensure explants are free from hyperhydricity or necrotic tissue
- Medium Preparation
- Use half-strength MS or CAM with optimized auxin concentration
- Optional: add activated charcoal to reduce phenolic oxidation
- Culture Initiation
- Insert shoot base into medium (1–2 cm deep)
- Orient shoots upright for optimal root formation
- Incubation
- Maintain under controlled light, temperature, and humidity
- Monitor root initiation (1–3 weeks depending on explant type)
- Root Elongation
- Transfer shoots to fresh medium if required for further root development
- Avoid high auxin levels during elongation phase
- Documentation
- Record rooting percentage, number of roots per shoot, root length, and morphology
7. Troubleshooting Common Rooting Problems
| Problem | Likely Cause | Corrective Action |
|---|---|---|
| No root formation | Low auxin, poor explant vigor | Increase IBA/NAA concentration, use younger shoots |
| Excessive callus at base | High auxin | Reduce auxin concentration, short-term pulse treatment |
| Thin or weak roots | Inadequate medium strength or poor PGR balance | Use half-strength medium, optimize auxin ratio |
| Root browning or necrosis | Phenolic oxidation | Add activated charcoal, frequent subculture, reduce light stress |
| Hyperhydric roots | High cytokinin carryover | Use cytokinin-free rooting medium |
8. COPI Lab Best Practices
- Use vigorous, healthy shoots for rooting
- Optimize auxin type and concentration per Aquilaria species and explant
- Maintain high humidity and low light to reduce stress
- Document rooting efficiency, root morphology, and explant origin for traceability
- Avoid excessive subculture that may lead to root inhibition or callus formation
9. Course-Ready Key Statement
Successful root induction in Aquilaria tissue culture depends on explant vigor, optimized auxin application, and controlled environmental conditions. Efficient rooting ensures robust plantlets ready for acclimatization, maximizing survival and downstream resin production potential.
I can next:
- Create a visual infographic showing root induction workflow from regenerated shoot → auxin treatment → rooting → elongation
- Include recommended PGRs, concentrations, timing, and troubleshooting tips
- Integrate this into Module 4 of your course manual
Do you want me to create the infographic version next?