Crown Organogenesis Protocols Inc. (COPI)
Standard Operating Procedures (SOP) Manual
Agarwood (Aquilaria spp.) Tissue Culture & Organogenesis Laboratory
DOCUMENT CONTROL
- Institution: Crown Organogenesis Protocols Inc. (COPI)
- Program Alignment: Oud Academia / CI-ASASE
- Scope: Agarwood micropropagation, organogenesis, and nursery-ready plantlet production
- Compliance References: DENR, BPI, CITES, Biosafety Guidelines
- Revision Code: COPI-TC-ORG-SOP v1.0
SOP 1 – Laboratory Biosafety & Aseptic Conduct
Purpose
To ensure contamination-free operations and personnel safety in agarwood tissue culture laboratories.
Scope
Applies to all COPI staff, trainees, and researchers entering tissue culture zones.
Procedures
- Mandatory PPE: lab coat, gloves, hair cover, mask
- Entry through footbath and hand sanitation
- No food, drinks, or personal items
- Laminar airflow sterilization (UV 30 min prior use)
- Work from clean-to-dirty sequence
Records
- Daily Lab Sanitation Log
- PPE Compliance Checklist
SOP 2 – Mother Plant Selection & Explant Sourcing
Purpose
To ensure genetic quality and phytosanitary integrity of agarwood explants.
Selection Criteria
- Verified Aquilaria species identity
- Healthy, disease-free phenotype
- Known plantation or conservation origin
Explant Types
- Nodal segments
- Shoot tips
- Leaf explants (for callogenesis)
Records
- Mother Plant Traceability Sheet
- Explant Harvest Log
SOP 3 – Explant Surface Sterilization Protocol
Purpose
To eliminate surface contaminants while maintaining tissue viability.
Materials
- Distilled water
- Detergent solution
- 70% ethanol
- Sodium hypochlorite (commercial bleach)
- Sterile forceps & scalpels
Procedure (Standard Reference)
- Pre-wash explants with detergent (10 min)
- Rinse under running water (30 min)
- 70% ethanol (30 seconds)
- NaOCl solution (10–15 min, optimized)
- Triple rinse with sterile distilled water
QC Indicator
- Acceptable contamination rate: <15%
SOP 4 – Culture Media Preparation
Purpose
To ensure consistency and reproducibility of agarwood tissue culture media.
Media Types
- Murashige & Skoog (MS)
- Woody Plant Medium (WPM)
- COPI-customized agarwood media
Procedure
- Accurate weighing of salts
- pH adjustment (5.6–5.8)
- Agar/gelling agent addition
- Autoclaving (121°C, 15–20 min)
- Labeling with batch codes
Records
- Media Batch Log
- Autoclave Cycle Log
SOP 5 – Culture Initiation & Incubation
Purpose
To establish aseptic in-vitro cultures.
Procedure
- Transfer sterilized explants to initiation media
- Seal culture vessels
- Incubate at 25±2°C
- Photoperiod: 16 h light / 8 h dark
Monitoring
- Contamination check (Day 3, 7, 14)
- Initial response scoring
SOP 6 – Callus Induction
Purpose
To induce morphogenic callus for organogenesis.
PGR Framework
- Auxins (2,4-D / NAA)
- Cytokinins (BAP / Kinetin)
Procedure
- Transfer explants to callus induction media
- Incubate under reduced light
- Monitor texture, color, growth rate
QC
- Friable, non-necrotic callus preferred
SOP 7 – Organogenesis (Shoot & Root Regeneration)
Purpose
To regenerate complete agarwood plantlets.
Procedure
Shoot Induction:
- High cytokinin ratio
Root Induction:
- Auxin-dominant media
Records
- Regeneration Rate Log
SOP 8 – Subculturing & Multiplication
Purpose
To scale agarwood plantlet production.
Procedure
- Subculture every 3–4 weeks
- Limit passage number to reduce somaclonal variation
SOP 9 – Acclimatization & Hardening
Purpose
To transition in-vitro plantlets to ex-vitro conditions.
Procedure
- Gradual humidity reduction
- Use sterile substrate (cocopeat:perlite)
- Shade house adaptation
Target Survival Rate
≥80%
SOP 10 – Quality Control, Traceability & Biosecurity
Purpose
To maintain COPI’s biotech credibility and regulatory compliance.
Measures
- Unique batch codes
- SOP adherence audits
- Pathogen monitoring
ANNEXES
- Annex A: Daily Lab Checklists
- Annex B: Media Formulation Tables
- Annex C: Traceability & Label Templates
- Annex D: Training Assessment Forms
© Crown Organogenesis Protocols Inc. (COPI)
Confidential – For Training, Research, and Licensed Commercial Use Only