Micropropagation SOPs
Contents
- Workspace Setup
- Sterilization Protocols
- Explant Preparation
- Subculturing Techniques
- Acclimatization Methods
- Troubleshooting Guide
Workspace Setup
A proper workspace is critical for successful micropropagation with minimal contamination.
Essential Equipment
| Equipment | Purpose | Specifications | Notes |
|---|---|---|---|
| Laminar Flow Hood | Sterile workspace | HEPA filter (99.99% at 0.3μm) | Professional option, 24-36" width ideal |
| DIY Flow Hood | Budget sterile workspace | HEPA filter + box fan setup | Entry-level, less reliable |
| Glove Box | Ultra-budget option | Clear plastic container with hand holes | Limited space, less effective |
| Autoclave/Pressure Cooker | Media sterilization | 15-22 PSI capacity | Pressure cooker works for hobbyists |
| Alcohol Lamps/Bunsen Burner | Tool sterilization | Any laboratory grade | Alcohol lamps safer for home use |
| 70-95% Isopropyl Alcohol | Surface sterilization | Laboratory grade | 70% more effective than higher concentrations |
| Bleach Solution | Explant sterilization | 5-10% sodium hypochlorite | Dilute household bleach works (5.25%) |
| Sterile Tools | Precise work | Stainless steel | Forceps, scalpels, blade holders |
| Sterile Containers | Culture vessels | Glass/plastic with vented lids | Mason jars or specialized vessels |
Workspace Preparation Checklist
24 Hours Before
- Clean entire room (vacuum, dust, mop)
- Reduce airflow (close vents, seal windows)
- Prepare and run HEPA air purifier
4 Hours Before
- Sterilize workspace surfaces with 10% bleach solution
- Follow with 70% isopropyl alcohol wipedown
- Turn on laminar flow hood to equilibrate
1 Hour Before
- UV sterilize laminar flow hood (if equipped)
- Gather all necessary equipment and materials
- Prepare 70% alcohol spray bottles fresh
Immediately Before
- Wash hands thoroughly with antibacterial soap
- Wear clean lab coat or fresh clothes
- Put on mask and nitrile gloves
- Spray work area with 70% alcohol
Sterilization Protocols
Media Sterilization
| Method | Equipment | Procedure | Notes |
|---|---|---|---|
| Autoclaving | Laboratory autoclave | 121°C, 15 PSI, 15-20 minutes | Gold standard, most reliable |
| Pressure Cooking | Kitchen pressure cooker | 15 PSI, 20-25 minutes | Effective alternative, allow extra time |
| Filter Sterilization | 0.22μm syringe filters | For heat-sensitive components | Use for PGRs, antibiotics, some vitamins |
Tool Sterilization Methods
| Method | Procedure | Duration | Notes |
|---|---|---|---|
| Flame Sterilization | Heat tool tip until red hot, cool briefly | 5-10 seconds | Quick between cuts, but can damage tools |
| Alcohol + Flame | Dip in 70% alcohol, flame briefly, cool | 10-15 seconds | Less damaging to tools than direct flame |
| Alcohol Soak | Submerge in 70% alcohol | 5+ minutes | Less effective but gentler on tools |
| Autoclave/Pressure Cook | Wrap tools in aluminum foil, sterilize | Full cycle | Most thorough, prepare multiple sets |
| Bead Sterilizer | Heat tools in glass bead sterilizer | 15-30 seconds | Quick and effective, specialized equipment |
Explant Sterilization Protocol
| Plant Material | Primary Sterilant | Concentration | Duration | Secondary Treatment | Notes |
|---|---|---|---|---|---|
| Soft Tissue (young shoots) | Bleach solution | 5-10% (v/v) | 5-10 minutes | 70% alcohol rinse (30 sec) | 3-5 sterile water rinses after |
| Mature Tissue (older stems) | Bleach solution | 10-15% (v/v) | 10-15 minutes | 70% alcohol rinse (60 sec) | Longer sterilization needed |
| Field Material | Bleach solution | 15-20% (v/v) | 15-20 minutes | Fungicide pre-soak | Most contamination risk |
| Seeds | Hydrogen peroxide | 3-10% | 10-15 minutes | Optional scarification | Can soak overnight in lower concentration |
Standard Cannabis Explant Sterilization Procedure
Pre-sterilization
- Remove large debris by washing under running water (5 minutes)
- Soak in water + 2-3 drops dish soap (10 minutes)
- Rinse thoroughly with clean water
Primary Sterilization
- Prepare fresh 10% bleach solution (1 part 5.25% bleach : 9 parts water)
- Add 1-2 drops of Tween-20 or dish soap as surfactant
- Submerge tissue completely (8-12 minutes)
- Agitate occasionally
Secondary Sterilization
- Transfer to 70% ethanol or isopropyl alcohol (30-60 seconds)
- Do not exceed recommended time to avoid tissue damage
Rinse Process
- Transfer to sterile distilled water (2 minutes)
- Repeat with fresh sterile water 3 times
- Use sterile forceps for all transfers
Explant Preparation
Optimal Explant Selection
| Source Material | Size | Advantages | Disadvantages | Success Rate |
|---|---|---|---|---|
| Shoot Tips | 0.5-1.0 cm | Fast growth, true-to-type | Higher contamination risk | High (70-80%) |
| Nodal Segments | 1.0-2.0 cm | High multiplication rate | Medium contamination risk | Very High (80-90%) |
| Axillary Buds | 0.3-0.8 cm | Compact, less space needed | Slower initial growth | Medium (60-70%) |
| Meristems | 0.1-0.3 cm | Virus elimination | Technical difficulty, slower | Low-Medium (40-60%) |
| Leaf Sections | 0.5-1.0 cm² | Abundant material | Low regeneration in cannabis | Very Low (10-30%) |
Mother Plant Preparation Protocol
2 Weeks Before Explant Collection
- Move mother plants to clean environment
- Remove all soil/media from vicinity
- Apply preventative fungicide (if needed)
- Begin watering with distilled water only
1 Week Before Explant Collection
- Withhold fertilizer completely
- Continue watering with only distilled water
- Remove any unhealthy/pest-damaged tissues
- Apply systemic fungicide if pest concerns
3 Days Before Explant Collection
- Maintain plants in low-humidity environment
- Avoid overhead watering
- Bag selected branches if outdoor plants
Day of Explant Collection
- Collect material early in the day
- Select newest growth from vigorous branches
- Cut segments longer than needed
- Place in sterile container with moist paper
- Keep cool and process within 2-4 hours
Explant Cutting Procedure
Preparation
- Sterilize workspace and tools
- Prepare sterile petri dish with filter paper
- Have culture media vessels ready
Initial Trimming
- Remove all leaves from nodal segments
- Trim stems to appropriate size
- Remove any damaged or discolored tissue
Final Cutting
- Make clean 45° cuts 2-3mm below nodes
- Cut upper portion 5-10mm above node
- Ensure at least one viable bud per segment
Immediate Transfer
- Transfer to culture medium within 5 minutes
- Insert cut end into medium
- Ensure good contact with medium surface
Subculturing Techniques
Optimal Timing
| Culture Stage | Visual Indicators | Typical Timeline | Consequences of Delay |
|---|---|---|---|
| Initial Explant | Nodes, new growth | 3-4 weeks | Tissue browning, media depletion |
| Multiplication | 3-4 new shoots, 2-3cm height | 4-6 weeks | Hyperhydricity, vitrification |
| Elongation | Shoots reach jar height | 3-4 weeks | Stunting, leaf damage |
| Pre-rooting | Multiple nodes developed | 2-3 weeks | Poor rooting potential |
| Rooting | Visible root development | 2-4 weeks | Shoot deterioration |
Multiplication Stage Protocol
Preparation
- Sterilize workspace and tools
- Prepare fresh multiplication media
- Allow vessels to reach room temperature
Transfer Process
- Remove entire culture from vessel
- Place on sterile petri dish
- Identify multiplication units (nodes/shoots)
- Make clean cuts to separate units
- Remove any callus or discolored tissue
Explant Placement
- Place 3-5 uniform explants per vessel
- Ensure good medium contact
- Space evenly to allow air circulation
- Insert base 2-3mm into medium
Post-Transfer Care
- Seal vessel with proper closure
- Label with date, generation, cultivar
- Transfer to growth room within 1 hour
Rooting Protocol
Pre-rooting Preparation
- Select shoots 3-5cm long with 3+ nodes
- Transfer to elongation media 1 week before rooting
- Reduce cytokinin concentration
Rooting Induction
- Transfer to rooting medium (½MS + IBA/NAA)
- Alternative: 24h dark treatment on induction medium
- Then transfer to hormone-free medium
Root Development
- Maintain under reduced light (50-70%)
- Optimal temperature: 23-25°C
- First roots visible in 7-14 days
- Allow 2-3 weeks for adequate root system
Acclimatization Methods
Hardening Process
| Stage | Duration | Environment | Watering | Light | Success Indicators |
|---|---|---|---|---|---|
| Pre-transplant | 1 week | In vitro with vented lids | Normal | 16h, standard intensity | Thicker leaves, waxy cuticle |
| Initial | 7-10 days | Humidity dome (90-95%) | Mist 2-3x daily | 14-16h, 50% intensity | New growth, leaf expansion |
| Intermediate | 7-10 days | Partially vented dome (70-80%) | Daily light watering | 14-16h, 70% intensity | Continued growth, firm stems |
| Final | 7-10 days | Open trays (50-60%) | Every 2-3 days | Full light intensity | New leaves, visible growth |
Transfer to Soil Protocol
Medium Preparation
- Use sterile, well-draining mix (coco:perlite 70:30)
- Pre-moisten and pre-warm medium (24°C)
- Fill cell trays or small pots (2-3")
- Pre-make holes for plantlets
Plantlet Preparation
- Remove from culture vessel carefully
- Gently wash all medium from roots
- Trim any damaged/dead roots or leaves
- Optional: Dip in rooting hormone
Transplanting
- Place plantlet in pre-made hole
- Gently firm medium around roots
- Water immediately with ¼ strength nutrient
- Apply fungicide drench if available
Critical Care Period
- Maintain humidity dome for 7-10 days
- Gradually increase ventilation daily
- Protect from direct sun for 2 weeks
- Begin fertilizing at ¼ strength after 7 days
Acclimatization Environment
| Parameter | Initial Stage (Days 1-7) | Intermediate Stage (Days 8-14) | Final Stage (Days 15-21) |
|---|---|---|---|
| Humidity | 90-95% | 70-80% | 50-60% |
| Temperature | 22-24°C | 22-24°C | 20-26°C |
| Light Intensity | 50-100 μmol/m²/s | 100-200 μmol/m²/s | 200-400 μmol/m²/s |
| Photoperiod | 16 hours | 16-18 hours | 18 hours |
| Air Movement | Minimal | Gentle | Moderate |
| Watering | Misting 2-3x daily | Light watering daily | Normal watering |
Troubleshooting Guide
Common Contamination Issues
| Contaminant | Visual Signs | Common Sources | Prevention Strategies |
|---|---|---|---|
| Bacteria | Cloudy, slimy growth around explant | Poor sterilization, water sources | Longer sterilization, PPMâ„¢ addition |
| Fungi/Mold | Fuzzy/cottony growth, often colored | Air contamination, explant surface | HEPA filtration, fungicide pretreatment |
| Yeast | Shiny, creamy colonies | Tool contamination | Proper tool sterilization between transfers |
| Mites/Thrips | Tiny moving insects, webbing | Mother plants | Preventative treatments, isolation |
| Endogenous | Delayed contamination from inside plant | Systemic infection | Meristem culture, antibiotics |
Culture Development Problems
| Problem | Symptoms | Possible Causes | Solutions |
|---|---|---|---|
| Browning/Blackening | Tissue turns brown/black after transfer | Phenolic oxidation | Add activated charcoal, PVP, or antioxidants |
| Hyperhydricity | Glassy, waterlogged appearance | Excessive humidity, cytokinin | Reduce humidity, decrease cytokinin levels |
| Vitrification | Brittle, translucent growth | Hormonal imbalance | Transfer to lower cytokinin medium |
| No Growth | Explant remains unchanged | Dormancy, medium issues | Change medium formulation, cold treatment |
| Stunted Growth | Limited, abnormal growth | Contamination, toxicity | Check for latent contamination, reduce nutrients |
| Leaf Abscission | Leaves fall off | Ethylene accumulation | Use vented lids, adsorbents like silver thiosulfate |
| Poor Rooting | Few or no roots develop | Insufficient auxin, timing | Increase auxin concentration, dark treatment |
| Callus Formation | Undifferentiated tissue growth | Hormone imbalance | Reduce auxin levels, increase cytokinin |
| Shoot Tip Necrosis | Dying shoot tips | Calcium deficiency | Increase calcium in medium |
| Albinism | White/pale shoots | Light issues, genetic | Adjust light quality, discard |
Emergency Response Protocols
| Scenario | Immediate Action | Follow-up Measures | Prevention |
|---|---|---|---|
| Widespread Contamination | Isolate affected cultures | Disinfect entire workspace | Review sterilization protocols |
| Equipment Failure | Transfer cultures to backup environment | Repair or replace equipment | Maintain backup systems |
| Power Outage | Minimize opening incubator/growth room | Restore optimal conditions ASAP | Install battery backup |
| Temperature Extremes | Move cultures to temperature-controlled area | Assess damage, subcultured unaffected areas | Install monitoring systems |
| Accidental Contamination | Seal affected area, increase air filtration | Deep clean all surfaces | Improve workflow procedures |
Critical Note: Document all issues, actions taken, and outcomes to improve protocols over time.