Troubleshooting Guide
Solutions to Common Problems

Bacteria, fungi, molds, and yeasts invading your sterile cultures, leading to complete culture failure. Contaminants can enter from external sources or exist internally within plant tissue as Endophytes .

Prevention Strategies

• Maintain Clean Culture Room: Regularly disinfect all surfaces with 70% ethanol
• Use Proper Work Environment: Laminar flow hood or SAB for all manipulations
• Sterilize Everything: All media, vessels, tools, and water via autoclaving (121°C, 15 psi, 15-20 minutes)
• Perfect Surface Sterilization: Use 1-10% sodium hypochlorite solution followed by multiple sterile water rinses
• Consider PPM™ ™: Plant Preservative Mixture can suppress contamination (follow dosage carefully)

Solution Steps

1. Immediate Isolation: Remove contaminated cultures from clean area
2. Assess Damage: Determine if any clean tissue remains salvageable
3. Re-sterilization: If tissue remains healthy, re-sterilize and transfer to fresh media
4. Protocol Review: Examine your aseptic technique for potential breach points
5. Environmental Check: Inspect workspace for contamination sources

Plants develop thick, glassy, brittle leaves and stems with translucent appearance. This physiological disorder results from oxidative stress and disrupted cell wall formation.

Causes

• High salt or sugar concentrations in medium
• Excessive humidity within culture vessel
• Insufficient light intensity
• Ethylene gas accumulation
• Prolonged subculture intervals
• High Cytokinin levels
• Excessive agar concentration

Control Solutions

1. Improve Ventilation: Use gas-permeable lids or periodically open vessels under sterile conditions
2. Increase Light: Boost intensity or adjust light spectrum
3. Reduce Agar: Lower concentration or switch to gellan gum
4. Shorten Subcultures: Transfer to fresh media more frequently
5. Balance Hormones: Reduce Cytokinin concentrations
6. Add Activated Charcoal: Absorbs excess gases and inhibitory compounds

Tissue and media turn brown due to Phenolic oxidation compound oxidation, particularly common in woody plant species. This inhibits cell division and reduces regeneration capacity.

Prevention Solutions

1. Activated Charcoal: Add to medium to absorb phenolic compounds
2. Antioxidants: Include ascorbic acid and citric acid to prevent oxidation
3. Polyphenol Inhibitors: Use compounds like sulfur dioxide or sodium chloride
4. Frequent Subculturing: Regular transfers prevent compound accumulation

More Solutions

5. Light Optimization: Reduce exposure to minimize phenolic synthesis
6. Pre-treatment: Soak explants in water to leach phenolic compounds
7. Explant Orientation: Try vertical placement to reduce browning

Recalcitrant plant cells or tissues fail to adapt to in vitro conditions due to free radical damage to cellular macromolecules, causing complete dysfunction.

Overcoming Strategies

1. Use Juvenile Material: Young plant tissues are more responsive to culture
2. Pre-treatment: Spray donor plants with cytokinins to rejuvenate tissues
3. Media Experimentation: Try different formulations with varying hormone concentrations

More Strategies

4. Explant Diversity: Test different tissue types (shoot tips, nodes, embryos)
5. Environment Optimization: Adjust light, temperature, and humidity conditions
6. Patience and Persistence: Some species require multiple attempts with slight modifications

Genetic or phenotypic variations arise from chromosomal alterations or DNA sequence changes during culture, affecting plant stability and uniformity.

Minimization Techniques

1. Limit Culture Duration: Minimize time in culture and number of subcultures
2. Add Salicylic Acid: Growth regulator shown to reduce variation occurrence
3. Gentle Handling: Prevent physical damage during transfers (avoid hot instruments)
4. Avoid 2,4-D : High synthetic Auxin concentrations increase variation

More Techniques

5. Use Axillary Buds: More genetically stable than callus culture
6. Minimize Callus Phase: If using Callus , keep this stage as short as possible
7. Fresh Starts: Periodically reinitiate cultures from new explants

Plants grown in sterile, controlled environments experience shock and die when transferred to natural greenhouse or soil conditions.

Success Strategies

1. Strong Root Development: Optimize rooting medium before transplanting
2. Gradual Humidity Reduction: Progressively open culture vessels over days/weeks
3. Proper Potting Mix: Use well-draining, sterile growing medium

More Strategies

4. Initial Protection: Maintain high humidity and shade during transition
5. Gradual Exposure: Slowly introduce higher light intensity and lower humidity
6. Monitor Closely: Watch for wilting signs and adjust conditions accordingly

Severe, rapid phenolic oxidation occurring immediately after explant excision—a critical form of tissue damage.

Prevention Steps

1. Work Quickly: Minimize time between cutting and culture placement
2. Antioxidant Solution: Immediately immerse explants in chilled ascorbic acid or citric acid

More Steps

3. Activated Charcoal: Include in initial culture medium
4. Cold Treatment: Keep explants and solutions chilled during preparation

Improper media formulation leads to essential nutrient deficiencies, severely affecting growth and development.

Prevention Solutions

1. Balanced Media: Ensure proper macro- and micronutrient ratios
2. Proven Formulations: Start with tested formulations like MS medium

More Solutions

3. Species-Specific Adjustments: Modify standard media for particular plant needs
4. Quality Control: Use fresh, high-quality chemicals and distilled water

Excessive undifferentiated callus formation hinders shoot or root development, leading to poor-quality plants.

Control Methods

1. Adjust Hormone Balance: Increase Cytokinin -to- Auxin ratio for shoot development
2. Fresh Medium Transfer: Move callus to medium with adjusted hormone levels
3. Regular Monitoring: Track hormone ratios and adjust based on response

More Methods

4. Ethylene Management: Use inhibitors or reduce ethylene production
5. Antioxidants: Add compounds to enhance cell wall integrity

Tissue cultures grow too slowly, significantly delaying the propagation process and reducing efficiency.

Acceleration Solutions

1. Environment Optimization: Perfect temperature (22°C-25°C) and light conditions
2. Growth Stimulants: Add gibberellic acid ( GA3 ) to accelerate development

More Solutions

3. pH Adjustment: Ensure media pH is correctly adjusted (pH 5.6-pH 5.8)
4. Fresh Media: Replace old or degraded medium with fresh preparations

Some species struggle to develop healthy, functional root systems in vitro, preventing successful acclimatization.

Solutions

1. Hormone Adjustment: Include IBA or other rooting hormones in medium
2. Aeration: Provide root aeration in liquid culture systems
3. Dedicated Rooting Medium: Transfer shoots to high- Auxin , low- Cytokinin medium

More Solutions

4. Environmental Modification: Darken culture vessels during rooting phase
5. Gelling Agent Changes: Try different agents or reduce agar concentration

Step 1: Observation and Documentation

• Record all symptoms with photos and detailed notes
• Track environmental conditions and media compositions
• Note timing of problem appearance

Step 2: Systematic Analysis

• Review recent protocol changes or environmental shifts
• Check equipment functionality and calibration
• Examine chemical quality and storage conditions

Step 3: Controlled Testing

• Make single variable changes to isolate causes
• Test solutions on small culture batches first
• Compare with control cultures when possible

Step 4: Implementation and Monitoring

• Apply solutions systematically across affected cultures
• Monitor progress with regular observations
• Adjust approaches based on response