🌱 Plant Tissue Culture Troubleshooting Manual (Gellan Gum Edition)

🌱 Plant Tissue Culture Troubleshooting Manual (Gellan Gum Edition)

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🌐 www.cinogel.com | www.gellangum.net

📚 Table of Contents

🧪 1.Introduction to Gellan Gum in Tissue Culture

Low acyl gellan gum, a high-purity polysaccharide, plays an important role in plant tissue culture by providing a reliable, firm, and clear medium for growing plantlets. This manual will guide you through common issues and effective solutions for optimal results.

🧪 2.Common Problems in Plant Tissue Culture

🔍 Problem ❗ Symptoms
Gel too soft or fails to set Medium remains semi-liquid or runs
Gel too firm or brittle Gel is too hard to root or easily cracks
Gel syneresis Water separates from the gel after autoclaving
Vitrification Translucent, weak plants
Microbial contamination Mold, bacteria, or yeast growth
Medium pH instability Gel degrades or fails to set
Precipitation/cloudiness White particles or hazy medium
Uneven gel in vessels Irregular depth or inconsistent texture

🛠️ 3.Troubleshooting Guide

3.1 🧊 Soft or Non-Solidified Gels

Symptoms: Medium is watery or remains unset.

Causes:

  • Gellan gum concentration too low
  • Incorrect autoclaving conditions (time/temperature)
  • Inadequate calcium ions
  • pH too low before sterilization

Solutions:

  • Use 2.5–3.5 g/L of LA gellan gum
  • Ensure autoclaving at 121°C for 15–20 minutes
  • Add 1–2 mM calcium salts like CaCl₂
  • Adjust medium pH to 5.6–5.8 before autoclaving

3.2 🪨 Too Firm or Brittle Gels

Symptoms: Medium is too hard or cracks easily when touched.

Causes:

  • High gellan gum concentration
  • Excess calcium or divalent ions
  • Wrong type of gellan gum (use of LA instead of HA)

Solutions:

  • Reduce gellan gum to 2.0–2.5 g/L
  • Lower calcium levels
  • Use high acyl (HA) gellan gum for a softer texture

3.3 💧 Excessive Gel Syneresis (Water Leakage)

Symptoms: Water separates from gel after cooling or storage.

Causes:

  • Too high gellan concentration
  • Autoclaving too long
  • Incorrect pH or salt interaction

Solutions:

  • Use optimal gellan gum concentration (2.5–3.5 g/L)
  • Autoclave for recommended time only
  • Adjust medium pH before autoclaving

3.4 🌫️ Poor Plant Growth or Vitrification

Symptoms: Plants appear translucent, fragile, or hyperhydrated.

Causes:

  • Excess water availability due to soft gel
  • High relative humidity in culture vessels
  • Wrong growth regulator balance

Solutions:

  • Increase gellan gum concentration for firmer gel
  • Improve vessel ventilation
  • Adjust plant growth regulators accordingly

3.5 🦠 Contamination Issues

Symptoms: Presence of mold, bacteria, or yeast in culture vessels.

Causes:

  • Poor sterilization techniques
  • Contaminated explants or media components
  • Insufficient sealing of culture vessels

Solutions:

  • Sterilize all tools and media properly
  • Use aseptic technique during explant transfer
  • Seal vessels effectively but allow gas exchange

3.6 🧪 pH-Related Gel Instability

Symptoms: Gel fails to set or degrades after autoclaving.

Causes:

  • Medium pH outside optimal range
  • Buffer salts interacting adversely with gellan gum

Solutions:

  • Adjust medium pH to 5.6–5.8 before autoclaving
  • Use compatible buffer salts

3.7 ⚪ Precipitation or Cloudiness in Medium

Symptoms: White particles or haziness appear in the medium.

Causes:

  • Excess divalent cations
  • Improper mixing before autoclaving

Solutions:

  • Reduce calcium or magnesium salts
  • Ensure thorough mixing before sterilization

3.8 ⚖️ Uneven Gel Distribution in Containers

Symptoms: Gel level or firmness varies within the same container.

Causes:

  • Uneven pouring or setting
  • Improper temperature during solidification

Solutions:

  • Pour medium evenly and quickly
  • Allow gel to set at room temperature undisturbed

3.9 🪨 Gel Cracking

Symptoms: Visible cracks appear in the gel surface.

Causes:

  • Excessive drying
  • Too firm gel consistency

Solutions:

  • Use sealed culture vessels to prevent evaporation
  • Reduce gellan gum concentration slightly

3.10 🦠 Microbial Contamination Due to Poor Handling

Symptoms: Unexpected microbial growth in culture vessels.

Causes:

  • Handling without sterile gloves or tools
  • Exposure to open air too long during transfer

Solutions:

  • Practice strict aseptic techniques
  • Minimize exposure time during culture transfers

3.11 🔄 Inconsistent Gelation Between Different Batch Sizes

Symptoms: Gel strength varies when preparing different batch volumes.

Causes:

  • Unequal mixing during media preparation
  • Temperature differences during autoclaving

Solutions:

  • Use thorough mixing procedures
  • Maintain consistent autoclaving conditions

3.12 🔍 Reduced Gel Clarity or Transparency

Symptoms: Medium appears cloudy or less transparent.

Causes:

  • Use of low purity gellan gum
  • Excess salts or impurities

Solutions:

  • Source high purity gellan gum
  • Adjust salt concentrations carefully

3.13 🌿 Non-Uniform Plant Growth

Symptoms: Some explants grow well, others poorly.

Causes:

  • Uneven nutrient or gel distribution
  • Variable explant quality

Solutions:

  • Ensure uniform media preparation and pouring
  • Use explants of similar physiological state

3.14 🌱 Poor Rooting Performance

Symptoms: Shoots form but fail to root.

Causes:

  • Low auxin concentration in medium
  • Inappropriate gelling agent texture
  • Excessive gel firmness limiting root penetration

Solutions:

  • Increase auxin levels such as IBA or NAA
  • Use lower concentration of gellan gum for softer gel
  • Try high acyl (HA) gellan gum for more elastic gel texture

📏 4.Tips for Optimal Gellan Gum Use

  • Always measure gellan gum precisely for consistency.
  • Use high purity gellan gum from trusted suppliers like CINOGEL BIOTECH.
  • Adjust calcium ion concentration to control gel firmness.
  • Maintain medium pH between 5.6 and 5.8 before autoclaving.
  • Store gellan gum powder in a cool, dry place away from humidity.

🧴 5.Recommended Concentrations and Combinations

Gellan Gum Type Typical Concentration (g/L) Calcium Concentration (mM) Notes
Low Acyl (LA) 2.5 – 4 0.5 – 2 Firm, brittle,transparent gels, widely preferred for plant tissue culture media. Supports explants well and provides clarity.

🧠 6.Final Checklist for Success

  • ☑️ Confirm gellan gum type and purity
  • ☑️ Measure accurate gellan gum and calcium salt quantities
  • ☑️ Adjust medium pH to 5.6–5.8 before sterilization
  • ☑️ Autoclave medium at 121°C for 15–20 minutes
  • ☑️ Pour medium evenly and allow to set undisturbed
  • ☑️ Use aseptic techniques for culture handling
  • ☑️ Monitor culture vessels regularly for contamination or gel issues

✅ 7.Summary

Proper use of gellan gum in plant tissue culture is essential for healthy plant development and reproducible results. By carefully selecting the type and concentration of gellan gum, controlling calcium ions, maintaining correct pH, and following strict aseptic techniques, most common issues can be avoided or resolved.

CINOGEL BIOTECH offers high-quality gellan gum products designed specifically to meet the demanding needs of plant tissue culture laboratories worldwide.

For more information, visit www.cinogel.com or www.gellangum.net.

Plant Tissue Culture Troubleshooting