🌱 Plant Tissue Culture Troubleshooting Manual (Gellan Gum Edition)

🌱 Plant Tissue Culture Troubleshooting Manual (Gellan Gum Edition)

🔬 Powered by CINOGEL BIOTECH

🌐 www.cinogel.com | www.gellangum.net

📚 Table of Contents

🧪 1.Introduction to Gellan Gum in Tissue Culture

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. Whether using high acyl (HA) or low acyl (LA) gellan gum, 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: Glassy, translucent, water-soaked plantlets.

Causes:

  • High relative humidity in containers
  • Medium too soft or too rich in nutrients
  • Overuse of cytokinins

Solutions:

  • Use high acyl gellan gum to create softer, more breathable gels
  • Reduce cytokinin levels
  • Improve airflow or open culture lids periodically

3.5 🦠 Contamination Issues

Symptoms: Presence of mold, yeast, or bacteria.

Causes:

  • Poor sterilization of tools or media
  • Improper aseptic techniques

Solutions:

  • Ensure all equipment is autoclaved properly
  • Disinfect workspace thoroughly
  • Use laminar flow hood and sterile gloves

3.6 ⚖️ pH-Related Gel Instability

Symptoms: Gel degrades quickly or never sets.

Causes:

  • pH outside ideal range (5.4–5.8)
  • pH drops during autoclaving

Solutions:

  • Always check and adjust pH before autoclaving
  • Use a pH buffer if needed

3.7 ⚗️ Precipitation or Cloudiness in Medium

Symptoms: White particles or turbidity after sterilization.

Causes:

  • Incompatible salts or vitamins
  • Incorrect pH before autoclaving

Solutions:

  • Double-check media formulation
  • Filter-sterilize sensitive ingredients separately

3.8 🥄 Uneven Gel Distribution in Containers

Symptoms: Gel thickness varies across plates or tubes.

Causes:

  • Improper pouring techniques
  • Surface not level during cooling

Solutions:

  • Place culture vessels on a flat surface
  • Use automatic media dispensers if possible

3.9 🔥 Gel Cracking

Symptoms: Cracks appear on gel surface during incubation.

Causes:

  • Gel too dry due to low humidity
  • Too high gellan concentration

Solutions:

  • Maintain proper humidity in growth chamber
  • Adjust gel formulation for better elasticity

3.10 🧼 Microbial Contamination Due to Poor Handling

Symptoms: Repeated mold or bacterial outbreaks.

Causes:

  • Inadequate aseptic training
  • Improper handling during subcultures

Solutions:

  • Train staff in sterile technique
  • Use disposable sterile tools where possible

3.11 ⚙️ Inconsistent Gelation Between Different Batch Sizes

Symptoms: Gels vary from batch to batch.

Causes:

  • Manual weighing errors
  • Uneven mixing of gellan gum powder

Solutions:

  • Use precision balances
  • Pre-mix dry ingredients thoroughly before dissolving

3.12 🪟 Reduced Gel Clarity or Transparency

Symptoms: Gel appears hazy or milky.

Causes:

  • Too much calcium or phosphate salts
  • Overheating during sterilization

Solutions:

  • Optimize calcium/phosphate ratio
  • Follow proper sterilization guidelines

3.13 🌿 Non-Uniform Plant Growth

Symptoms: Some explants thrive while others lag.

Causes:

  • Uneven media composition
  • Variation in explant quality

Solutions:

  • Ensure homogeneity during media preparation
  • Use similar size and age explants

3.14 🌱 Poor Rooting Performance

Symptoms: Shoots form but fail to root.

Causes:

  • Lack of auxins in rooting media
  • Overly firm or dry gel base

Solutions:

  • Include IBA or NAA in the rooting medium
  • Use HA gellan gum for a moister environment

📏 4.Tips for Optimal Gellan Gum Use

  • Always use deionized water for media preparation.
  • Adjust pH to 5.6–5.8 before autoclaving.
  • Add calcium or magnesium salts for better gel strength.
  • Autoclave at 121°C for 15–20 minutes only.
  • Avoid reheating gellan-based media repeatedly.

🧴 5.Recommended Concentrations and Combinations

  • LA Gellan Gum: 2.5–3.5 g/L (clear, firm gel)
  • HA Gellan Gum: 2.0–3.0 g/L (soft, elastic gel)
  • With Calcium Chloride: 0.5–1.0 mM for enhanced setting

🧠 6.Final Checklist for Success

  1. Use the correct gellan gum type for your crop.
  2. Always weigh ingredients precisely.
  3. Check autoclave pressure and time settings.
  4. Inspect gel texture and clarity before inoculation.
  5. Keep records of batch performance for each medium.

✅ 7.Summary

Mastering plant tissue culture requires consistency, sterility, and a thorough understanding of your gelling agent. Gellan gum—when properly used—offers superior transparency, tunable firmness, and excellent plant compatibility. This manual helps you troubleshoot, improve, and standardize results. For premium-quality gellan gum, choose CINOGEL BIOTECH.

💼 Visit us at: www.cinogel.com | www.gellangum.net

Plant Tissue Culture Troubleshooting