Why Kanamycin Loses Activity in Gellan Gum Media

Understanding antibiotic–gelling agent interactions in plant tissue culture

Gellan gum is a popular alternative to agar in modern plant tissue culture due to its clarity, consistency, and low required dosage.
However, one well-documented issue is that kanamycin often loses activity when used in gellan gum–solidified media, leading to weak or failed selection.

This guide explains why this happens and what antibiotics work better in gellan-based culture systems.


What Is Gellan Gum?

Gellan gum is a microbial polysaccharide used as a gelling agent in plant tissue culture, microbiology, and food applications.
It forms a firm, transparent gel at low concentrations and provides higher consistency than agar.

Two types are commonly used:

  • Low-acyl (LA) gellan gum – firm, brittle gels
  • High-acyl (HA) gellan gum – soft, elastic gels

Both types can influence antibiotic behavior under certain conditions.

Why Kanamycin Loses Activity in Gellan Gum Media

1. Gellan gum carries negatively charged groups

The polymer contains carboxyl groups, which introduce negative charges within the gel matrix.

2. Kanamycin is a positively charged aminoglycoside

Kanamycin includes multiple amino groups, making it strongly cationic.

3. Charge interactions reduce the free antibiotic concentration

When kanamycin is added to a gellan-based medium:

  • Ionic attraction occurs
  • Part of the kanamycin binds to the gellan structure
  • Less "free" kanamycin remains available for selection

This reduces the antibiotic's effective concentration even when the correct dosage is used.

Practical Problems in Plant Tissue Culture

Researchers frequently observe the following issues:

  • Weakened selection pressure
  • Kanamycin escape (surviving non-transformants)
  • False-positive regenerants
  • Slow or inconsistent selection results
  • Differences compared to agar-based media

The problem becomes more pronounced as gellan gum concentration increases, because more binding sites are available.

Recommended Alternatives to Kanamycin

✔ Hygromycin

Very potent; unaffected by gellan gum.

✔ G418 (Geneticin)

Reliable selection agent for many transformation systems.

✔ Glufosinate (Basta / Phosphinothricin)

Stable and effective in gellan-based media.

✔ Cefotaxime / Timentin

Used for eliminating Agrobacterium; both remain active in gellan media.

These alternatives provide more predictable results in plant transformation and regeneration experiments.

Best Practices When Using Gellan Gum in Selection Media

To avoid antibiotic activity loss:

Avoid kanamycin when using gellan gum

Use hygromycin, G418, or glufosinate for strong selection

Keep gellan gum concentration at the lowest level that still forms a firm gel

Monitor for "kanamycin escape" if forced to use it

Reselect questionable regenerants on fresh plates

These steps help ensure consistent, reliable transformation outcomes.

Conclusion

Kanamycin loses activity in gellan gum media due to charge-based interactions that reduce its availability.
For successful plant selection and transformation work, researchers should choose antibiotics that remain stable in gellan-based systems—especially hygromycin, G418, glufosinate, and cefotaxime.

This understanding can prevent failed experiments and save significant time in plant biotechnology workflows.

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