Surface Sterilization Technique of Explants

Surface sterilization is a crucial step in plant tissue culture, as it removes microorganisms present on the surface of the explant (the plant material used for culture). The presence of contaminants like bacteria or fungi can lead to culture failure by outgrowing and consuming the nutrients meant for plant tissue growth. Here’s a step-by-step guide on how the surface sterilization process is generally carried out:



1. Selection and Preparation of Explant:

  • Type of Explant: The type of plant part (such as leaves, stems, or seeds) chosen for tissue culture affects the sterilization process. Explants collected from younger, actively growing parts of plants are typically preferred since they are less likely to harbor contaminants.
  • Collection: Explants are collected from healthy plants under sterile conditions to minimize microbial contamination.
  • Pre-rinse: The explants are initially rinsed under running tap water to remove dust, soil, or any visible debris.

2. Use of Detergents:

  • Detergent Wash: A mild detergent (such as liquid soap or Tween-20) is often used for 10-15 minutes to remove surface debris and reduce microbial load. The explants are stirred or agitated gently to enhance the effect.
  • Rinsing: After the detergent wash, explants are thoroughly rinsed with distilled water or sterile water multiple times to remove detergent residues.

3. Sterilizing Agents:

Various chemical agents are used for sterilization, depending on the type of explant and the contaminant load. Commonly used sterilizing agents include:

  • Sodium Hypochlorite (NaOCl): This is the most commonly used sterilant. A solution of 0.5% to 1% sodium hypochlorite is typically used for 5 to 15 minutes. Commercial bleach (containing 5.25% sodium hypochlorite) is often diluted to the desired concentration.
  • Ethanol (70%): Ethanol is effective for surface sterilization but is usually used for a very short duration (30 seconds to 1 minute) because it can be toxic to plant tissues if exposed for too long.
  • Hydrogen Peroxide (H₂O₂): A 3-6% hydrogen peroxide solution can be used for about 10 minutes, especially for soft tissues or sensitive explants.
  • Mercuric Chloride (HgCl₂): While effective, this chemical is toxic and hazardous, and its use has decreased. It is usually applied in a 0.1% to 0.2% solution for 3 to 5 minutes, followed by thorough washing.
  • Silver Nitrate (AgNO₃): Sometimes used as an alternative sterilizing agent for certain plant species.

4. Washing with Sterile Water:

After treatment with sterilizing agents, it is crucial to wash the explants multiple times (3-5 times) with sterile distilled water to remove any residual sterilizing chemicals that might harm the explant tissue.

5. Handling and Transfer to Media:

  • Sterile Conditions: The sterilized explants are handled under sterile conditions, usually in a laminar airflow hood, to avoid post-sterilization contamination.
  • Transfer to Growth Media: Once sterilized, explants are immediately transferred to sterile growth media for tissue culture. The media should be free of any contaminants to promote healthy growth.

6. Post-Sterilization Observations:

After transferring the explants to the media, it is important to monitor for any signs of contamination (such as bacterial or fungal growth). If contamination is observed, it suggests either incomplete sterilization or post-sterilization contamination.

Factors to Consider in Surface Sterilization:

  • Type of Explant: More delicate tissues, such as meristematic tissues, require gentler sterilizing treatments, while tougher explants can tolerate harsher chemicals.
  • Duration of Treatment: Longer exposure to sterilizing agents increases the chance of complete sterilization but also raises the risk of damaging the plant tissue.
  • Concentration of Sterilants: Using a higher concentration of sterilants can be effective but needs careful monitoring to prevent tissue damage.

Conclusion:

Surface sterilization is a delicate balance between killing contaminants and maintaining the viability of the explant. A well-executed surface sterilization technique is essential for successful plant tissue culture and further in vitro propagation or genetic studies.