Sterilization is a critical step in plant tissue culture to prevent contamination by microorganisms and ensure the successful growth of plant tissues. Several sterilization techniques are applicable, each with specific purposes and protocols. Here are some of the key sterilization techniques used in plant tissue culture:



1. Surface Sterilization of Explants

Surface sterilization involves treating plant tissues (explants) to remove surface contaminants such as bacteria, fungi, and spores. Common agents and methods include:

  • Sodium Hypochlorite (Bleach): A widely used sterilant, typically in a concentration of 10-20% (commercial bleach) diluted in water. Explants are immersed for 5-20 minutes, depending on the tissue type, followed by thorough rinsing with sterile water.

  • Ethanol: Usually used at 70-95% concentration for a short duration (30 seconds to 5 minutes). This is often followed by another sterilant like bleach or hydrogen peroxide for more effective sterilization.

  • Hydrogen Peroxide: Typically used at concentrations of 3-10%, with immersion times varying based on the explant's sensitivity.

  • Mercuric Chloride: Effective but highly toxic, used at concentrations of 0.1-0.2% for 1-10 minutes, followed by extensive rinsing with sterile water.

2. Sterilization of Culture Media and Equipment

Culture media and equipment must be sterilized to create a contamination-free environment for plant tissue culture. Methods include:

  • Autoclaving: The most common method, involving steam sterilization at 121°C and 15 psi for 15-30 minutes. This effectively kills all microorganisms, including spores. It is used for sterilizing media, glassware, and some types of plasticware.

  • Filtration: Used for heat-sensitive components of culture media (e.g., certain vitamins and hormones). The solution is passed through a 0.22-micron filter to remove microorganisms.

  • Dry Heat Sterilization: Involves heating glassware and metal instruments in an oven at 160-180°C for 2-4 hours. Suitable for items that can withstand high temperatures.

3. Sterilization of Work Area

Maintaining a sterile work environment is crucial. Techniques include:

  • Laminar Flow Hood: Provides a sterile working environment by filtering air through HEPA filters, creating a continuous flow of sterile air over the work surface.

  • UV Light: Ultraviolet light is used to sterilize the work area and equipment surfaces. The UV lamp should be turned on for 15-30 minutes before starting the work.

4. Chemical Sterilization

Certain tools and surfaces can be sterilized using chemical disinfectants:

  • 70% Ethanol: Commonly used to wipe down work surfaces, equipment, and gloves before starting tissue culture work.

  • Sodium Hypochlorite Solutions: Used for surface sterilization of laminar flow hoods and work areas.

5. Flame Sterilization

  • Bunsen Burner or Alcohol Lamp: Used to sterilize metal instruments (e.g., scalpels, forceps) by passing them through the flame until they are red hot. This is often done immediately before using the instrument to ensure it is sterile.

General Tips for Sterilization in Plant Tissue Culture

  • Work Quickly and Efficiently: Minimize the time that sterile items are exposed to the air.
  • Use Sterile Technique: Always handle sterile items with sterile gloves or tools to prevent contamination.
  • Regular Monitoring: Regularly monitor cultures for signs of contamination and maintain good laboratory practices.

By employing these sterilization techniques meticulously, the risk of contamination in plant tissue culture can be significantly minimized, leading to more successful and reproducible results.