Tissue culture is a method of propagating plants under sterile conditions, often used for rapid multiplication of plant material. Here is a step-by-step guide on how to propagate tissue culture plants:

1. Materials Needed

• Sterile containers (Petri dishes, jars, or bottles)
• Sterile growth medium (Murashige and Skoog (MS) medium is commonly used)
• Plant tissue (explants) (leaf, stem, or root sections)
• Growth regulators (like auxins, cytokinins)
• Sterile environment (laminar flow hood or clean area)
• Scalpel or blade (sterilized)
• Sterilizing agents (ethanol, bleach)
• Tweezers or forceps (sterilized)

2. Prepare the Growth Medium

• Use a standard plant growth medium like MS medium. The medium should contain all essential nutrients, including vitamins, minerals, and sugar for energy.
• Add growth regulators like auxins and cytokinins depending on the type of plant and the part of the plant used (explants). For example:
  • Auxins (e.g., IAA, NAA) promote root development.
  • Cytokinins (e.g., BAP, Kinetin) encourage shoot formation.
• Adjust the pH of the medium to around 5.6 – 5.8 using a pH meter.
• Sterilize the medium by autoclaving at 121°C for 20-30 minutes.

3. Sterilize the Explants

• Select small pieces of plant tissue (e.g., leaf, stem, or root sections).
• Wash the explants under running water for 10-15 minutes to remove any surface debris.
• Sterilize the explants by submerging them in a sterilizing solution, such as:
  • 70% ethanol for 30 seconds.
  • Then in 1-2% bleach (sodium hypochlorite) for 5-10 minutes, depending on the tissue type.
• Rinse the explants thoroughly 3-4 times with sterile distilled water to remove any sterilizing agent.

4. Inoculate the Explants

• Work in a sterile environment (e.g., laminar flow hood) to avoid contamination.
• Using sterilized tweezers and scalpel, place the sterilized explants onto the sterile growth medium inside the containers.
• Seal the containers with parafilm or sterile lids to prevent contamination.

5. Incubate the Explants

• Place the containers in a growth chamber or under controlled conditions:
  • Maintain temperature between 22-28°C (depending on plant type).
  • Provide light (about 12-16 hours a day).
  • Ensure high humidity if required.
• After some days or weeks, callus formation (a mass of undifferentiated cells) or direct shoot/root formation should begin.

6. Subculture the Plantlets

• Once the plantlets or shoots appear, transfer them to fresh medium if needed.
• For rooting, transfer shoots to a medium with higher auxin concentration.
• Maintain sterile conditions while transferring to prevent contamination.

7. Acclimatization (Harden Off the Plants)

• When the plantlets have developed roots and shoots, they need to be hardened off before planting in soil.
• Transfer the plantlets from the tissue culture medium to potting soil or a soilless mix in small pots.
• Gradually expose them to natural conditions (light, temperature, and humidity) over several days or weeks by:
  • Placing them in a high-humidity environment.
  • Reducing humidity slowly to acclimatize the plants to room or greenhouse conditions.

8. Planting in Soil

• Once the plants are acclimatized, they can be transplanted into larger pots or the field.

Tips for Success:

• Avoid contamination: Use a laminar flow hood or very clean space for handling explants.
• Monitor regularly: Check for any signs of contamination (fungal or bacterial growth) and remove any infected tissue.
• Use the correct hormones: Balance between auxins and cytokinins is key for shoot and root development.

By following these steps, you can propagate plants through tissue culture, producing large numbers of genetically identical plants.