Plant tissue culture is a technique used to grow plants under sterile conditions on a nutrient medium, from small tissue fragments or cells. This process is often used in research, agriculture, and horticulture for plant propagation and genetic modification. The key steps involved in plant tissue culture are:

1. Selection and Preparation of Explant

• Explant: A small piece of plant tissue (leaf, root, stem, meristem, etc.) is chosen.
• The explant should be disease-free and healthy, as its quality greatly influences the success of the culture.
• It is carefully cut into small, manageable pieces for culture.

2. Sterilization

• Surface Sterilization: The explant is sterilized to remove any contaminants (bacteria, fungi, etc.). Common sterilizing agents include:
  • Ethanol (70%)
  • Sodium hypochlorite (bleach)
  • Hydrogen peroxide
• Sterilization must be done in a sterile environment, usually in a laminar flow hood, to prevent contamination.

3. Preparation of Culture Medium

• The medium is prepared with essential nutrients like:
  • Macronutrients: Nitrogen, potassium, phosphorous, etc.
  • Micronutrients: Iron, zinc, copper, etc.
  • Vitamins and amino acids.
  • A carbon source (usually sucrose).
  • Plant growth regulators (auxins, cytokinins, gibberellins, etc.) to stimulate growth and development.
• The pH of the medium is adjusted (typically between 5.6 and 5.8).
• The medium is sterilized by autoclaving.

4. Inoculation

• The sterilized explant is transferred onto the sterile nutrient medium.
• This process is done in sterile conditions to prevent contamination, usually inside a laminar airflow hood.

5. Incubation

• Cultures are placed in controlled environmental conditions, often in growth chambers, where:
  • Temperature: Usually 22°C to 28°C.
  • Light: 12-16 hours of light and 8-12 hours of darkness (photoperiod).
• The explant begins to form a callus (undifferentiated mass of cells), or direct organogenesis occurs (shoots or roots form directly).

6. Subculturing

• After the explant develops a callus or new shoots, the growing tissue may be transferred to a fresh medium to:
  • Promote further growth.
  • Encourage root or shoot development.
  • This process may be repeated multiple times.

7. Regeneration

• Shoots and roots are developed from the callus or directly from the explant, depending on the plant species and medium composition.
• This stage involves differentiation where specialized plant organs form.

8. Hardening or Acclimatization

• Once plantlets are developed, they are transferred from the in vitro (lab) environment to soil or a greenhouse.
• Before being exposed to the external environment, plants are gradually acclimatized to reduce shock, ensuring their survival outside the sterile conditions.

9. Transfer to Soil

• Once the plantlets are sufficiently hardened, they are transplanted into soil or a hydroponic system for further growth and development under natural conditions.

Each of these steps requires precise control to ensure the successful growth and propagation of plants using tissue culture techniques.