Micropropagation: Techniques, Applications, and Challenges

Micropropagation: Techniques, Applications, and Challenges

Micropropagation is the clonal propagation of a selected genotype using in vitro culture techniques. It is generally done by culturing apical shoots, auxiliary buds, and meristematic tissues. This method ensures a faster rate of multiplication compared to conventional vegetative propagation.
George Morel (1960) pioneered the commercial-scale production of orchid plants using micropropagation. Today, this method is widely used for producing commercially important plants such as banana, apple, strawberry, and orchids.
Murashige (1974) recognized four stages of micropropagation:

  1. Stage I: Establishment of culture
  2. Stage II: Multiplication of shoots in culture
  3. Stage III: Rooting
  4. Stage IV: Hardening of tissue culture-raised plants

Stage I – Establishment of Culture

The objective of this stage is to initiate an axenic (sterile) culture. The process begins with excising meristem tissue from an identified stock plant, followed by antimicrobial treatments to remove contaminating organisms. The explants are then cultured in a nutrient medium under aseptic conditions.

Key considerations:

  • Microbial contamination and phenolic exudates are major constraints, especially in woody tissues.
  • Use of fungicides and antibiotics limits microbial infection.
  • Antioxidants like ascorbic acid, PVP, and charcoal help eliminate phenolic exudates.
  • The developmental stage of the explant significantly impacts its survival and response.
  • Young tissues, such as terminal or axillary shoot buds, regenerate better than mature tissues.
  • The culture medium is supplemented with cytokinins (e.g., BA, TDZ) for axillary shoot formation and auxins (e.g., NAA, 2,4-D) for callusing.
  • Sucrose is the most commonly used carbon source.
  • Murashige and Skoog’s medium is the most widely used basal medium.

Stage II – Proliferation of Shoots in Culture

During this stage, shoot multiplication is achieved by subculturing the shoots at regular intervals in an appropriate medium.

Key considerations:

  • Shoot cultures serve as both propagation material and stock maintenance.
  • Long-term maintenance may result in genetic variations.
  • To ensure genetic fidelity, shoots from cultures with excessive subculturing should be avoided.

Stage III – Rooting


Shoots from the multiplication stage are transferred to conditions that promote root initiation and shoot elongation. Rooting can be induced either in vitro or ex vitro.

In vitro rooting:

  • Shoots are cultured in growth regulator-free medium or subjected to pulse treatment with auxins before being transferred to auxin-free medium.

Ex vitro rooting:

  • Shoots (micro cuttings) are treated with commercial rooting mixtures and planted in soil under high humidity conditions.

Advantages of ex vitro rooting:

  • Easier to establish cuttings in soil than to transfer rooted plantlets.
  • Reduces labor-intensive operations.
  • The root system develops naturally in soil.
  • Avoids root damage during transplantation.
  • More economical for difficult-to-root plants.

Stage IV – Hardening of Tissue Culture-Raised Plants


This stage involves transferring plantlets from aseptic culture conditions to a greenhouse and eventually to the field.

Key considerations:

  • In vitro plantlets are heterotrophic and require gradual acclimatization.
  • A high relative humidity environment is essential to prevent desiccation.
  • The plantlets must develop new leaves adapted to natural growing conditions before field transfer.

Advantages of Micropropagation

  1. Millions of plants can be propagated within a year.
  2. Propagation of difficult-to-multiply species: Some species resistant to conventional propagation can be efficiently propagated.
  3. Disease-free plants: Meristem tip culture produces virus-free plants in species like potato, Dianthus, and Chrysanthemum.
  4. Year-round propagation: Micropropagation can be performed independent of seasonal constraints.
  5. Germplasm exchange: Provides a disease-free method for the international exchange of plant materials, reducing quarantine periods.
  6. Seed production: Useful in F1 hybrid seed production (e.g., onion and asparagus breeding programs).
  7. Germplasm storage: Cryopreservation of meristem cells ensures long-term storage of valuable genetic material.
  8. Artificial seeds: Synthetic seeds produced via somatic embryogenesis serve as an alternative propagation method.

Disadvantages of Micropropagation

  1. High initial costs: Requires sophisticated facilities and trained personnel.
  2. Contamination risk: Despite aseptic conditions, cultures may become infected with pathogens, leading to high losses.
  3. Genetic variability: Some propagation methods may induce undesirable genetic variations.
  4. Vitrification (Hyperhydration): A physiological disorder causing translucent, water-soaked leaves, ultimately leading to plant death.

Preventive measures:

  • Increase agar concentration.
  • Overlay medium with paraffin.
  • Use desiccants like CuSO4.
  • Improve aeration through bottom cooling.
  • Adjust cytokinin levels and manipulate NH4+ or salt concentrations in the culture medium.

Conclusion

Micropropagation is a powerful technique that has revolutionized commercial plant propagation. It enables large-scale production of disease-free, genetically uniform plants preserving valuable germplasm. However, challenges such as high costs, contamination risks, and genetic variability must be carefully managed to optimize the benefits of this technique.