Micropropagation is a controlled method for producing large numbers of identical plants under sterile, in vitro conditions.
It uses small plant parts called explants and grows them on defined nutrient media that contain mineral salts, sugars, vitamins, and growth regulators.
This technique ensures disease-free, uniform, and rapid plant production throughout the year.
The process follows four to five major stages, each with distinct objectives and media formulations.
Understanding these stages and their media composition is critical for reproducibility and successful regeneration.
Stage I : Establishment (Culture Initiation)
The first stage begins with the selection and sterilization of the explant.
Typical explants include shoot tips, nodes, leaves, or meristems.
Goal
- To establish contamination-free, viable cultures on nutrient medium.
Procedure
- Surface-sterilize explants using ethanol and sodium hypochlorite.
- Place them on a basal nutrient medium such as Murashige & Skoog (MS) or Gamborg’s B5.
- Add appropriate antioxidants (ascorbic acid or citric acid) if browning occurs.
Medium Composition
- Basal salts : MS medium provides nitrogen (NH₄⁺/NO₃⁻), phosphorus, potassium, calcium, magnesium, sulfur.
- Carbon source: Sucrose (2–3 %) as an energy supply.
- Gelling agent : 0.7 % agar (Supreme or tissue-culture grade for clarity).
- Vitamins : Thiamine, pyridoxine, nicotinic acid, myo-inositol.
- Plant growth regulators (PGRs): Low cytokinin concentration (0.5–1 mg/L BAP or kinetin) promotes initial bud growth.
Notes
- The success of Stage I depends on the sterility of culture and physiological age of the explant.
- Juvenile tissues usually respond better than mature ones.
Stage II : Multiplication (Shoot Proliferation)
Once the explant establishes, the next goal is to multiply shoots rapidly.
Goal
To achieve maximum shoot proliferation without morphological abnormalities.
Medium
- Base medium: MS or modified MS with reduced ammonium concentration.
- Cytokinin: BAP (6-benzylaminopurine) or kinetin (1–3 mg/L).
- Auxin (optional): Small amounts of NAA (0.05–0.1 mg/L) to balance growth.
- Sucrose: 30 g/L; agar 6–8 g/L.
Procedure
- Excise and subculture new shoots every 3–4 weeks.
- Each node forms multiple shoots, increasing the propagation rate exponentially.
- Maintain light intensity ~3000 lux, 25 ± 2 °C, and a 16-hour photoperiod.
Remarks
- High cytokinin promotes shoot multiplication, but excess causes vitrification or abnormal leaf morphology.
- Testing various cytokinin levels helps optimize multiplication for each species.
Stage III : Rooting (In Vitro Root Induction)
Rooting converts the regenerated shoots into complete plantlets.
Goal
To induce strong, healthy root systems that will support acclimatization.
Medium
- Basal medium: Half-strength MS (reduces nitrogen and osmotic stress).
- Auxin: Indole-3-butyric acid (IBA, 0.5–2.0 mg/L) or naphthalene acetic acid (NAA, 0.5 mg/L).
- Carbon source: Sucrose 2 %.
- Agar: 0.7 % (use high-purity agar for transparent roots).
Conditions
Keep cultures in low light (~2000 lux) to enhance root elongation.
Roots typically emerge within 2–4 weeks, depending on species.
Notes
Some species root better ex vitro using auxin dips rather than in vitro rooting, saving time and cost.
Stage IV : Acclimatization (Hardening)
This is the transition from laboratory to greenhouse conditions.
Goal
To help plantlets adapt to lower humidity, higher light, and non-sterile soil environments.
Procedure
- Remove plantlets carefully and wash away agar residues with sterile water.
- Transfer to soil, peat moss, coco-peat, or a sand–vermiculite mixture.
- Cover with transparent lids or plastic domes to maintain humidity.
- Gradually expose to ambient conditions over 1–2 weeks.
Key Parameters
- Humidity reduction: 95 % → 70 % → 50 %.
- Temperature: 25–28 °C.
- Light intensity: 4000–6000 lux.
Media for Hardening
- No artificial medium is used; instead, substrates rich in organic matter with good drainage are preferred.
- Adding mild fungicides (0.1 % carbendazim) helps prevent contamination.
Stage V : Field Transfer (Optional Stage)
In large-scale commercial micropropagation, Stage V involves moving hardened plantlets to open soil or controlled greenhouses.
Fertilization with half-strength nutrient solution supports early growth.
At this stage, plants are considered identical to donor plants and ready for distribution or research trials.
| Stage | Basal Medium | Cytokinin (mg/L) | Auxin (mg/L) | Carbon Source | Agar (%) | Notes |
| I – Initiation | MS full strength | BAP 0.5 | 3 % Sucrose | 0.7 | Sterile explant establishment | |
| II – Multiplication | MS | BAP 2.0 | NAA 0.05 | 3 % Sucrose | 0.7 | Multiple shoot induction |
| III – Rooting | ½ MS | IBA 1.0 | 2 % Sucrose | 0.7 | Root elongation | |
| IV – Acclimatization | Soil substrate | Gradual humidity reduction |
Factors Affecting Success
- Genotype of the plant : Different species require distinct hormonal balances.
- Explant age : Young tissues respond better than older ones.
- Medium strength : Reducing macro-salts during rooting improves root quality.
- Light and temperature : Constant 25 °C and 16-hour photoperiod are standard.
- Subculture timing : Regular subculture prevents nutrient depletion and tissue browning.
- Agar quality : Use tissue-culture-grade agar with high gel strength and clarity.
Micropropagation bridges classic plant biology and modern biotechnology.
Each stage, from initiation to acclimatization, demands precision in media composition, hormone balance, and environmental control.
By understanding the sequence of stages and optimizing the nutrient media, scientists can ensure reproducible, large-scale plant production with genetic fidelity.
This technology remains indispensable for research, agriculture, and global plant conservation.