1. Introduction

Micropropagation is structured into four distinct stages, each with its own protocols and objectives. Mastering these stages allows you to manage large-scale propagation efforts effectively while minimizing losses.

2. Stage I: Initiation

Goal: Establish a clean culture from explant tissue.

• Choose healthy, actively growing plant material

• Surface sterilize with ethanol + bleach + rinses

• Place explants on initiation medium (often MS + low cytokinin)

• Observe for:

  • Contamination (usually within 1–2 days)

  • Swelling, callus, or initial bud break

📌 Tissue Culture Initiation Protocol

3. Stage II: Multiplication

Goal: Increase the number of shoots or growing points.

• Shoots are exposed to cytokinins (e.g., BAP, TDZ)

• Subculturing every 3–4 weeks keeps growth fresh and uniform

• Monitor for:

  • Hyperhydricity (excess moisture)

  • Browning or necrosis

  • Uneven shoot growth

📌 Shoot Multiplication in Micropropagation

4. Stage III: Rooting

Goal: Develop a root system capable of nutrient uptake.

• Transfer shoots to rooting medium
  (MS or ½ MS + auxin like IBA or NAA)

• Adjust:

  • Light levels

  • Auxin concentration

  • Agar content for support

• Rooting takes 2–3 weeks on average

• Some species root better in peat plugs or perlite (ex vitro)

5. Stage IV: Acclimatization

Goal: Transition the plantlet from in vitro to ex vitro conditions.

• Remove residual agar (inhibits root function)

• Transfer to potting mix under high humidity

• Cover with transparent dome or place in misting chamber

• Reduce humidity gradually over 7–14 days

• Move to greenhouse or outdoors for full acclimation

Common losses occur here, so gentle handling and gradual changes are critical.

📌 Hardening-Off Procedures for Micropropagated Plants