Part 08 : Micropropagation & Tissue Culture

Part 1: Media Formulations & Plant Growth Regulators (PGRs)

1. Basal Salts & Nutrient Media

• Murashige & Skoog (MS) Media: The universal standard basal salt mixture containing macro-nutrients, micro-nutrients, and vitamins.
• Full-Strength vs. Half-Strength: Full-strength MS is often too concentrated in total salts > 2000 PPM for sensitive carnivorous explants.
  • 1/2 MS or 1/3 MS: Ideal for most carnivorous genera (Dionaea, Drosera, Sarracenia, Pinguicula).
  • 1/4 MS or Woody Plant Medium (WPM): Preferred for sensitive epiphytic species (Nepenthes, Heliamphora).
• Carbon Source: Sucrose at 20 – 30 g/L acts as the primary energy source for non-photosynthesizing explants inside culture vessels.
• Gelling Agent: Agar 6 – 8 g/L or Gellan Gum / Gelrite 2 – 2.5 g/L for solid media setups.
• Media pH: Adjust media pH to 5.6 – 5.8 using 0.1M HCl or 0.1M KOH prior to autoclaving/sterilization.

2. PGR Dynamics: Cytokinins vs. Auxins

• Cytokinins (Multiplication & Shoot Proliferation):
  • BAP (6-Benzylaminopurine), Kinetin, TDZ (Thidiazuron).
  • Promotes lateral bud break and shoot organogenesis. High cytokinin-to-auxin ratios drive rapid multiplication phases.
• Auxins (Rooting & Cell Elongation):
  • IBA (Indole-3-butyric acid), NAA (1-Naphthaleneacetic acid), IAA (Indole-3-acetic acid).
  • Induces adventitious root formation. High auxin-to-cytokinin ratios drive rhizogenesis and root initiation.

Part 2: Four-Stage Micropropagation Protocol

Stage I: Explant Selection & Surface Sterilization (Initiation)

• Explant Sources: Clean flower scapes (Dionaea, Drosera), un-opened spore capsules, unripe seed pods, or immature leaf pullings. Avoid dirty subterranean rhizomes whenever possible due to high systemic fungal loads.
• Aseptic Sterilization Sequence (Inside Laminar Flow Hood):
  1. Rinse explants under running tap water with a drop of liquid dish soap for 10 minutes.
  2. Submerge in 70% Ethanol for 30–60 seconds.
  3. Submerge in a 10% – 15% Commercial Bleach solution (0.5% – 1.0% Sodium Hypochlorite) with a surfactant (Tween-20) for 10–15 minutes with continuous agitation.
  4. Triple-rinse in sterile, autoclaved deionized/RO water (1 minute per rinse) to eliminate residual chlorine.
  5. Trim bleached edges with a sterile scalpel and place onto Stage I initiation media (1/2 MS zero-PGR or low-cytokinin).

Stage II: Shoot Multiplication & Proliferation

• Transfer clean, uncontaminated Stage I explants onto multiplication media containing a higher cytokinin ratio (e.g., 1/2 MS} + 0.5 – 2.0 mg/L BAP).
• Explants proliferate into dense clusters of multiple shoots every 4–6 weeks.
• Temporary Immersion Systems (TIS): Utilizing liquid media immersion cycles (e.g., BioTilt / BioCoupler systems) dramatically speeds up tissue multiplication and biomass growth by providing periodic liquid nutrient exposure alongside optimum vessel gas exchange.

Stage III: In Vitro Rooting & Elongation

• Separate shoot clusters into individual plantlets.
• Transfer shoots onto rooting media containing low auxins and reduced sucrose (e.g., 1/2 MS} + 0.5 mg/L IBA} + 15 g/L Sucrose, or hormone-free 1/2 MS).
• Shoots elongate and form functional in vitro root systems within 3–5 weeks.

Stage IV: Ex Vitro Acclimatization & Deflasking

This is the most critical phase. Plants growing in vitro have non-functional stomata and a thin cuticular wax layer. Removing them directly into ambient air results in rapid desiccation.

1. Deflasking Wash: Carefully pull plantlets from vessels and wash away all agar/gel residue from roots using warm pure RO water (0 PPM). Residual agar left on roots fosters rapid fungal mold growth (Botrytis).
2. Substrate Setup: Plant into pre-hydrated, sterile media (70% Shredded Long-Fiber Sphagnum Moss / 30% Fine Perlite).
3. Humidity Management: Place potted deflasked plantlets under high humidity (95% – 100% RH) under sealed propagation domes or fogging systems.
4. Hardening Off: Slowly decrease relative humidity over 14–21 days by venting domes incrementally, encouraging the plantlets to develop epicuticular wax and functional stomatal control.

Genus-Specific Tissue Culture Media Guidelines

Genus	Primary Explant	Initiation Media	Multiplication Media	Rooting Media
Dionaea	Flower Scape (1 – 2 cm)	1/2 MS Hormone-Free	1/2 MS + 0.5 mg/L BAP + 0.1 mg/L NAA	1/2 MS Hormone-Free or 0.2 mg/L IBA
Drosera	Leaf Segment / Flower Scape	1/2 MS Hormone-Free	1/2 MS + 0.2 mg/L Kinetin	1/2 MS Hormone-Free (Spontaneous Rooting)
Sarracenia	Seed / Immature Scape	1/2 MS Hormone-Free	1/2 MS + 1.0 mg/L BAP	1/2 MS + 0.5 mg/L IBA
Nepenthes	Nodal Stem / Seed	1/4 MS or WPM	1/2 MS + 0.5 mg/L BAP + 0.2 mg/L Kin	1/2 MS} + 1.0 mg/L IBA
Pinguicula	Fleshy Leaf Pulling	1/2 MS Hormone-Free	1/2 MS + 0.2 mg/L BAP	1/2 MS Hormone-Free

Troubleshooting Common Laboratory Issues

• Bacterial / Fungal Contamination: Visible cloudy halos around explant bases (bacterial) or white/gray fuzzy threads (fungal).
  • Solution: Improve surface sterilization protocols, verify autoclave pressure (121°C at 15 PSI for 20 minutes), and sterilize tool tips in a glass bead sterilizer or flame inside the laminar flow hood.
• Hyperhydricity (Vitrification): Plantlets appear translucent, glassy, brittle, and water-logged.
  • Solution: Increase agar/gelling agent concentration, reduce cytokinin levels, and improve vessel gas exchange using microporous membrane caps.
• Explant Browning / Phenolic Leaching: Explant releases brown or black tannins into the media, poisoning the tissue.
  • Solution: Add antioxidants (e.g., Ascorbic Acid 100 mg/L or Citric Acid) to wash solutions, add Activated Charcoal (1 – 2 g/L) to initial culture media, and subculture explants onto fresh media every 24–48 hours until leaching stops.