Persea americana

Here’s a detailed tissue culture protocol for Persea americana (avocado), a high-value tropical fruit tree that is notoriously difficult to propagate in vitro due to its high phenolic content, low regenerative capacity, and genotype dependency. However, with careful media formulation and explant handling, success is possible—especially with embryo culture or juvenile shoot tips.

???? Tissue Culture Protocol for Persea americana (Avocado)

1. Explant Selection

  • Best sources:
  • Immature zygotic embryos (from green fruits 3–5 months old)
  • Juvenile shoot tips or nodal segments
  • Avoid mature tissue (high phenolic exudation and poor response)

2. Surface Sterilization

  • Wash under running tap water (20–30 minutes)
  • 70% ethanol dip for 30 seconds
  • Sterilize with:
  • 0.1% HgCl₂ or 1% NaOCl (10–15 minutes) + 1 drop Tween-20
  • Rinse 3–5 times with sterile distilled water

3. Culture Medium

A. Embryo Germination / Callus Induction

  • Basal medium: MS or WPM (Woody Plant Medium)
  • Supplements:
  • 2,4-D (1.0–2.0 mg/L) or Picloram – for callus induction
  • Sucrose (30 g/L)
  • Ascorbic acid (50–100 mg/L) to reduce browning
  • Agar (7–8 g/L)
  • Incubate in darkness (25 ± 2°C)

B. Shoot Induction (organogenesis)

  • Transfer to MS or WPM + BAP (1.0–2.0 mg/L)
  • Add Kinetin (0.5–1.0 mg/L) or GA₃ (0.1 mg/L) for elongation
  • Reduce 2,4-D or omit auxin for shoot development
  • Frequent subculturing (every 2–3 weeks) is essential

4. Shoot Multiplication

  • BAP (1.0 mg/L) + Kinetin (0.5 mg/L) gives better multiplication
  • Shoots are slow-growing—maintain for 4–6 weeks per cycle
  • High browning? Add:
  • Activated charcoal (0.2%)
  • Polyvinylpyrrolidone (PVP) to bind phenolics

5. Rooting

  • Half-strength MS or WPM
  • Auxins:
  • IBA (1.0–2.0 mg/L) or
  • NAA (0.5–1.0 mg/L)
  • Use liquid medium or vermiculite overlays for better rooting
  • Rooting success is variable; some protocols recommend in vitro grafting onto rootstock

6. Acclimatization

  • Transfer rooted shoots to cocopeat:sand or vermiculite:peat (sterilized)
  • Maintain high humidity (mist chamber/plastic dome) for 3–4 weeks
  • Gradual hardening to outdoor conditions over 6–8 weeks

✅ Key Challenges & Tips

  • Phenolic oxidation is the biggest challenge; use:
  • Ascorbic acid, citric acid, activated charcoal, or PVP
  • Avocado tissue browns rapidly after cutting—work quickly
  • Genotype matters – West Indian types respond better than Guatemalan types
  • In vitro grafting onto seedling rootstock is an emerging workaround for poor rooting

Would you like a flowchart diagram for this Persea americana protocol, or a PDF combining this with other tropical tree protocols like Cinnamomum, Magnolia, and Citrus?

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