Citrus aurantifolia

Here’s a full tissue culture protocol for Citrus aurantifolia (Key lime or dayap), a highly valued citrus species used for juice, essential oils, and rootstock breeding. Citrus species generally respond well to tissue culture, especially when juvenile explants are used.

???? Tissue Culture Protocol for Citrus aurantifolia (Key Lime)

1. Explant Selection:

  • Best source: Immature seeds (for somatic embryogenesis) or shoot tips/nodal segments (for clonal propagation)
  • Use from healthy, disease-free mother plants

2. Surface Sterilization:

  • Wash seeds or shoot segments under running water for 20–30 minutes
  • Dip in 70% ethanol for 30 seconds
  • Sterilize with:
  • 0.1% HgCl₂ for 5–7 minutes or
  • 1.0% NaOCl + 1–2 drops Tween-20 for 10–15 minutes
  • Rinse 3–4 times with sterile distilled water

3. Culture Media:

A. For Shoot Induction (from nodal explants):
  • Basal medium: MS (Murashige & Skoog) or MT (Murashige & Tucker)
  • Growth regulators:
  • BAP (1.0–2.0 mg/L) – for shoot induction
  • NAA (0.1 mg/L) – to support shoot formation
  • Additives:
  • Ascorbic acid (50–100 mg/L) to prevent browning
  • Sucrose (30 g/L)
  • Agar (7 g/L)
B. For Somatic Embryogenesis (from immature embryos):
  • Medium: MT medium with:
  • 2,4-D (1.0–3.0 mg/L) for callus initiation
  • BAP or kinetin (0.5–1.0 mg/L) for embryo maturation
  • Embryos develop in 3–5 weeks under low light or dark conditions

4. Shoot Multiplication:

  • Transfer shoots to fresh MS + BAP (1.0–2.0 mg/L)
  • Subculture every 3–4 weeks for continuous multiplication
  • Kinetin or GA₃ (0.1–0.5 mg/L) may enhance elongation

5. Rooting:

  • Use half-strength MS medium with:
  • IBA (0.5–2.0 mg/L) or
  • NAA (0.5–1.0 mg/L)
  • Activated charcoal (0.2%) may be added to support root growth
  • Rooting takes 2–4 weeks

6. Acclimatization:

  • Transfer rooted plantlets to pots containing cocopeat:sand or peat moss:vermiculite
  • Keep under plastic cover or in a mist chamber
  • Gradual exposure to ambient humidity over 3–4 weeks

✅ Special Notes:

  • Browning due to phenolic leaching is common—use antioxidants like ascorbic acid or activated charcoal.
  • Juvenile tissues respond better in vitro than mature tissues.
  • Somatic embryogenesis is highly effective for virus-free plant production and large-scale propagation.

Would you like a visual protocol flowchart or comparison chart between all citrus tissue culture methods (clonal vs somatic)?

Spread the love