Aechmea fasciata
Temporary Immersion System
Aechmea fasciata is propagated using Temporary Immersion Systems (TIS), which have proven highly effective for mass propagation of ornamental bromeliads. The reference workflow follows the Laboratory of Plant Developmental Physiology and Genetics (LFDGV/CCA/UFSC) benchmark described in Jain & Ochatt (2010).
TIS benefits include increased multiplication rates (up to 30:1), improved responses during acclimatization, and decreased production costs through reduced manipulation, labor, space requirements, and fewer containers.
This species requires a higher Paclobutrazol concentration (6 μM) compared to Vriesea brusquensis (2 μM), demonstrating species-specific optimization within the TIS framework.
What is a Temporary Immersion System (TIS)?
TIS is a semi-automated bioreactor system where plant cultures are periodically immersed in liquid medium and then drained back, combining the benefits of liquid culture (better nutrient uptake) with gas exchange of conventional culture.
Key advantages:
- 30:1 multiplication rate (vs 10-15:1 in conventional culture)
- Reduced labor and manipulation
- Lower contamination risk
- Better plantlet quality
- Automated nutrient delivery
Stage 0: Donor Plant Conditioning
Duration
Benchmark Tasks
- Fertilize donor clumps weekly with chelated 30-10-10 (NPK) plus Nitrofoska® to maintain vigorous offshoots.
- Syringe-feed each rosette with 10 mL NAA (5 mM) + Vitamin B₁ to stimulate axillary bud formation.
- Spray benomyl + mineral oil before moving plants to a phyto-tron (25 ± 2 °C, 16 h photoperiod at 300 μmol/m²/s) for sanitation.
Healthy donor plants drive aseptic success—follow the fertilizer, phyto-tron, and sanitation cadence before excising buds.
TIS Equipment Requirements
- TIS containers: 300-500 mL capacity bioreactor vessels (e.g., RITA® system or custom apparatus)
- Air pump: Compressed air source for immersion/drainage
- Timer: Programmable for 3h:3min cycles
- Tubing & filters: Sterile air delivery system with 0.22 μm filters
- Culture room: Standard tissue culture environment (25°C ± 2°C, 16h light)
Basal Medium Reference (MS + Morel)
The PDF lists the full Murashige & Skoog macro/micro salts plus Morel vitamins; keep this reference handy when preparing multi-liter batches.
| Component | Stock | Volume | Final in 1 L |
|---|---|---|---|
| NH₄NO₃ | 82.5 g/L | 20 mL | 1,650 mg/L |
| KNO₃ | 95.0 g/L | 20 mL | 1,900 mg/L |
| CaCl₂·2H₂O | 88.0 g/L | 5 mL | 440 mg/L |
| KH₂PO₄ | 34 g/L | 5 mL | 170 mg/L |
| MgSO₄·7H₂O | 74 g/L | 5 mL | 370 mg/L |
| Fe-EDTA solution | 37.3 & 27.8 g/L | 10 mL | Fe chelate pair |
| Micronutrient mix | 1.24–0.0055 g/L | 5 mL | Boron, iodine, molybdenum, cobalt |
| Morel vitamins | 1–100 mg/L | full strength | Thiamine, pyridoxine, nicotinic acid, Ca pantothenate, myo-inositol, glycine |
| Sucrose | - | - | 30 g/L |
Operational Timeline (PDF Benchmark)
Stage 0 · Weeks -4–0
Donor Conditioning
Fertilizer regime + NAA/Thiamine syringe feeds, benomyl sanitation
Stage 1 · Weeks 0–8
Buds on Filter Bridges
15 mL induction medium per tube, PBZ-free
Stage 2 · Weeks 8–20
TIS Multiplication
300–500 mL medium, immersion 3h : 3 min, refresh at week 6
Stage 3 · Weeks 20–26
TIS Elongation
GA₃ replaces PBZ; shoots reach ≥3 cm
Stage 4 · Weeks 26–32
Acclimatization
2:1:1 Plantmax® HA : pine bark : carbonized rice coat + weekly ¼ MS foliar feed
Stage 1: Initial Induction (8 weeks)
This stage uses conventional liquid culture before TIS transfer
- Step 1 Inoculate buds over filter paper bridges in test tubes (25 mm × 150 mm).
- Step 2 Use 15 mL liquid medium per tube.
- Step 3 Medium: MS salts + Morel vitamins + Sucrose 30 g/L + NAA (2 μM) + BAP (4 μM)
- Step 4 Conditions: 25°C ± 2°C, 16h Photoperiod (50-60 μmol/m²/s), 60% ± 5% RH
- Step 5 After , each bud produces clusters of 5-8 shoots (average 0.5 cm).
Stage 2: TIS Multiplication (12 weeks)
Ensure all TIS components are properly sterilized. The timer must be calibrated precisely for 3h stationary : 3min immersion cycles. Compressed air must be filtered through 0.22 μm filters to prevent contamination.
- Step 1 Transfer 8-10 shoot clusters to each TIS unit.
- Step 2 Add 300-500 mL liquid medium per container (volume depends on container type).
- Step 3 Medium composition: MS salts + Morel vitamins + Sucrose 30 g/L + Paclobutrazol (PBZ) (6 μM)
- Step 4 Immersion cycle: Set timer for 3 hours stationary : 3 minutes immersion
- Step 5 After , replace culture medium (becomes dark brown).
- Step 6 Continue for with fresh medium (total 12 weeks in TIS).
TIS Medium Matrix
Use the PDF matrix as the authoritative reference for hormone swaps inside the TIS.
| Stage | Window | Intent | Formulation |
|---|---|---|---|
| Induction | Weeks 0-8 | Axillary bud awakening on filter bridges |
|
| Multiplication | Weeks 8-20 | TIS nodular clumps and microshoot proliferation |
|
| Medium Refresh | Week 14 | Replace oxidized medium to maintain nutrient flow |
|
| Elongation | Weeks 20-26 | Synchronize shoots before ex vitro transfer |
|
Stage 3: TIS Elongation (6 weeks)
- Step 1 Replace culture medium in TIS containers.
- Step 2 Medium: MS salts + Morel vitamins + Sucrose 30 g/L + GA₃ (10 μM)
- Step 3 Note: GA₃ replaces PBZ to synchronously elongate microshoots
- Step 4 Continue same immersion cycle (3h : 3min).
- Step 5 After , regeneration rate reaches 30:1.
- Step 6 Shoots should be ≥3 cm long for acclimatization.
Stage 4: Acclimatization (6 weeks)
- Step 1 Remove shoots ≥3 cm from TIS containers.
- Step 2 Gently rinse shoots in tap water to remove residual medium.
- Step 3 Transfer to trays of 128 cells (60 cm³ each).
- Step 4 Substrate: 2:1:1 (v:v:v) Plantmax® HA : pine bark : carbonized rice coat
- Step 5 Place trays in greenhouse with controlled mist and 50% shade.
- Step 6 Weekly spray with ¼ MS salts solution.
- Step 7 Duration:
- Step 8 Expected survival: High rate due to improved TIS-grown plantlet quality
PBZ & Hormone Comparison
Directly from the PDF Table 6.2—use this to adjust PBZ or TDZ loads when adapting the TIS for other bromeliads.
| Species | Induction | Multiplication | Elongation |
|---|---|---|---|
| A. fasciata (TIS) | MS + NAA 2 μM + BAP 4 μM | MS + PBZ 6 μM (liquid) | MS + GA₃ 10 μM |
| V. brusquensis (TIS) | Same as A. fasciata | MS + PBZ 2 μM (lower compaction) | MS + GA₃ 10 μM |
| V. fosteriana (encapsulated) | MS + NAA 2 μM + BAP 4 μM + PBZ 4 μM | TDZ 0.1 μM boosts clusters | Hormone-free MS before acclimatization |
TIS Maintenance & Troubleshooting
| Issue | Cause | Solution |
|---|---|---|
| Medium turns dark brown | Normal phenolic oxidation | Replace medium after 6 weeks |
| Hyperhydricity (glassy shoots) | Too much immersion | Reduce immersion time to 2 min or check drainage |
| Slow growth | Insufficient nutrients | Replace medium more frequently (4 weeks) |
| Contamination | Air filter failure | Check 0.22 μm filters, replace if needed |
| Uneven multiplication | Poor air distribution | Ensure shoots not clumped, redistribute evenly |
| Roots forming too early | Low cytokinin | Normal - continue protocol, prune to 1cm at transplant |
Protocol Timeline & Multiplication
| Stage | Duration | System | Result |
|---|---|---|---|
| Induction | 8 weeks | Conventional liquid | 5-8 shoots per bud |
| TIS Multiplication | 12 weeks (2×6) | TIS with PBZ (6 μM) | Massive proliferation |
| TIS Elongation | 6 weeks | TIS with GA₃ (10 μM) | 30:1 multiplication rate |
| Acclimatization | 6 weeks | Ex vitro | High survival |
| Total | 32 weeks | 30-fold increase per cycle | |
Key Success Factors
- Precise timing: 3h:3min cycle is critical - do not alter without testing
- PBZ concentration: 6 μM produces compact microshoots ideal for mass multiplication
- Medium renewal: Replace every 6 weeks to prevent nutrient depletion
- GA₃ synchronization: Switching to GA₃ synchronizes elongation of all shoots
- Air filtration: Always use 0.22 μm filters to prevent contamination
- Drainage verification: Ensure complete drainage between cycles to prevent waterlogging
- Cluster distribution: Evenly distribute shoots in TIS container for uniform growth
- Economic advantage: 30:1 rate makes TIS highly cost-effective for commercial production