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Vriesea brusquensis
Temporary Immersion System

TIS ProtocolPBZ: 2 μMMass Propagation

Vriesea brusquensis is propagated using Temporary Immersion Systems (TIS), an advanced technique for mass propagation of bromeliads. The reference workflow follows the Laboratory of Plant Developmental Physiology and Genetics (LFDGV/CCA/UFSC) benchmark described in Jain & Ochatt (2010).

TIS offers significant advantages over conventional tissue culture including increased multiplication rates, improved acclimatization responses, and decreased production costs through reduced manipulation, labor, and container requirements.

This species requires a lower Paclobutrazol concentration (2 μM) compared to Aechmea fasciata (6 μM), demonstrating species-specific optimization within the TIS framework.

Temporary Immersion System Overview

TIS Technology provides automated periodic immersion of plant cultures in liquid medium, combining optimal nutrient delivery with excellent gas exchange. The system drastically improves multiplication efficiency.

Key Benefits for V. brusquensis:

  • High multiplication rates (up to 30:1)
  • Synchronized shoot development
  • Reduced labor and contamination risk
  • Superior plantlet quality for acclimatization
  • Economically viable for commercial production

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 Setup

  • Bioreactor containers: 300-500 mL capacity TIS vessels
    • RITA® apparatus (commercial)
    • Custom LFDGV/CCA/UFSC apparatus
    • Standard TIS systems
  • Air delivery system: Compressed air with 0.22 μm sterile filtration
  • Programmable timer: Precise 3h stationary : 3min immersion cycles
  • Culture environment: Standard tissue culture room (25°C ± 2°C, 16h photoperiod)

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 0–9

Buds on Filter Bridges

15 mL induction medium per tube, PBZ-free

Stage 2 · Weeks 9–18

TIS Multiplication

300–500 mL medium, immersion 3h : 3 min, refresh at week 6

Stage 3 · Weeks 18–24

TIS Elongation

GA₃ replaces PBZ; shoots reach ≥3 cm

Stage 4 · Weeks 24–33

Acclimatization

2:1:1 Plantmax® HA : pine bark : carbonized rice coat + weekly ¼ MS foliar feed

Stage 1: Initial Induction (8 weeks)

Conventional liquid culture before TIS transfer

  1. Step 1 Inoculate buds over filter paper bridges in test tubes (25 mm × 150 mm).
  2. Step 2 Use 15 mL liquid medium per tube.
  3. Step 3 Medium composition: MS salts + Morel vitamins + Sucrose 30 g/L + NAA (2 μM) + BAP (4 μM)
  4. Step 4 Culture conditions: 25°C ± 2°C, 16h light (50-60 μmol/m²/s), 60% ± 5% RH
  5. Step 5 After , clusters containing 5-8 shoots (average 0.5 cm) are obtained.

Stage 2: TIS Multiplication (12 weeks)

Species-Specific PBZ Concentration

Vriesea brusquensis requires only 2 μM Paclobutrazol, compared to 6 μM for Aechmea fasciata. This lower concentration prevents over-compaction while still promoting healthy microshoot proliferation.

  1. Step 1 Transfer 8-10 shoot clusters to each TIS unit under aseptic conditions.
  2. Step 2 Add 300-500 mL liquid medium per container (volume varies by container type).
  3. Step 3 Medium composition: MS salts + Morel vitamins + Sucrose 30 g/L + Paclobutrazol (PBZ) (2 μM)
  4. Step 4 Immersion cycle: Program timer for 3 hours stationary : 3 minutes immersion
  5. Step 5 After , replace culture medium (which normally acquires a dark-brown color due to phenolic oxidation).
  6. Step 6 Continue for with fresh medium (total 12 weeks in multiplication).

TIS Medium Matrix

Use the PDF matrix as the authoritative reference for hormone swaps inside the TIS.

Stage Window Intent Formulation
Induction Weeks 0-9 Axillary bud awakening on filter bridges
  • MS salts + Morel vitamins
  • Sucrose 3%
  • NAA 2 μM
  • BAP 4 μM
Multiplication Weeks 9-18 TIS nodular clumps and microshoot proliferation
  • MS salts + Morel vitamins
  • Sucrose 3%
  • PBZ 2 μM (V. brusquensis)
  • Immersion 3h : 3 min
Medium Refresh Week 18 Replace oxidized medium to maintain nutrient flow
  • Fresh multiplication medium
  • Maintain 3h : 3 min cycle
Elongation Weeks 18-24 Synchronize shoots before ex vitro transfer
  • MS salts + Morel vitamins
  • Sucrose 3%
  • GA₃ 10 μM
  • Continue 3h : 3 min

Stage 3: TIS Elongation (6 weeks)

  1. Step 1 Replace culture medium in TIS containers.
  2. Step 2 Medium composition: MS salts + Morel vitamins + Sucrose 30 g/L + GA₃ (10 μM)
  3. Step 3 GA₃ replaces PBZ to synchronously elongate the microshoots.
  4. Step 4 Continue same immersion cycle (3h stationary : 3min immersion).
  5. Step 5 After , regeneration rate may reach 30:1.
  6. Step 6 Shoots should be ≥3 cm long and ready for acclimatization.

Stage 4: Acclimatization (6 weeks)

  1. Step 1 Carefully remove shoots ≥3 cm from TIS containers.
  2. Step 2 Gently rinse shoots in tap water to remove residual culture medium.
  3. Step 3 If roots are present, prune to approximately 1 cm length (roots not essential for success).
  4. Step 4 Transfer to trays of 128 cells (60 cm³ per cell).
  5. Step 5 Substrate composition: 2:1:1 (v:v:v) Plantmax® HA : pine bark : carbonized rice coat
  6. Step 6 Place trays in greenhouse with controlled mist system.
  7. Step 7 Provide 50% shade using shade cloth or greenhouse screening.
  8. Step 8 Weekly spray with ¼ MS salts solution for nutrition.
  9. Step 9 Duration: in controlled mist environment
  10. Step 10 Expected outcome: High survival rate due to superior TIS-produced 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
V. brusquensis (TIS) MS + NAA 2 μM + BAP 4 μM MS + PBZ 2 μM (liquid) MS + GA₃ 10 μM
Aechmea fasciata (TIS) Same as V. brusquensis MS + PBZ 6 μM (higher compaction tolerance) 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

Comparison: V. brusquensis vs A. fasciata TIS

Parameter V. brusquensis A. fasciata
Induction 8 weeks, NAA + BAP 8 weeks, NAA + BAP
PBZ Concentration 2 μM 6 μM
Multiplication Duration 12 weeks 12 weeks
GA₃ Elongation 10 μM, 6 weeks 10 μM, 6 weeks
Immersion Cycle 3h : 3min 3h : 3min
Multiplication Rate Up to 30:1 Up to 30:1
Total Time 32 weeks 32 weeks

TIS Operation & Troubleshooting

Normal Observations

  • Medium color: Dark brown after 6 weeks is normal (phenolic oxidation)
  • Shoot clusters: Will increase in density during multiplication phase
  • Root formation: Some adventitious roots may form - this is normal
  • Shoot size variation: GA₃ treatment synchronizes elongation

Common Issues & Solutions

  • Hyperhydricity: Reduce immersion time or check drainage system
  • Slow growth: Replace medium more frequently (every 4 weeks)
  • Contamination: Check air filter (0.22 μm) integrity, replace if needed
  • Uneven development: Redistribute shoots evenly in container
  • Chlorosis: Increase light intensity or check nutrient levels

Protocol Timeline Summary

Week Stage Action Medium
0-8 Induction Conventional liquid culture MS + NAA (2 μM) + BAP (4 μM)
8-14 TIS Multiplication 1 First TIS cycle MS + PBZ (2 μM)
14 Medium Change Replace dark medium Fresh MS + PBZ (2 μM)
14-20 TIS Multiplication 2 Second TIS cycle MS + PBZ (2 μM)
20-26 TIS Elongation Synchronous elongation MS + GA₃ (10 μM)
26-32 Acclimatization Ex vitro establishment 2:1:1 substrate, mist system

Key Success Factors

  • PBZ optimization: 2 μM is species-specific for V. brusquensis - do not use A. fasciata concentration
  • Cycle precision: Maintain exact 3h:3min timing for optimal results
  • Medium replacement: Essential at 6-week intervals to prevent nutrient depletion
  • GA₃ synchronization: Switching to GA₃ ensures uniform shoot elongation across entire culture
  • Sterile air filtration: Always use 0.22 μm filters to prevent airborne contamination
  • Complete drainage: Verify system drains fully between cycles to prevent waterlogging
  • Shoot distribution: Evenly space clusters in TIS container for uniform access to medium
  • Minimum size: Only transfer shoots ≥3 cm to acclimatization for maximum survival
  • Economic viability: 30:1 multiplication rate makes TIS highly cost-effective for commercial scale
Guerra & Dal Vesco (2010) in Jain & Ochatt (2010), Methods in Molecular Biology, vol. 589