Vriesea hieroglyphica
Hieroglyphic Bromeliad
Vriesea hieroglyphica is a bromeliad native to southern Brazil. Its ornamental importance relies mainly on extremely handsome foliage with glossy, bright green leaves featuring wide and irregular horizontal bands of dark green. The reference workflow follows the LFDGV/CCA/UFSC benchmark described in Jain & Ochatt (2010).
The leaves are 90 cm long and 7.5 cm wide, forming an impressive rosette. The inflorescence with long green bracts and yellow flowers is modest compared to the spectacular foliage.
This protocol uses Thidiazuron (TDZ) for multiplication, which is significantly more cost-effective than traditional BAP/NAA combinations while providing reliable regeneration rates.
Basal Medium Reference (MS + Morel)
Murashige & Skoog salts plus Morel vitamins form the basal medium for all stages.
| 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 1 · Weeks 0–28
Induction
NAA 2 μM + BAP 4 μM, 3× subculture at 6-week intervals
Stage 2 · Variable
Multiplication
TDZ 0.1 μM, 6:1 multiplication rate
Stage 3 · Weeks 28–38
Elongation
PGR-free MS medium
Stage 4 · Weeks 38–47
Acclimatization
2:1:1 substrate, 95% survival
Stage 1: Induction (28 weeks total)
- Step 1 Inoculate buds over filter paper bridges in test tubes (25 mm × 150 mm).
- Step 2 Use 15 mL liquid culture medium per tube.
- Step 3 Medium composition: MS salts + Morel vitamins + Sucrose 30 g/L + NAA (2 μM) + BAP (4 μM)
- Step 4 Conditions: 25°C ± 2°C, 16h light (50-60 μmol/m²/s), 60% ± 5% RH
- Step 5 After , nodule cluster cultures arise from explants.
- Step 6 Subculture 3 times, every 6 weeks, in the same medium.
- Step 7 Total induction period: 10 weeks + (3 × 6 weeks) = 28 weeks
Stage 2: Multiplication with Thidiazuron
At only 0.1 μM concentration, TDZ provides effective multiplication at a fraction of the cost of traditional cytokinin/auxin combinations. This makes large-scale production economically viable.
- Step 1 Subculture 5-8 microshoot clusters per 300 mL flask.
- Step 2 Use 15 mL liquid medium per flask.
- Step 3 Medium: MS salts + Morel vitamins + Sucrose 30 g/L + Thidiazuron (TDZ) (0.1 μM)
- Step 4 Multiplication rate: 6:1 per cycle
Stage 3: Elongation (10 weeks)
- Step 1 Subculture 8-10 microshoot clusters per 300 mL flask.
- Step 2 Use 15 mL liquid medium per flask.
- Step 3 Medium: MS salts + Morel vitamins + Sucrose 30 g/L, free of PGR
- Step 4 Culture for until shoots reach ≥3.0 cm.
Stage 4: Acclimatization (9 weeks)
- Step 1 Transfer shoots ≥3.0 cm to trays of 128 cells (60 cm³ each).
- Step 2 Substrate composition: 2:1:1 (v:v:v) Plantmax® HA : pine bark : carbonized rice coat
- Step 3 Place trays in greenhouse with controlled mist.
- Step 4 Provide 50% shade using shade cloth or greenhouse screening.
- Step 5 Weekly spray with ¼ MS salts solution for nutrition.
- Step 6 Duration:
- Step 7 Survival rate: 95%
TDZ Protocol Comparison
Compare TDZ-based protocols across species from the PDF benchmark.
| Species | Multiplication Rate | Survival | Elongation |
|---|---|---|---|
| V. hieroglyphica | 6:1 | 95% | PGR-free |
| V. fosteriana | 14:1 | 98% | PGR-free |
| A. imperialis | 6:1 | 98% | PGR-free |
| V. splendens | Variable | 95% | GA₃ 10 μM |
Culture Conditions
In Vitro Stages
- Temperature: 25°C ± 2°C
- Light: 50-60 μmol/m²/s
- Photoperiod : 16 hours light
- Humidity: 60% ± 5% RH
- Container: Test tubes (induction), 300 mL flasks (multiplication)
- Support: Filter paper bridges for liquid cultures
Acclimatization Stage
- Container: 128-cell trays (60 cm³/cell)
- Environment: Greenhouse with mist
- Shade: 50% light reduction
- Irrigation: Controlled mist system
- Nutrition: Weekly ¼ MS spray
- Minimum size: 3.0 cm shoots
Key Success Factors
- Extended induction: 28-week induction period ensures stable nodule cluster formation
- TDZ concentration: Very low 0.1 μM concentration is optimal - higher concentrations may cause abnormalities
- PGR-free elongation: Hormone-free medium prevents hyperhydricity and promotes natural shoot development
- Shoot size critical: Minimum 3 cm essential for 95% survival rate
- Substrate drainage: Three-component mix ensures excellent drainage for epiphytic species
- Gradual acclimation: Controlled mist and 50% shade prevent stress during transition
- Ornamental value: Foliage quality maintained through tissue culture process