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Vriesea reitzii
Endemic South Brazil

Threatened Species2 Protocol OptionsMultiplication: 20:1

Vriesea reitzii is an endemic bromeliad in South Brazil with excellent potential as an ornamental plant. This species presents red and yellow colored inflorescences and occurs in regions with altitudes from 750 to 1,200 m. The reference workflow follows the LFDGV/CCA/UFSC benchmark described in Jain & Ochatt (2010).

The species name honors Raulino Reitz, the "father of bromeliads" in Brazil. The ecosystem where this bromeliad is distributed, the Araucarian Mixed Forest, was severely devastated during the last century.

Two protocols are provided: Protocol A uses adventitious shoots from nodule clusters, while Protocol B focuses on regeneration from nodule cluster cultures with different hormone combinations.

Basal Medium Reference (MS + Morel)

Both protocols use Murashige & Skoog salts plus Morel vitamins as the basal medium.

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

Protocol A: Adventitious Shoots from Nodule Cluster

Stage 1: Induction (10 weeks)

  1. Step 1 Inoculate buds over filter paper bridges in test tubes (25 mm × 150 mm).
  2. Step 2 Use 15 mL liquid medium: MS + Morel vitamins + Sucrose 30 g/L + NAA (2 μM) + BAP (4 μM)
  3. Step 3 After , subculture with NAA (1 μM) + BAP (2 μM) for another 10 weeks.

Stage 2: Multiplication

  1. Step 1 Inoculate 5-8 microshoot clusters in 300 mL flasks with 20 mL liquid medium.
  2. Step 2 Medium: MS + Morel vitamins + Sucrose 30 g/L + NAA (2 μM) + BAP (4 μM)
  3. Step 3 Yellow and green nodular clumps develop at basal region within .
  4. Step 4 Subculture 0.05 g nodular clumps twice at 9-week intervals in same medium.
  5. Step 5 Nodular clumps evolve to nodular cluster cultures bearing microshoots.

Long-term Maintenance

Maintain nodular clumps in 300 mL flasks with 15 mL liquid medium free of PGR .

Multiplication Option 1

  1. Step 1 Subculture 0.05 g nodule clusters in 300 mL flasks.
  2. Step 2 Use 10 mL liquid medium with NAA (2 μM) + BAP (4 μM).

Multiplication Option 2

  1. Step 1 Inoculate previously elongated shoots (1.0-2.5 cm) in 300 mL flasks.
  2. Step 2 Use 20 mL liquid medium with NAA (1 μM) + BAP (2 μM).
  3. Step 3 Multiplication rate: Up to 20:1 after .

Stage 3: Elongation (16 weeks total)

  1. Step 1 Subculture 10 microshoot clusters per 300 mL flask with 15 mL liquid medium.
  2. Step 2 First 8 weeks: Medium with GA₃ (10 μM).
  3. Step 3 Next 8 weeks: Transfer to same medium free of PGR .

Stage 4: Acclimatization (9 weeks)

  1. Step 1 Transfer shoots >3 cm to trays of 72 cells (120 cm³ each).
  2. Step 2 Substrate: 1:1 (v:v) carbonized rice coat + Turfa Fértil® (N:4%, P₂O₅:14%, K₂O:8%)
  3. Step 3 Greenhouse with controlled mist until shoots reach 4-5 cm.

Protocol B: Regeneration from Nodule Cluster Cultures

Stage 1: Induction (7 weeks)

  1. Step 1 Remove 0.5 mm segments from basal region of young leaves from established microshoots.
  2. Step 2 Inoculate 5-8 segments in Petri dishes with 25 mL culture medium.
  3. Step 3 Medium: MS + Morel vitamins + Sucrose 30 g/L + Agar 7 g/L + 2,4-D (20 μM) + Kinetin (1 μM)
  4. Step 4 After , nodular cluster cultures arise from basal region.

Stage 2: Establishment & Development (8 weeks)

  1. Step 1 Subculture 0.25 g nodular cluster cultures.
  2. Step 2 Medium: MS + Morel vitamins + Sucrose 30 g/L + 2-iP (2.5 μM) + NAA (0.5 μM)

Stage 3: Multiplication (6 weeks)

  1. Step 1 Subculture 0.5 g nodular clusters for .
  2. Step 2 Medium: Same as above but free of PGR .
  3. Step 3 Clusters of microshoots evolve from nodular cultures.

Stage 4: Elongation

  1. Step 1 Subculture 8-10 clusters of microshoots in 300 mL flasks.
  2. Step 2 Use 15 mL liquid medium with GA₃ (10 μM).

Stage 5: Acclimatization (9 weeks)

  1. Step 1 Transfer shoots >3 cm to trays of 128 cells (60 cm³ each).
  2. Step 2 Substrate: 1:1 (v:v) carbonized rice coat + Turfa Fértil®
  3. Step 3 Nebulization tunnel, then transfer to pots after .
  4. Step 4 Survival rate: 95%

Protocol A Timeline (PDF Benchmark)

Stage 1 · Weeks 0–20

Induction

NAA 2 μM + BAP 4 μM → subculture with NAA 1 μM + BAP 2 μM

Stage 2 · Weeks 20–38

Multiplication

Nodular clumps in 300 mL flasks, 9-week subculture intervals

Stage 3 · Weeks 38–54

Elongation

GA₃ 10 μM for 8 weeks → PGR-free for 8 weeks

Stage 4 · Weeks 54–63

Acclimatization

1:1 carbonized rice coat + Turfa Fértil®, nebulization tunnel

Protocol B Timeline (PDF Benchmark)

Stage 1 · Weeks 0–7

Induction

2,4-D 20 μM + Kinetin 1 μM on agar medium

Stage 2 · Weeks 7–15

Establishment

2-iP 2.5 μM + NAA 0.5 μM

Stage 3 · Weeks 15–21

Multiplication

PGR-free medium

Stage 4 · Weeks 21–29

Elongation

GA₃ 10 μM in liquid medium

Stage 5 · Weeks 29–38

Acclimatization

95% survival rate

Protocol Comparison

Choose Protocol A for higher multiplication rates or Protocol B for faster turnaround.

Aspect Protocol A Protocol B
Explant Source Buds from shoots 0.5 mm leaf segments
Induction Hormones NAA + BAP 2,4-D + Kinetin
Medium Type Liquid throughout Agar → Liquid
Multiplication NAA + BAP or PGR-free PGR-free only
Max Rate 20:1 High rate
Total Time ~63 weeks ~38 weeks
Survival High 95%

Key Success Factors

  • Protocol selection: Choose based on explant availability and timeline requirements
  • Nodule development: Critical phase requiring proper hormone balance
  • PGR-free maintenance: Allows sustainable long-term culture storage
  • Elongation timing: Two-phase elongation (with/without GA₃) ensures proper shoot development
  • Cell size for acclimatization: Protocol A uses larger cells (120 cm³) for better initial development
Guerra & Dal Vesco (2010) in Jain & Ochatt (2010), Methods in Molecular Biology, vol. 589