Vriesea friburgensis
var. paludosa

Threatened Species2-iP: 12 μMNodule Cluster Culture

Vriesea friburgensis var. paludosa is found in epiphytic or terrestrial habitats in southern and southeastern Brazilian Atlantic Forest. They grow preferentially as terrestrials in sandy, dry seacoast soils with sand dune vegetation. The reference workflow follows the LFDGV/CCA/UFSC benchmark described in Jain & Ochatt (2010).

This species forms dense groups and is typically helophytic but capable of surviving under diffuse light with few air humidity requirements. In its natural habitat, this bromeliad is threatened by extinction.

The protocol uses nodule cluster regeneration with 2,4-D/Kin induction followed by 2-iP multiplication, providing an efficient system for conservation and mass propagation.

Operational Timeline (PDF Benchmark)

Stage 1 · Weeks 0–13

Induction

2,4-D 5 μM + Kinetin 1 μM on agar

Stage 2 · Variable

Establishment

PGR-free liquid medium

Stage 3 · Weeks 13–22

Multiplication

2-iP 12 μM, microshoot clusters

Stage 4 · Weeks 22–30

Elongation

GA₃ 5 μM, 2× 4-week subcultures

Stage 5 · Weeks 30–39

Acclimatization

1:1 substrate, 9 weeks in mist

Hormone Matrix

Stage-by-stage hormone requirements from the PDF benchmark.

Stage	Hormone	Medium	Duration
Induction	2,4-D 5 μM + Kinetin 1 μM	Agar (solid)	13 weeks
Establishment	PGR-free	Liquid	Variable
Multiplication	2-iP 12 μM	Liquid	9 weeks
Elongation	GA₃ 5 μM	Liquid	8 weeks (2×4)
Acclimatization	N/A	1:1 substrate	9 weeks

Explant Source

Use microshoots in proliferation from established cultures as explant source.

Stage 1: Induction (13 weeks)

Step 1 Inoculate microshoots in Petri dishes containing 25 mL culture medium.
Step 2 Medium composition: MS salts + Morel vitamins + Sucrose 30 g/L + Agar Sigma® 7 g/L + 2,4-D (5 μM) + Kinetin (1 μM)
Step 3 Culture conditions: 25°C ± 2°C, 16h light (50-60 μmol/m²/s), 60% ± 5% RH
Step 4 After , yellow-colored nodular cluster cultures arise.

Stage 2: Establishment & Development

Step 1 Subculture 0.05 g nodular cluster cultures over paper filter bridges.
Step 2 Use test tubes (25 mm × 150 mm) containing 15 mL liquid culture medium.
Step 3 Medium: Same as induction stage but free of PGR (plant growth regulators).

Stage 3: Multiplication (9 weeks)

Step 1 Subculture 0.5 g nodular cultures in 300 mL glass flasks.
Step 2 Use 15 mL liquid culture medium per flask.
Step 3 Medium: MS + Morel vitamins + Sucrose 30 g/L + 2-iP (12 μM)
Step 4 Clusters of microshoots develop after .

Stage 4: Elongation

Step 1 Subculture twice at 4-week intervals.
Step 2 Use 300 mL glass flasks with 15 mL liquid medium per flask.
Step 3 Medium: MS + Morel vitamins + Sucrose 30 g/L + GA₃ (5 μM)
Step 4 Shoots elongate to ≥3 cm for Acclimatization .

Stage 5: Acclimatization

Step 1 Transfer shoots ≥3 cm to trays with 128 cells (60 cm³ each).
Step 2 Substrate: 1:1 (v:v) carbonized rice coat + Turfa Fértil® mineral supplement (N:4%, P₂O₅:14%, K₂O:8%)
Step 3 Keep in greenhouse with controlled mist for .
Step 4 Success rate: High survival when shoots reach minimum 3 cm size.

Key Success Factors

Nodule induction: 2,4-D and Kinetin combination critical for yellow nodular cluster formation
PGR-free establishment: Allows natural development before multiplication
2-iP multiplication: High concentration (12 μM) promotes microshoot cluster proliferation
GA₃ elongation: Ensures adequate shoot size for successful acclimatization
Minimum size: 3 cm shoots essential for acclimatization success
Conservation value: Protocol enables propagation of this threatened species