Begonia tuberous
Thin Cell Layer Protocol

TCL SystemTDZ: 0.2 mg/L210 shoots/segment

Begonias demonstrate exceptional responsiveness to tissue culture across multiple species. This protocol utilizes the Thin Cell Layer (TCL) system for high-frequency shoot regeneration from petiole, stem, and floral stalk explants. The reference workflow follows Nhut et al. (2005) in Jain & Ochatt (2010).

The TCL system produces several thousand shoots per sample. By optimizing tissue size and applying improved selection procedures, shoots elongate in 8 weeks with an average of 210 ± 9.7 shoots per segment.

On average, the best treatment allows production of about 10,000 plantlets from the axillary buds of one plant with five petioles within 8 months.

Operational Timeline (PDF Benchmark)

Stage 1 · Day 0

Sterilization

0.1% HgCl₂ for 6 min after 70% EtOH

Stage 2 · Weeks 0–3

TCL Culture

TDZ 0.2 mg/L, shoot initiation

Stage 3 · Weeks 3–8

Shoot Elongation

PGR-free MS medium

Stage 4 · Weeks 8–10

Further Elongation

BA 1.0 mg/L

Stage 5 · Weeks 10–16

Rooting

BA 0.5 mg/L + NAA 0.1 mg/L + AC

Stage 6 · Weeks 16–22

Acclimatization

1:1 sand:soil, then greenhouse

TCL Culture Parameters

Critical parameters for successful Thin Cell Layer culture from the PDF benchmark.

Parameter	Value	Notes
TCL Thickness	0.2–1.0 mm (3 mm optimal)	Affects regeneration rate
Culture Vessel	100 mm × 20 mm Petri dish	40 mL semi-solid medium
Temperature	25 ± 2°C	Growth chamber
Humidity	75–80% RH	Controlled environment
Light	45 μmol/m²/s PPFD	10 h photoperiod
pH	5.8	Adjusted with 1 M KOH
Gelling Agent	Phytagel 2.5 g/L	Sigma brand

Sterilization Protocol

Step 1 Wash explants with tap water and liquid soap. Duration: .
Step 2 Rinse thoroughly with running tap water. Duration: .
Step 3 Surface sterilization with 1.0-1.5% Sodium hypochlorite plus 2-3 drops Tween 20 . Duration: .
Step 4 Rinse with sterile water multiple times. Washes: 3-4 times, each.
Step 5 Optional: Brief 70% ethanol rinse for for additional sterilization.

Media Formulations

Stage	Components & Conditions
Stage I Callus /Shoot Induction	MS 4.44 g/L • Sucrose 30 g/L • BAP 1.0-3.0 mg/L • NAA 0.1-0.5 mg/L • Agar 7 g-8 g/L • pH pH 5.7-pH 5.8 Begonias respond particularly well to Cytokinin -heavy medium
Stage II Shoot Multiplication	MS 4.44 g/L • Sucrose 30 g/L • BAP 1.0-2.0 mg/L • NAA 0.1 mg/L • Agar 7 g/L • pH pH 5.7-pH 5.8 Lower hormone concentrations promote shoot elongation
Stage III Rooting	MS 2.22 g/L (half-strength) • Sucrose 20 g/L • NAA 0.5-1.0 mg/L • Agar 7 g/L • pH pH 5.7-pH 5.8 Begonias root easily with low Auxin concentration

TCL Explant Comparison

Compare different explant types and hormone combinations from the PDF benchmark.

Explant Type	Hormone	Shoot Rate	Notes
Petiole TCL	TDZ 0.2 mg/L	High (210/segment)	Best for mass propagation
Petiole TCL	BA 0.2 + NAA 0.2 mg/L	>75%	Alternative combination
Stem TCL	BA + NAA	Moderate	2 mm thick sections
Floral Stalk TCL	TDZ 0.2 mg/L	High	Position 2 optimal

Rooting & Acclimatization

Step 1 Adventitious shoots 1–1.5 cm high with 3–4 leaves are transferred to rooting medium.
Step 2 Rooting medium: MS + BA 0.5 mg/L + NAA 0.1 mg/L + activated charcoal 1 g/L
Step 3 Roots develop after of culture.
Step 4 Transfer plantlets to sterile moistened sand:soil (1:1), pH 5.8, in jam bottles.
Step 5 After , transfer to polybags with same potting mixture.
Step 6 Keep in greenhouse at 25°C and 80% humidity.
Step 7 Plantlets adapt well and flower after .

Key Success Factors

TCL thickness: 3 mm recommended for high shoot formation frequency and uniform shoots
TDZ concentration: Low concentration (<0.2 mg/L) effective for caulogenesis from petiole tTCLs
BA alone: Highest shoot formation (56.67%) recorded with 1.0 mg/L BA
BA + NAA: Shoot formation over 75% when both used at low concentrations
Floral stalk position: Position 2 gives highest regeneration rate
Mass propagation: ~10,000 plantlets from one plant with 5 petioles in 8 months