Surfactants (Tween 20/Tween 80 for wetting)

Surfactants (Tween 20/Tween 80) in Plant Tissue Culture

Safety Note: Always consult the SDS for Surfactants (Tween 20/Tween 80) and follow institutional safety procedures; treat unknowns conservatively. Tweens are generally considered low hazard, but appropriate PPE (gloves, eye protection) should be used during handling and preparation.

Overview and Identity

Tween 20 and Tween 80 are non-ionic surfactants frequently used in plant tissue culture to improve the wetting of explants and the distribution of media components. Their primary function is to reduce surface tension, facilitating better contact between the explant and the culture medium.

Common Names, Synonyms, and Abbreviations

• Tween 20: Polysorbate 20, polyoxyethylene (20) sorbitan monolaurate
• Tween 80: Polysorbate 80, polyoxyethylene (20) sorbitan monooleate

Chemical Identity

Both Tweens are polyethoxylated sorbitan esters. The precise chemical formula varies slightly depending on the degree of polymerization of the polyoxyethylene chain (indicated by the number “20”). Tissue culture grade Tweens are generally available as solutions but can be purchased as solids. The exact form should be specified by the supplier.

Functional Role(s) in Plant Tissue Culture

In plant tissue culture, Tween 20 and Tween 80 function solely as surfactants. They are not macronutrients, micronutrients, vitamins, PGRs, buffers, chelators, gelling agents, sterilants, solvents, or mutagens.

Mechanism and Rationale In Vitro

Tweens reduce the surface tension of the liquid medium, allowing for better penetration into the plant tissues. This improved wetting enhances nutrient and hormone uptake by the explants, particularly important for recalcitrant species or when using large, dense explants. It promotes more uniform contact between the explant and the medium, resulting in more consistent and efficient growth.

Stage-Specific Relevance

Tweens can be beneficial at all stages of plant tissue culture: callus induction, shoot proliferation, rooting, somatic embryogenesis, and protoplast culture. Their use is particularly advantageous with recalcitrant species or when working with dense explants.

Interactions or Compatibility/Antagonism with Other Agents

Tweens have generally been shown to exhibit good compatibility with most commonly used plant tissue culture components. However, potential interactions might arise if using highly cation-sensitive gelling agents, and therefore testing is often required to determine optimal compatibility. No significant interactions with plant growth regulators (PGRs) have been widely documented, though this needs to be considered throughout the experimentation process.

Preparation and Stock Solutions

Solubility: Tweens are readily soluble in water, ethanol, and other common organic solvents.

Stock Concentration: Typically, 5-10% (v/v) stock solutions are prepared.

Preparation: Accurately weigh the required amount of Tween 20 or Tween 80. Slowly add the solvent while stirring gently to ensure complete dissolution. Filter sterilize (0.22 µm) the stock solutions; autoclaving is not recommended as it may degrade the surfactant.

Example Stock Recipe (10% Tween 20):

1. Weigh out 10g of Tween 20.
2. Add 90ml of sterile distilled water.
3. Stir gently until completely dissolved.
4. Filter sterilize (0.22 µm).
5. Store in an amber glass bottle at 4°C.

Light/Oxygen Sensitivity: Tweens are relatively stable but are best stored in amber glass bottles to minimize photodegradation.

Working Concentrations and Usage in Media

Typical working concentrations range from 0.01% to 0.1% (v/v). The optimal concentration is dependent on the species, explant type, and culture medium components. It is crucial to optimize this empirically for each system. The addition of surfactant should ideally occur after the other medium components are autoclaved and cooled to ~50°C in order to avoid heat-mediated degradation.

Notes on Species/Explant Variability: The optimal Tween concentration is highly variable and needs to be determined empirically for specific species and explant types via dose-response assays.

Storage and Stability

Storage Conditions: Store stock solutions in amber glass bottles at 4°C, protected from light and desiccation.

Shelf Life: Stock solutions are generally stable for several months under proper storage conditions. However, always visually inspect stock solutions, and discard them if cloudiness, precipitation, or significant pH changes occur.

Quality, Sourcing, and Compatibility

Use tissue culture-grade Tween 20/80. Inspect for clarity and absence of precipitates before use. It’s vital to record lot number and results thoroughly for repeatability. Compatibility issues are rare but can sometimes manifest when used with gelling agents.

Safety and Precautions

Tweens are generally considered to be of low toxicity. However, skin and eye irritation is possible; therefore, appropriate personal protective equipment (PPE), including gloves and eye protection, should be used during handling and preparation. In case of spillage, follow institutional safety protocols relevant to surfactant spills.

Troubleshooting and Optimization

Issues rarely arise due to surfactants. If problems occur, confirm that the stock solution is free of precipitates and that the working concentration is optimal. Adjusting the Tween concentration based on the results of dose-response experiments is a common approach to refining any tissue culture protocol for efficiency and consistency.

Example Protocols and Parameters

• Example 1 (Shoot Proliferation): Murashige and Skoog (MS) medium supplemented with 1mg/L BAP, 0.1% Tween 20, solidified with 2 g/L agar, pH 5.8. Add Tween 20 after autoclaving and cooling the base medium.

• Example 2 (Callus Induction): MS medium supplemented with 2 mg/L 2,4-D, 0.05% Tween 80, solidified with 6g/L agar, pH 5.7. Sterilize the Tween by membrane filtration before addition to the cooled autoclaved medium.

(Note: These are merely examples; concentrations must always be tailored to the specific plant species and explant.)

Documentation and Labeling

Clearly label all stock and working solutions with:

• Chemical name (Tween 20/80).
• Lot number.
• Preparation date.
• Stock concentration (% v/v).
• Solvent used.
• pH (if applicable).
• Storage conditions.
• Expiration date.

Meticulous record keeping in lab notebooks is essential, including cross-referencing media batch, plate/bottle IDs, and treatment matrices.

Key Takeaways

• Tweens 20 and 80 are non-ionic surfactants improving explant wetting and nutrient uptake.
• Typical working concentrations range from 0.01% to 0.1% (v/v), but optimization is crucial.
• Prepare stock solutions by dissolving in water or ethanol; filter sterilize; avoid autoclaving.
• Store stock solutions in amber glass bottles at 4°C, protected from light.
• Always consult the SDS and institutional safety protocols before working with any chemicals.