Ferric chloride hexahydrate

Ferric Chloride Hexahydrate in Plant Tissue Culture

Safety Note: Ferric chloride hexahydrate is a corrosive substance. Always consult the SDS for Ferric chloride hexahydrate and follow institutional safety procedures; treat unknowns conservatively.

Overview and Identity

Ferric chloride hexahydrate, a common iron source in plant tissue culture media, provides iron (Fe), an essential micronutrient for plant growth and development. Its hexahydrate form indicates six water molecules are bound to each ferric chloride molecule.

Common Names, Synonyms, and Abbreviations

• Ferric chloride hexahydrate
• Iron(III) chloride hexahydrate
• FeCl₃·6H₂O

Chemical Identity

• Formula: FeCl₃·6H₂O
• Relevant Forms/Grades: Tissue-culture-grade ferric chloride hexahydrate is preferred to minimize the presence of contaminants that could interfere with plant growth or sterilization processes. The hexahydrate form is most commonly used due to its higher solubility compared to anhydrous ferric chloride.

Functional Role(s) in Plant Tissue Culture

Ferric chloride hexahydrate serves as a source of iron (Fe), a crucial micronutrient for various enzymatic processes involved in chlorophyll synthesis, electron transport, and nitrogen metabolism. Iron deficiency leads to chlorosis and stunted growth in vitro.

Mechanism and Rationale in vitro

Iron from ferric chloride hexahydrate is chelated (typically with EDTA or similar chelating agents) to enhance its solubility and bioavailability for plant cells. Chelation prevents iron precipitation at the physiological pH of the culture media, ensuring its uptake by plant cells. The chelated iron is absorbed via specific transporters on the plant cell membrane.

Stage-Specific Relevance

Iron is essential at all stages of plant tissue culture: callus induction, shoot proliferation, rooting, and somatic embryogenesis. Iron deficiency can limit growth and development at any of these stages. Protoplasts require readily available iron for cell wall regeneration and division. Adequate iron is crucial to prevent chlorosis and maintain healthy cell cultures.

Interactions or Compatibility/Antagonism with Other Agents

• Chelation: Ferric chloride hexahydrate is often complexed with EDTA (ethylenediaminetetraacetic acid) or other chelators to improve solubility and prevent precipitation. The choice of chelator and the molar ratio are crucial for optimal iron availability.
• pH: Iron solubility is pH-dependent; precipitation can occur at high pH.
• Cation Interactions: High concentrations of divalent cations (e.g., Ca²⁺, Mg²⁺) can interfere with iron chelation and may lead to precipitation.
• Light Sensitivity: While not as photosensitive as some other compounds, prolonged exposure to intense light may influence iron oxidation state and bioavailability.

Preparation and Stock Solutions

• Solubility: Ferric chloride hexahydrate is highly soluble in water.

• Solvents: Distilled or deionized water is typically used.

• Typical Stock Concentrations: 100g/L (10%) is a common stock concentration.

• Preparation: Weigh the required amount of ferric chloride hexahydrate, add the solvent, and stir until completely dissolved. Adjust pH as needed(see below). Filter sterilize solution through a 0.22 µm filter or sterilize through autoclaving (See below)

• Filtration/Autoclaving: Ferric chloride hexahydrate solutions can be autoclaved; however, repeated autoclaving may lead to slight degradation. Sterile filtration (0.22 µm) is a safer alternative especially for long-term storage. 0.22µm filter is recommended for all ferric chloride solutions. Always filter sterilize after all components of the culture media are combined to avoid further precipitation.

• Light/Oxygen Sensitivity: Store stock solutions in amber glass bottles to minimize light exposure.

Example Stock Recipe:

To prepare 100ml of a 100g/L stock solution:

1. Weigh out 10g of tissue culture grade ferric chloride hexahydrate.
2. Add 90ml of distilled water.
3. Stir until completely dissolved and filter sterilize.

Working Concentrations and Usage in Media

Working concentrations of iron vary widely depending on the plant species, explant type, and stage of culture. Typical ranges are from 0.1-70mg/L Fe. Ranges are species- and explant-dependent; optimize empirically. Iron is often incorporated into the base media preparation with other macronutrients before autoclaving or sterile filtration.

Stage-Specific Examples:

• Callus induction: Iron concentration can be within the typical range (0.1-70mg/L).
• Shoot proliferation: Iron levels stay consistent with callus.
• Rooting: Maintaining adequate iron levels is important for robust root development.

Notes on Species/Explant Variability: Iron requirements vary significantly amongst species and even different explant types within a species. Dose–response experiments are necessary to determine optimal concentrations.

Storage and Stability

• Storage Conditions: Store stock solutions in tightly sealed amber glass bottles at room temperature, protected from light.
• Container Type: Amber glass bottles are recommended to protect against light degradation.
• Shelf Life: Prepared stock solutions should be used within a month for optimal stability. Always note the preparation date and test the quality before use after a month of preparation. Assess for precipitation and pH drift.

Dry Chemical Stability and Handling of Hydrates vs. Anhydrous Forms: The hexahydrate form is hygroscopic; careful handling and storage in a desiccator is recommended. Anhydrous ferric chloride is less stable generally avoided.

Quality, Sourcing, and Compatibility

• Recommended Grade: Tissue culture tested grade. This guarantees sufficient purity and minimal contaminants.
• Lot-to-Lot Variability: Lot-to-lot variability is possible; it’s advisable to perform quality checks like assessing for clarity, precipitate formation, and pH.
• Compatibility Issues: Ferric chloride hexahydrate can interact with other salts and chelators; correct molar ratios ensuring appropriate chelation is crucial.

Troubleshooting and Optimization

• Precipitation: Adjust pH with a base or change the iron chelator..
• Iron Deficiency Symptoms: Increase iron concentration within optimal ranges.
• Other Issues: Interactions of iron concentrations and other supplements such as growth hormones must be tested empirically for optimal growth conditions.

Example Protocols and Parameters

• Protocol 1: Callus induction in Arabidopsis thaliana: MS media supplemented with 2,4-D (1mg/L), BAP (0.5mg/L), and ferric chloride hexahydrate (20mg/L Fe); pH 5.8, solidified with agar (8g/L).

• Protocol 2: Shoot proliferation in Nicotiana tabacum: MS media supplemented with BAP (2mg/L), and ferric chloride hexahydrate (30mg/L Fe); pH 5.7; solidified with agar (8g/L).

Remember, these ranges are examples. Optimal concentrations must be determined empirically and will vary based on the specific plant species and explant.

Documentation and Labeling

Labels and notebooks should include:

• Chemical name and form (FeCl₃·6H₂O)
• Lot number
• Preparation date
• Stock concentration (g/L or %)
• Solvent
• pH
• Storage conditions
• Expiry date

Always cross-reference media batch, plate/bottle IDs, and treatment matrices.

Key Takeaways

• Ferric chloride hexahydrate is a crucial iron source in plant tissue culture, providing an essential micronutrient.
• Chelation is vital to improve solubility and bioavailability.
• Optimal concentrations vary greatly among plant species and explants; empirical optimization via dose-response experiments are necessary.
• Careful attention to preparation, storage, and compatibility with other media components is crucial for successful tissue culture.
• Always consult the SDS and adhere to institutional safety protocols.