Folic acid

Folic Acid in Plant Tissue Culture

Safety Note: Always consult the SDS for Folic acid and follow institutional safety procedures; treat unknowns conservatively. Folic acid is generally considered non-toxic at the concentrations used in plant tissue culture, but appropriate PPE (gloves, lab coat, eye protection) should still be used during handling and preparation.

Overview and Identity

Folic acid, a B vitamin crucial for plant growth and development, plays a supporting role in plant tissue culture media. Its primary function is not as a primary macronutrient or growth regulator, but rather as a vital micronutrient facilitating various metabolic processes. Although not always essential for successful culture, its inclusion can improve growth and development in some species and explant types.

Common Names, Synonyms, and Abbreviations

Folic acid

Vitamin B9

Pteroylglutamic acid

Chemical Identity

Formula: C₁₉H₁₉N₇O₆

Relevant Forms/Grades: Tissue culture grade is preferred. The anhydrous form or the monosodium salt form may be used. Hydrate states should be considered, impacting the calculation of required mass when preparing stock solutions.

Functional Role(s) in Plant Tissue Culture

Folic acid functions primarily as a micronutrient and vitamin in plant tissue culture. It’s a cofactor in various metabolic pathways, including those related to one-carbon metabolism, crucial for DNA and amino acid synthesis. It does not act as a PGR, buffer, chelator, gelling agent, sterilant, solvent, or mutagen in plant tissue culture media.

Mechanism and Rationale in Vitro

Folic acid’s mechanism involves its participation in enzymatic reactions related to the biosynthesis of purines, pyrimidines, and amino acids. These are essential building blocks for nucleic acids, proteins, and other crucial components of plant cells. Supplementation ensures adequate availability of these metabolites for cell division, growth, and differentiation during in vitro culture.

Stage-Specific Relevance

While not universally required, folic acid may positively influence all stages of plant tissue culture: callus induction, shoot proliferation, rooting, and somatic embryogenesis. Its effects are usually subtle and often species-dependent. Improved growth and reduced stress during these stages are possible benefits of its inclusion. Its relevance to protoplast culture or contamination control is not established.

Interactions or Compatibility/Antagonism with Other Agents

Folic acid generally exhibits good compatibility with other media components. However, its solubility can be influenced by pH and the presence of certain cations. No significant antagonistic interactions with auxins or cytokinins are reported. Light exposure may cause degradation.

Preparation and Stock Solutions

Solubility: Folic acid is sparingly soluble in water. Enhanced solubility can be achieved using dilute alkali (NaOH or KOH) solutions. Avoid using strong acids/bases as they may degrade the vitamin. Consider using 0.1N NaOH to dissolve the acid.

Suitable Solvents: Water (with base adjustment), ethanol, or a mixture of both can be used. DMSO is less preferable due to its toxicity concerns at high concentrations.

Typical Stock Concentrations: 1000 mg/L or 1000 µg/mL to 10000 mg/L are commonly prepared.

Preparation: Accurately weigh out the required amount of folic acid (consider hydrate state for calculations). Gently dissolve in a small volume of solvent while stirring, add adjusted pH water to reach final volume.

Filtration/Autoclaving: Folic acid is heat-labile. Sterile filtration (0.22 µm) is recommended after preparing the stock solutions. Add the filter-sterilized Folic acid to the media after cooling it to 45-50°C.

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

Example Stock Recipe (1000 mg/L): Weigh 100 mg of folic acid, dissolve it in 5 mL of 0.1N NaOH, then gradually add sterile distilled water to reach 100 mL final volume. Filter sterilize (0.22µm).

Working Concentrations and Usage in Media

Common working concentrations range from 0.1 – 10 mg/L (0.1 – 10 µM), but are highly species- and explant-dependent. Empirical testing is essential to optimize concentrations for different plant species and developmental stages. Add the working concentration to the cooled, but still liquid, media.

Storage and Stability

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

Container Type: Amber glass bottles are preferred.

Stock Solution Shelf-Life: Stock solutions generally remain stable for several months under appropriate storage conditions (re-test monthly, discarding upon any signs of degradation such as precipitation or discoloration). Use prepared media quickly after adding folic acid.

Dry Chemical Stability: Folic acid is relatively stable when stored dry in a cool, dark, dry place. Ensure you label properly and account for water molecule changes in the hydrate vs anhydrous form.

Quality, Sourcing, and Compatibility

Recommended Grade: Tissue culture tested grade folic acid is recommended to minimize variability.

Lot-to-Lot Variability: Check for clarity, precipitates, and measure pH.

Compatibility Issues: Precipitation may occur if incompatible salts are present.

Safety and Precautions

Folic acid, at the used concentration in plant tissue culture, presents minimal hazard in terms of acute toxicity, but appropriate safety measures are recommended. Wear gloves, eye protection, and a lab coat while handling. Any spill must be cleaned promptly. Follow institutional waste disposal protocols.

Troubleshooting and Optimization

Issues like callus browning, hyperhydricity, or inconsistent regeneration may indicate problems with media formulation, including folic acid concentration or interactions. Empirical optimization is crucial. Consider adjusting concentration, solvent, pH, or adding antioxidants.

Example Protocols and Parameters

Example 1 (Callus Induction): Folic acid = 0.5 mg/L; 2,4-D = 2 mg/L; Kinetin = 0.5 mg/L; Gellan gum = 2 g/L; pH 5.7; Autoclave base media; Filter sterilize PGRs, add at 45°C.

Example 2 (Shoot Proliferation): Folic acid = 1 mg/L; BAP = 1 mg/L; Agar = 8 g/L; pH 5.8.

Example 3 (Rooting): Folic acid = 0.1 mg/L; IBA = 2 mg/L; Agar = 8 g/L; pH 5.8.

Note: All ranges and concentrations are given as examples; they must be empirically determined for each species and explant.

Documentation and Labeling

Meticulous records are crucial. Labels should include: Chemical form of Folic acid, lot number, preparation date, stock concentration, solvent, pH, storage conditions, expiry date, and media batch number. Notebooks should cross-reference media batch IDs, treatment matrices, and observations (growth, morphology, phenotype).

Key Takeaways