Oryzalin

Oryzalin in Plant Tissue Culture: A Practical Guide

Safety Note: Oryzalin is a herbicide; handle with care. Always consult the SDS for Oryzalin and follow institutional safety procedures; treat unknowns conservatively.

Overview and Identity

Oryzalin is a dinitroaniline herbicide commonly used in plant tissue culture to inhibit microtubule assembly, thereby affecting cell division and growth. Its precise role varies depending on concentration and stage of culture. While not a standard component of basal media, it finds niche applications for specific manipulations.

Common Names, Synonyms, and Abbreviations

Oryzalin; 3,5-dinitro-N4,N4-dipropylsulfanilamide; Surflan. No standard abbreviation is widely used.

Chemical Identity

• Formula: C₁₂H₁₇N₃O₄S
• Relevant Forms/Grades: Tissue-culture-grade is preferred; purity and absence of plant growth-inhibiting contaminants should be verified. Typically supplied as a solid powder. Hydrate forms may exist, but the anhydrous form is common.

Functional Role(s) in Plant Tissue Culture

Oryzalin functions primarily as a microtubule-disrupting agent, impacting cell division and growth. It is not a nutrient, vitamin, buffer, chelator, gelling agent, sterilant, solvent, or mutagen in the traditional sense of these terms in tissue culture.

Mechanism and Rationale in vitro

Oryzalin binds to tubulin dimers, preventing their polymerization into microtubules. Microtubules are crucial for cell division (cytokinesis, spindle formation), cell wall synthesis and expansion, and other cytological processes. By disrupting microtubule function, Oryzalin arrests cell division and alters cell morphology, which can be exploited to regulate development.

Stage-Specific Relevance

• Callus Induction: Low concentrations might enhance callus formation in some species by triggering stress responses that stimulate cell proliferation. But high concentrations are inhibitory.
• Shoot Proliferation: Primarily used to control hyperhydricity and promote more compact, healthier shoot growth in some species.
• Rooting: Not typically used directly for rooting.
• Somatic Embryogenesis: Its effect is species-dependent and may influence embryo development based on controlled inhibition of cell division.
• Protoplasts: Not commonly employed with protoplasts.
• Contamination Control: Oryzalin is not a sterilant.

Interactions or Compatibility/Antagonism with Other Agents

Oryzalin’s effects can interact with plant growth regulators (PGRs). The optimal concentration is highly species- and explant-specific. Its interaction with other media components requires empirical determination. For example, extreme pH may affect solubility. Certain gelling agents might be less compatible than others.

Preparation and Stock Solutions

• Solubility: Oryzalin is sparingly soluble in water. DMSO is a common solvent, but ethanol or a mixture of DMSO and water can also be used.
• Typical Stock Concentrations: 1000–10,000 mg/L (1–10 g/L) stock solutions are common. This allows for easy dilution to working concentrations.
• Procedure: Weigh out the required amount of Oryzalin. Add the solvent slowly, stirring continuously to ensure complete dissolution. The process might need gentle heating (avoid exceeding 40°C). Do not adjust pH significantly.
• Filtration/Autoclaving: Oryzalin is generally heat-stable and can be autoclaved. However, filter-sterilization (0.22 µm) is recommended to avoid potential heat-induced degradation and precipitation.
• Light/Oxygen Sensitivity: Oryzalin is relatively stable but should be stored in amber glass bottles to minimize light exposure.

Example Stock Recipe: To prepare 100 mL of a 10,000 mg/L Oryzalin stock solution in DMSO:

1. Weigh 1 g of Oryzalin powder.
2. Add 90 mL of DMSO.
3. Stir continuously until completely dissolved.
4. Bring the volume to 100 mL with DMSO.
5. Store in an amber glass bottle at 4°C.

Working Concentrations and Usage in Media

Working concentrations are highly species and explant-dependent. Ranges of 0.1–10 mg/L are commonly explored. The concentration should be carefully chosen based on preliminary experiments and dose-response trials. Begin with lower concentrations and incrementally increase until the desired effect is achieved.

Storage and Stability

• Storage Conditions: Store stock solutions at 4°C in amber glass bottles. Protect from light and moisture.
• Container Type: Amber glass bottles are preferred due to light sensitivity.
• Shelf Life: Stock solutions are generally stable for several months under appropriate storage conditions. However, it is advisable to periodically check for signs of degradation like precipitation or changes in color or clarity.
• Dry Chemical Stability: Oryzalin is relatively stable as a solid, but should be stored in a cool, dry place in a closed container, away from light and oxidizing agents.

Quality, Sourcing, and Compatibility

• Recommended Grade: Tissue culture-grade Oryzalin is highly recommended to ensure acceptable purity and the absence of growth inhibitors.
• Lot-to-Lot Variability: Lot-to-lot variability in purity and performance may exist. QC checks (solubility, clarity, pH) are essential.
• Compatibility Issues: It’s crucial to test compatibility with other media components, such as salts, chelators and gelling agents. Precipitation or decreased gel strength may indicate incompatibility.

Safety and Precautions

Oryzalin is a herbicide; it may cause skin irritation, eye irritation, respiratory problems and ingestion is extremely hazardous. Wear appropriate PPE (gloves, goggles, lab coat), work in a well-ventilated area (fume hood recommended), and follow all institutional regulations concerning disposal of chemical waste.

Troubleshooting and Optimization

Issues include precipitation, hyperhydricity, and inconsistent development. Troubleshooting requires careful assessment of the media composition, concentration of Oryzalin, solvent choice, and the interaction of Oryzalin with other components.

Example Protocols and Parameters

• Example 1 (Callus Induction): Oryzalin at 0.5–1 mg/L in combination with 2,4-D (1–2 mg/L) and kinetin (0.5 mg/L), in a Murashige and Skoog (MS) basal medium solidified with 2 g/L gellan gum, adjusted to pH 5.7, autoclave base, filter-sterilize PGRs and Oryzalin, add to cooled media, incubate in the dark at 25°C.

Documentation and Labeling

All working and stock solutions must be meticulously labeled with the date of preparation, the concentrations of all components (with units), the solvent used, the pH, and storage conditions. Cross-reference this information in your lab notebook with media batch numbers, plate/bottle IDs, and treatment matrices.

Key Takeaways

• Oryzalin is a microtubule-inhibitor used to regulate plant cell division and development in tissue culture.
• Its concentration and use are highly species- and explant-dependent; empirical optimization is essential.
• DMSO is a common solvent for Oryzalin; filter-sterilization is recommended.
• Careful attention must be paid to potential interactions with other media components, including PGRs, chelators and salts.
• Always consult the SDS and follow institutional safety procedures before using Oryzalin.