N‑methyl‑N’‑nitro‑N‑nitrosoguanidine (MNNG)

N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) in Plant Tissue Culture

Overview and Identity

N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) is a potent chemical mutagen used in plant tissue culture to induce genetic variability. It is not a nutrient, growth regulator, or component of standard media formulations. Rather, it’s employed specifically to generate somaclonal variation for breeding programs or research purposes focused on identifying useful mutations.

Common names, synonyms, and abbreviations for N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)

MNNG is primarily known by its full chemical name. No widely used common names or significant abbreviations exist within the plant tissue culture field.

Chemical identity: formula, relevant forms/grades used in tissue culture

Chemical formula: C₃H₅N₅O₃

Relevant forms: MNNG is typically available as a crystalline solid. Tissue culture grade is recommended, ensuring low levels of contaminating substances detrimental to plant cells. The anhydrous form is preferred due to the unpredictable effects of varying hydration levels. Avoid the use of other forms unless absolutely necessary, and verify their purity prior to use.

Functional role(s) in plant tissue culture

MNNG functions solely as a mutagen in plant tissue culture. It does not serve any other role in conventional media formulations.

Mechanism and rationale in vitro

MNNG is a potent alkylating agent that primarily modifies guanine bases in DNA. This leads to base pair mismatches during replication, resulting in point mutations and chromosomal aberrations. In plant tissue culture, this is exploited to introduce genetic variation into regenerating plantlets. The rationale is to create a diverse population of plants from which individuals with desirable traits (e.g., disease resistance, improved yield) can be selected.

Stage-specific relevance:

MNNG is most commonly applied during the callus induction or early stages of shoot proliferation. Its effectiveness in later stages (rooting, somatic embryogenesis) is less consistent and often less desirable. It is not used with protoplasts due to potentially higher toxicity, and would never be used as a contamination control method.

Interactions or Compatibility/Antagonism with other agents

MNNG’s interaction with other media components is less well-studied compared to established plant growth regulators (PGRs). However, it’s crucial to consider any potential reactions with buffers, chelators (like EDTA), and other compounds. MNNG’s mutagenicity is not known to be significantly affected by light or oxygen in typical tissue culture conditions.

Preparation and stock solutions

Solubility and suitable solvents: MNNG shows limited solubility in water. Dimethyl sulfoxide (DMSO) is generally the preferred solvent. Adjust pH is not needed.

Typical stock concentrations and how to prepare: Stock solutions are typically prepared at 10-100 mg/mL in DMSO. Prepare under a fume hood wearing appropriate PPE. Weigh the required amount of MNNG precisely, add the appropriate volume of DMSO, and gently dissolve it. Avoid vigorous shaking which may encourage premature degradation.

Filtration/autoclaving guidance: MNNG is heat-labile and should never be autoclaved. Always filter-sterilize (0.22 µm) the stock solution and add it to the cooled, autoclaved media aseptically.

Light/oxygen sensitivity: Minimize MNNG exposure to light and air by storing stock solutions in amber glass bottles.

Example stock recipe: To prepare a 100 mg/mL stock solution, dissolve 100 mg of tissue-culture-grade MNNG in 1 mL of DMSO.

Working concentrations and usage in media

Common working concentrations of MNNG typically range from 50 µg/mL to 1000 µg/mL (0.05 – 1 mg/mL), but the optimum concentration is highly species- and explant-dependent, requiring empirical testing.

Notes on species/explant variability: A dose–response curve should be generated for each plant species and explant type to determine the optimal concentration and exposure time. This typically involves setting up several treatment groups with different MNNG concentrations, alongside a control group.

Instruction on addition during media prep: Add the sterile-filtered MNNG solution to the cooled, autoclaved media aseptically, immediately before dispensing into culture vessels.

Storage and stability

Storage conditions: Store stock solutions at -20 °C in sealed, amber glass vials protected from light and moisture.

Container type: Amber glass vials are recommended.

Stock solution shelf-life: Prepare fresh stock solutions every 3-6 months, or sooner if signs of degradation (e.g., color change, precipitate formation) are observed.

Dry chemical stability: Store the dry chemical in a cool, dark, and dry place. Consult the SDS for information on appropriate storage duration.

Quality, sourcing, and compatibility

Recommended grade: Procure tissue-culture-tested MNNG to minimize the risk of contamination by impurities likely to interfere with plant growth.

Lot-to-lot variability: Always thoroughly check the received MNNG quantity and purity based on the received Certificate of Analysis (CoA). Monitor pH stability of the prepared stock.

Compatibility issues: MNNG’s compatibility with other salts and chelators (such as EDTA) needs evaluation on an experimental basis to confirm aseptic conditions and prevent precipitation.

Safety and precautions

Key hazards: MNNG is a potent mutagen and suspected carcinogen. Direct skin contact, inhalation, and ingestion should be avoided.

PPE: Wear appropriate personal protective equipment (PPE), including gloves, safety goggles, and a lab coat, at all times when handling MNNG.

Engineering controls: Perform all preparation and handling operations in a well-ventilated fume hood and biosafety cabinet when possible.

Safe handling: Follow good laboratory practices (GLP) and safety protocols. Always consult and comply with institutional safety guidelines. A detailed spill response plan should be available and practiced.

Waste disposal: Dispose of MNNG waste and contaminated materials according to institutional guidelines for hazardous chemical waste disposal.

Always consult the SDS for N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and follow institutional safety procedures; treat unknowns conservatively.

Troubleshooting and optimization

Common issues may include precipitation, reduced regeneration rates, callus browning, and a general decline in explant viabilty. Adjusting MNNG concentration, switching the solvent, or altering the pH might alleviate these problems. Conduct a dose-dependent study for optimization.

Example protocols and parameters

Example 1: MNNG (0.1-1 mg/L) in combination with 2,4-D (1-2 mg/L) for callus induction in Arabidopsis thaliana explants. Solidfy with 8 g/L agar; pH 5.8; filter sterilize and add MNNG solution to cooled-base media.

Example 2: MNNG (50-100 µg/mL) for shoot proliferation of Nicotiana tabacum, combined with auxin/cytokinin mixtures (optimise empirically).

Please note: These parameters are examples only, and actual working ranges must be determined empirically for each species and explant type (e.g., leaf disc versus shoot tip).

Documentation and labeling

All stock solutions and working media should be clearly labeled including:

• Chemical name (MNNG)
• Lot number
• Preparation date
• Stock concentration
• Solvent used
• pH (if applicable)
• Storage conditions
• Expiration date

Maintain detailed lab notebooks documenting preparation, working concentrations used, observations and results of experiments. Cross-reference media batch numbers, culture vessel IDs, and experimental treatment matrices meticulously.

Key takeaways:

• MNNG is a potent mutagen used to induce genetic variation in plant tissue culture.
• It’s crucial to use tissue-culture-grade MNNG and follow proper safety protocols as it’s a mutagen and suspected carcinogen.
• Optimal MNNG concentrations are highly species- and explant-dependent and must be determined empirically.
• MNNG solutions must be filter-sterilized; never autoclave.
• Always consult the SDS for MNNG and your institution’s safety guidelines.