Media Preparation
Complete Technical Guide

1. Macronutrients: The Building Blocks

Function: Provide essential elements for plant growth

• Nitrogen (N): Protein synthesis and chlorophyll production
• Phosphorus (P): Energy transfer and root development
• Potassium (K): Water regulation and disease resistance
• Standard Source: MS (Murashige and Skoog) salts provide optimal balance

2. Micronutrients: The Catalysts

Function: Enable enzymatic functions and metabolic processes

• Iron (Fe): Chlorophyll synthesis and electron transport
• Manganese (Mn): Photosynthesis and nitrogen metabolism
• Zinc (Zn): Enzyme activation and growth regulation
• Copper (Cu): Electron transport and cell wall formation
• Molybdenum (Mo): Nitrogen fixation processes
• Boron (B): Cell wall formation and carbohydrate transport

3. Vitamins: The Metabolic Enhancers

• Thiamine (Vitamin B1): Carbohydrate metabolism
• Pyridoxine (Vitamin B6): Amino acid metabolism
• Nicotinic Acid (Niacin): Energy production processes

4. Growth Regulators: The Control System

Cytokinin s (Promote Shoot Development):

• BAP (6-Benzylaminopurine): Most commonly used, highly effective
• Kinetin: Natural Cytokinin , gentler action
• TDZ (Thidiazuron): Very potent, use in lower concentrations

Auxin s (Promote Root Development):

• IBA (Indole-3-butyric acid): Stable and effective for rooting
• NAA (Naphthaleneacetic acid): Strong Auxin , use carefully
• IAA (Indole-3-acetic acid): Natural Auxin , less stable

5. Carbon Source: The Energy Provider

• Sucrose : Standard choice at 20 g-30 g/L concentration
• Alternative Options: Glucose or maltose for specific applications
• Function: Provides energy since photosynthesis is limited in culture vessels

6. Gelling Agent: The Support Matrix

• Agar : Traditional choice, 7 g-8 g/L concentration
• Gellan gum : Clearer medium, easier Contamination detection
• Phytagel : Alternative for species-sensitive to Agar

7. pH Adjusters: The Balance Keepers

• Target Range: pH 5.6-pH 5.8 for optimal nutrient uptake
• Basic Adjuster: Sodium hydroxide (NaOH) for raising pH
• Acidic Adjuster: Hydrochloric acid (HCl) for lowering pH

Multiplication Media Recipe ( MS -Based)

Base Components (per liter):

• MS basal salts: 4.4 g
• Sucrose : 30 g
• Agar : 8 g
• Distilled water : to 1000 ml

Hormone Addition:

• BAP ( Cytokinin ): 0.5-2.0 mg/L
• IAA ( Auxin ): 0.1-0.5 mg/L
• Ratio: High Cytokinin to low Auxin (4:1 to 10:1)

Vitamin Supplements (optional but recommended):

• Thiamine HCl: 0.1 mg/L
• Pyridoxine HCl: 0.5 mg/L
• Nicotinic acid : 0.5 mg/L
• Myo-inositol: 100 mg/L

Rooting Media Recipe ( MS -Based)

Base Components (per liter):

• MS basal salts: 4.4 g (sometimes half-strength: 2.2 g)
• Sucrose : 20 g (reduced from Multiplication media)
• Agar : 8 g
• Distilled water : to 1000 ml

Hormone Addition:

• IBA ( Auxin ): 1.0-5.0 mg/L
• Cytokinin : 0.1 mg/L or completely omitted
• Ratio: High Auxin to no/low Cytokinin

Alternative Formulations:

• Hormone-Free: Some species root better without added hormones
• NAA Alternative: 0.5-2.0 mg/L NAA instead of IBA for stubborn species

Step 1: Preparation of Stock Solutions

Purpose: Ensures accuracy and consistency across batches

1. Macronutrients Stock (10x concentration):
   • Dissolve 44 g MS salts in 1L distilled water
   • Store at 4°C for up to 6 months
2. Vitamin Stock (1000x concentration):
   • Prepare individual vitamin solutions
   • Store frozen in small aliquots
   • Use within 3 months
3. Hormone Stock Solutions (1000x concentration):
   • Dissolve hormones in small amount of 1N NaOH or ethanol
   • Dilute to final volume with distilled water
   • Store frozen, use within 1 year

Step 2: Base Media Preparation

1. Dissolve Basal Salts: Add stock solution to distilled water, stir until dissolved using magnetic stirrer
2. Add Sucrose: Add required amount, stir until no crystals remain
3. Add Vitamins: Add vitamin stock solutions and mix thoroughly

Step 3: Hormone Addition

1. Calculate exact volumes needed based on final concentrations
2. Add hormone stock solutions slowly while stirring, ensure complete dissolution

Step 4: pH Adjustment

1. Initial Measurement: Use calibrated pH meter (pH 5.6-pH 5.8 target)
2. Adjustment Process: Add 1N HCl to lower or 1N NaOH to raise pH dropwise, mix thoroughly between additions

Step 5: Volume Adjustment & Gelling Agent

1. Add distilled water to reach final volume
2. Weigh agar precisely (8 g per liter)
3. Heat in microwave in short bursts (30-60 seconds)
4. Stir between heating cycles until completely dissolved and clear

Step 6: Dispensing & Sterilization

Container Filling:

• 15-20 ml per culture tube
• 40-50 ml per culture jar

Autoclave Settings:

• Temperature: 121°C
• Pressure: 15 psi
• Time: 15-20 minutes

Storage:

• Cool to room temperature before handling
• Store at room temperature for immediate use
• Refrigerate for longer storage (up to 1 month)

Species-Specific Adaptations

Woody Plants:

• WPM (Woody Plant Medium): Often better than MS for trees and shrubs
• Lower Salt Concentrations: Reduce standard MS to half-strength
• Antioxidants: Add Ascorbic acid to prevent Browning

Orchids:

• VW (Vacin & Went) Medium: Specifically formulated for orchids
• Banana Extract: Natural supplement for enhanced growth
• Coconut Water: Natural Cytokinin source

Ferns:

• Modified MS : Reduced nitrogen concentrations
• Lower pH : Target pH 5-pH 5.5 instead of standard range
• Specialized Vitamins: Enhanced B-vitamin complex

Troubleshooting Common Media Issues

Problem	Cause	Solution
Poor Shoot Multiplication	Insufficient cytokinin	Increase BAP to 1.5-3.0 mg/L or switch Cytokinin type
Inadequate Rooting	Low auxin concentration	Increase IBA to 2-7 mg/L or try NAA
Contamination Issues	Poor sterilization	Add PPM, extend autoclave time by 5 minutes
Hyperhydricity (Vitrification)	Excessive moisture/agar	Reduce Agar to 6 g-7 g/L or switch to Gellan gum
Phenolic oxidation Browning	Oxidation of phenolics	Add Activated charcoal (0.5-2.0 g/L) or antioxidants

Batch Documentation

Essential Records:

• Date of preparation
• Exact formulation used
• pH before and after sterilization
• Sterilization cycle details
• Performance observations

Multiplication Media Success Metrics

• Number of shoots per explant
• Time to shoot development
• Overall health and vigor

Rooting Media Success Metrics

• Percentage of shoots that root
• Root length and density
• Time to root development

Safety and Best Practices

• Personal Protection: Always wear gloves and safety glasses
• Ventilation: Ensure adequate air circulation when handling chemicals
• Storage: Follow manufacturer's storage recommendations
• Cleanliness: Maintain spotless work environment
• Consistency: Follow protocols exactly for reproducible results