About Lesson
1. Importance of pH in Tissue Culture
pH influences:
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Nutrient solubility
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Uptake by explants
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Stability of the medium
Target Range:
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Most species: 5.6–5.8
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Orchid cultures: ~5.2
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Woody plants: up to 6.0
📌 The Importance of pH in Plant Tissue Culture
2. Equipment Needed
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pH Meter (digital, calibrated before each use)
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Beakers, flasks, stir bars
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Magnetic stirrer with hot plate
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NaOH (0.1N) and HCl (0.1N) for pH adjustments
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Balance for weighing powder
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Distilled or deionized water
3. Media Preparation Steps
| Step | Details |
|---|---|
| 1. Dissolve MS powder | In 80% of final water volume |
| 2. Add sucrose | 30 g/L unless specified otherwise |
| 3. Adjust pH | With 0.1N NaOH or HCl while stirring |
| 4. Add agar (optional) | 6–8 g/L (stir well) |
| 5. Top up volume | To 100% final volume with distilled water |
| 6. Dispense | Into autoclavable vessels or flasks |
| 7. Sterilize | Autoclave for 15–20 minutes at 121°C |
📌 Detailed Recipe Sheet – Phytotech Labs
4. Special Handling for Hormones and Additives
Many plant growth regulators degrade with heat. Instead:
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Dissolve them in ethanol or water
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Sterilize with 0.22 µm syringe filters
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Add them to cooled media (40–50°C) after autoclaving
This applies to:
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BAP, Kinetin, TDZ
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IAA, IBA, NAA
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ABA and GA₃
5. Troubleshooting Common Errors
| Error | Effect |
|---|---|
| pH too low (<5.2) | Iron and calcium availability drops |
| pH too high (>6.2) | Precipitation of phosphates |
| Not stirring properly | Lumpy agar, uneven nutrient mix |
| Autoclaving hormones | Loss of bioactivity |
| Tap water use | Contamination or heavy metal interference |
Always make notes of changes and observations for future optimization.