Introduction:
I am conducting an experiment to investigate the root phenotypes of Arabidopsis Col-0 plants grown under phosphate deficiency conditions. Here, I describe my current methodology and seek advice on potential improvements to observe a clear phenotypic difference between control and phosphate-deficient conditions.
Media Composition:
- Control Media: I am using a modified basal medium containing the following components: Macronutrients:0.5 mM NaH₂PO₄ (Sodium Phosphate Monobasic) - Phosphate Source (Consider mentioning the function of each component here) 2 mM KNO₃ (Potassium Nitrate) - Potassium Source 0.25 mM MgSO₄ (Magnesium Sulfate) - Magnesium Source 0.25 mM MgCl₂ (Magnesium Chloride) - Magnesium Source 2 mM CaCl₂ (Calcium Chloride) - Calcium Source 42.5 μM Fe(III)-EDTA (Iron (III) - Ethylenediaminetetraacetic Acid) - Iron Chelate Micronutrients: (List all micronutrients with their concentrations)1.8 μM MnSO₄ (Manganese Sulfate) 45 μM H₃BO₃ (Boric Acid) 0.38 μM ZnSO₄ (Zinc Sulfate) 0.16 μM CuSO₄ (Copper Sulfate)
- Phosphate-Deficient Media: The media composition is identical to the control media except for the omission of NaH₂PO₄. NaCl (Sodium Chloride) is added in its place to maintain similar ionic strength.
Media Preparation:
Sterilization and Growth Conditions:
- Seeds are sterilized using 70% ethanol followed by a solution of NaOCl (Sodium Hypochlorite) and Tween 20.
- Seeds are sown on the prepared media and grown vertically for 8-10 days.
Observations:
- I observe root growth in both control (+P) media containing NaH₂PO₄ and phosphate-deficient (-P) media without NaH₂PO₄ supplemented with NaCl.
- There are no clear phenotypic differences in root development between the control and phosphate-deficient conditions.
Question:
I am seeking advice from the ResearchGate community on potential reasons why I am not observing a clear phenotypic difference in root development between the control and phosphate-deficient conditions. Here are some areas I suspect might be contributing to the issue:
- Phosphate Deficiency Level: Is the current level of phosphate deficiency in the -P media sufficient to induce a clear phenotype?
- Media Components: Are there any additional components or adjustments needed in the media composition to enhance the response to phosphate deficiency?
- Sterilization Technique: Could the sterilization process be negatively affecting seed germination or root growth?
- Growth Duration: Is the growth period (8-10 days) long enough to observe significant differences in root phenotypes under phosphate deficiency?
Where did you get the numbers to decide on the phosphorus concentrations to use?
Also, unless the parent plants were grown on phosphorus-deficient growth conditions, the growing seed will have PLENTY of phosphorus for the first few days of growth (or even longer).
I think you'll need to grow the plants for a longer time to see any measurable difference (if you see one at all). Seedlings grown in the dark (no ability to do photosynthesis), can live 8 or more days depending on the temperature. No idea how long it would take a seedling to use up it's stored phosphorus, I just know they run out of carbohydrates after a week or so.
You might want to buy the really large, square petri plates. I remember them being about 6" x 6".
PS Do not add the sucrose. You're asking for mold.
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