The variation in growth between Arabidopsis plants of the same genotype grown on MS medium and in substrate can result from several factors. Here are the main reasons and suggestions to help minimize the variation:

1. Differences in Nutrient Availability:

• MS Medium: Nutrients are uniformly distributed and readily available. Plants on plates often experience optimal and consistent nutrient conditions.
• Substrate: The nutrient distribution can be uneven, depending on the type of soil or substrate used. This may result in localized deficiencies or excesses.
• Solution: Use a high-quality, homogeneously mixed substrate with consistent nutrient composition. Avoid substrates with uneven texture or organic matter.

2. Watering Practices:

• MS Medium: The agar in the medium provides uniform water availability, reducing variability.
• Substrate: Uneven watering can create patches of varying moisture, leading to differences in plant growth.
• Solution: Standardize watering methods, such as bottom watering or controlled irrigation, to ensure uniform moisture levels across all pots.

3. Root System Constraints:

• MS Medium: Roots grow on a flat surface, making nutrient and water uptake efficient and uniform.
• Substrate: Compaction or inconsistent drainage in the substrate can hinder root development, causing variability.
• Solution: Use a well-aerated substrate with good drainage properties. Avoid compacting the soil during pot filling.

4. Light Intensity and Distribution:

• MS Medium: Plates are typically grown in a highly controlled environment, with even light exposure.
• Substrate: Uneven light distribution in growth chambers or greenhouses can cause variability.
• Solution: Ensure uniform light intensity by placing pots in a growth chamber with well-distributed illumination. Rotate pots regularly if necessary.

5. Microbial Contamination or Competition:

• MS Medium: Sterile conditions minimize microbial interference.
• Substrate: Substrate can harbor microbes or fungi that compete with plants for nutrients or create stress.
• Solution: Use a sterilized substrate and pots. Alternatively, treat the substrate with a mild fungicide before planting.

6. Seed Quality and Sowing Practices:

• Seed Uniformity: Variation in seed vigor or maturity can lead to differences in germination and growth.
• Sowing Depth: Inconsistent sowing depth in substrate can cause differences in germination timing.
• Solution: Use seeds from the same batch, select evenly sized seeds, and ensure uniform sowing depth (just below the substrate surface).

7. Environmental Conditions:

• Temperature and Humidity: These are typically more controlled for MS plates but may vary more in substrate growth.
• Solution: Standardize growth conditions for both systems. Avoid fluctuations in temperature or humidity.

8. Pot Size and Density:

• Pot Size: Smaller pots can limit root growth, affecting individual plant performance.
• Density: Crowding plants can cause shading or root competition, especially in substrate.
• Solution: Use appropriately sized pots and maintain adequate spacing between plants.

9. Hormonal Differences in Growth:

• MS medium often has added hormones or consistent endogenous hormone levels due to uniform nutrient availability, which can differ in substrate.

Suggestions for Troubleshooting:

Conclusion: Variability is expected to some degree, but addressing substrate uniformity, environmental conditions, and watering practices can help reduce it.

André Arruda It is rather expected. Look at these publications:

Puy, Javier, et al. "Diversity of parental environments increases phenotypic variation in Arabidopsis populations more than genetic diversity but similarly affects productivity." Annals of botany 127.4 (2021): 425-436.

Walter, Greg M., et al. "Hidden genetic variation in plasticity provides the potential for rapid adaptation to novel environments." New Phytologist 239.1 (2023): 374-387.