Certainly! Here's a simple, step-by-step protocol for collecting root exudates from plants grown in soil-filled pots:

✅ Simple Protocol for Collecting Root Exudates from Soil-Grown Plants

🧪 Materials Needed:

• Soil-grown plants in pots (well-established)
• Distilled or deionized water (sterile if possible)
• Plastic tray or container
• Beakers or sterile collection tubes
• Aluminum foil or parafilm
• Gloves, scissors, forceps
• Optional: 0.22 µm syringe filter (if filtering exudates)

🔬 Step-by-Step Protocol:

1. Water the plants beforehand

• Water the plants 1 day before exudate collection to ensure healthy hydration but allow some soil drying.
• Avoid watering on the collection day to minimize soil water content.

2. Carefully remove the plant from the pot

• Gently take the plant out of the soil.
• Shake off loose soil without damaging the roots.

3. Wash the roots

• Rinse the roots gently with distilled water to remove soil particles.
• Avoid damaging root hairs or stripping root surfaces.

4. Incubate the roots in clean water

• Place the washed roots in a beaker with sterile distilled water (usually 50–200 mL depending on plant size).
• Ensure only the roots are submerged — no shoots or leaves in water.
• Cover the beaker with foil or parafilm to prevent contamination.

5. Incubation

• Leave the plant to incubate for 2 to 6 hours at room temperature (or under growth light conditions if needed).
• Avoid long incubation times to reduce microbial contamination.

6. Collect the exudate solution

• After incubation, collect the solution containing root exudates.
• Optionally, filter the solution through a 0.22 µm filter to remove microbes or debris.

7. Store or analyze

• Use the exudate solution immediately or store at –20°C or –80°C for later analysis (e.g., HPLC, LC-MS, etc.).

Apart from the ChatGPT-like nature of the answer above, (it frequently starts with "Certainly" or "Of course" or something like that), the answer is fairly good. I would question the use of distilled water in particular for extraction of the exudates (steps 4 & 5 - essentially the same action under 2 headings, another tell-tale of ChapGPT).

The osmotic difference between roots and water will just leach everything from the roots, rather than the highly specific exudates the plant was producing for its growing conditions. Maybe a pH buffered or at least osmotically balanced solution to reduce cell lysis? That will depend to some extent on the soil you grow the plants in, so you might want to do a quick pH test to match the wash solution to the soil.

Time of day is another aspect to consider: plants vary in production and uptake of exudates over the course of the day, so the morning samples may not line up as well with the afternoon/evening samples, unless you have a) lots of time-matched replicates, b) extremely distinct exudates for different treatments.

And syringe filtering 50mL of solution by hand through a 22um filter is hard work after 2 or 3 replicates. You start to lose consistency and the last few ml get left in the syringe, and then you don't have a comparable set of samples. 200mL is 4x harder. You may consider freeze drying (lyophilisation), or a buchner funnel type setup to maximise efficiency of this step.

The biggest issue for soil is separating the roots from the surrounding soil and then the particles stuck to the exudate. I wonder if cooling the pots to almost freezing would help keep the exudates in a semi-solid state that allows for more effective removal of the extraneous soil.

Collecting root exudates from soil-grown seedlings can indeed be challenging, as the soil environment makes it difficult to clearly separate root-derived compounds from background soil metabolites. Several approaches are available for this purpose, including hydroponic and sand culture systems, soil-based trap solutions, and more advanced in situ collection techniques. Each method has its own advantages and limitations in terms of sensitivity, representativeness, and potential contamination. For a detailed discussion of these methods, you may refer to the review article

Article Root exudation as a strategy for plants to deal with salt st...

where different approaches are outlined along with their respective pros and cons. This may help you in selecting the most suitable strategy for your experiment.

Simple Protocol for Collecting Root Exudates from Soil-Grown Plants

Materials Needed:

• Potted plants (grown in soil, at desired growth stage)
• Sterile distilled water or mild salt solution (e.g., 0.1 mM CaCl₂)
• Collection tubes (sterile, e.g., 50 mL Falcon tubes)
• Vacuum filtration setup (optional, with 0.22 µm filter)
• Ice or cold container (to keep samples cold)
• Growth chamber or greenhouse with controlled conditions
• Gloves and sterile tools (forceps, trowel)

Step-by-Step Procedure:

Important Notes:

• This method collects a mixture of root exudates, microbial metabolites, and soil leachates. Interpret results cautiously.
• For purer exudate profiles, consider hydroponic or sterile agar systems, though they lack soil interactions.
• The volume and composition of exudates depend on plant species, age, soil type, and environmental conditions.