Radial Water Loss Assay in Rice Roots Grown in Different Media

Materials and reagents

Biological materials

1. Rice line cv Nipponbare

Rice seeds were dehusked and surface-sterilized using 25% sodium hypochlorite with 0.01% Triton for 5 min, followed by five rinses in sterile water. The seeds were then incubated in moist filter paper at 28–30 °C in the dark for 48 h to allow for germination. Germinated rice seedlings were transferred into three-dimensionally printed soil columns (15 cm height, 3.3 cm diameter) for 3 days. The soil used was loamy sand soil, and the bulk density was 1.2 g/cm³. The soil was sprayed with water (roughly adding 7% of moisture to the dry soil) before preparing the soil columns. Then, the columns were hydrated via capillarity from the bottom of the column for at least 2 h and then gravity-drained to reach field capacity. For more details, see [3]. A small hole for each seed was made in the topsoil before transferring the seedlings to provide space for the already emerged radicle. Once transferred to the soil columns, rice seedlings grew under the following conditions: 12/12 h photoperiod, 300 μmol m²/s light, and 70% relative humidity. To calculate the bulk density, we used the following formula:

Bulk density (g/cm³) = dry soil (g) / volume of soil container (cm³).

In this case, as the soil container is a cylinder, the volume is:

Cylinder volume (cm³) = π × radius² (cm²) × height (cm)

Laboratory supplies

1. High-vacuum grease (Dow Corning, catalog number: 1597418); store at room temperature, in a cool, dry place, and ensure that the grease lid is tightly closed once finished using

2. Silica gel self-indicating (Fisherbrand^TM, catalog number: S/0761/60); store at room temperature, in a cool, dry place, and tightly close the lid of the container after using

Note: Any silica gel is acceptable as long as it can maintain the relative humidity constant inside the balance chamber throughout the measurements.

3. Aluminum foil (Scientific Laboratory Supplies, catalog number: ALU1011)

4. Weighing boat (Sigma-Aldrich, catalog number: HS1420AA)

5. Soft hairbrush 4 mm (Scientific Laboratory Supplies LTD, UK); as long as the brush is soft, other sizes can be used

6. Razor blades (Wilkinson Sword, UK)

7. Tweezers (Fisherbrand^TM Dumont #5 Fine Tip Tweezers, catalog number: 15483542)

8. Thin nylon mesh (nylon dress net fabric, Fabric Land, UK); any mesh of a material that does not absorb water (e.g., nylon, metal) can be used

9. Tea bags (round or square, e.g., Bird and Blend, UK, catalog number not available)

10. Tape (e.g., 3M Micropore Surgical Tape, Scientific Laboratory Supplies, catalog number: 4702)

11. Spatula (VWR International LTD, catalog number: RSGA830.500) (there is no specific spatula for this)

12. Ruler (Scientific Laboratory Supplies, catalog number: GW510) (there is no specific ruler for this)

13. Paper towels (e.g., Kimberly-Clark^TM Scott^TM Slimfold Hand Towels, Fischer Scientific, catalog number: 11715254); any paper towel is acceptable provided it is soft and kept moist while covering root segments

Equipment

1. Closed chamber, five-digit balance (Mettler Toledo, model: AX105DR)

Note: A five-digit balance allows for more precise measurements even if the root tissue mass is not ideal. Moreover, since water loss is gradual in small amounts of mass, a 4-digit balance might not capture these small changes within the timeframe of the assessment.

2. Pre-calibrated humidity and temperature indicator (Digi-sense, Fischer Scientific UK LTD, catalog number: 15890036)

3. Digital Timer (FisherBrand^TM, catalog number: 17951662)

4. Digital caliper 150 mm (RS PRO, UK, catalog number: 243-6808)

5. Drying oven (Mini 6L CLAD)

Software and datasets

1. Microsoft Excel, included in the Microsoft 365 Apps; any open-source spreadsheet editor will work

Procedure