Analysis of Cauline Leaf Development in Arabidopsis thaliana Using Time-Lapse Confocal Microscopy

Materials and reagents

Note: The original workflow used in Le Gloanec et al. [6] was developed by the author at the University of Montreal, Canada. For the present protocol paper, the workflow was independently re-implemented at the National University of Singapore, Singapore, in order to validate reproducibility and to generate the illustrative material presented here. The illustrative images shown in this protocol were generated from the Singapore re-implementation, whereas the time-lapse datasets and macroscopic measurements used for validation are from Le Gloanec et al. [6]. This manuscript therefore presents the protocol in a stand-alone format, with expanded methodological detail, updated figures, and implementation notes from both locations. Where materials and reagents differed between the two locations, both versions are listed in the following sections, separated by “or.”

Biological materials

1. Arabidopsis thaliana Columbia-0 (Col-0) carrying the pUBQ10::myr:YFP construct, which encodes a plasma membrane-localized fluorescent marker, was used for time-lapse experiments [7]; other plasma membrane marker lines can also be used, like pUBQ10::PM-tdTomato [8] or p35S::LTI6b-GFP [9]

Reagents

1. ½ MS medium

a. Murashige and Skoog (MS) basal salt mixture (Sigma, catalog number: M552450L)

b. Murashige and Skoog (MS) vitamin solution (Sigma, catalog number: M3900-50ML)

c. Sucrose (Fisher, catalog number: S5-3)

d. Agar (Fisher, catalog number: BP1423-2)

or

a. Murashige and Skoog (MS) medium, including vitamins (Duchefa Biochemie, catalog number: M0222.0100)

b. MES monohydrate (Duchefa Biochemie, catalog number: M1503.1000)

c. Sucrose crystallized (Duchefa Biochemie, catalog number: S0809.5000)

d. Plant agar biotechnology grade (1^st base, catalog number: 4020)

2. Plant preservative mixture (PPM) (Plant Cell Technology, catalog number: 71806-1)

3. 95% denaturated alcohol (Fisher, catalog number: HC-1100-1GL) or ethanol approx. 96% grade AR (QRëC, catalog number: E7045-1-2500)

Solutions

1. ½ MS medium (see Recipes)

2. 0.1% PPM solution (see Recipes)

3. 70% ethanol solution (see Recipes)

Recipes

1. ½ MS medium

2. 0.1% PPM immersion solution

1 L deionized H₂O

1 mL of PPM

3. 70% ethanol solution

737 mL of 95% denatured alcohol

263 mL of sterile deionized H₂O

Laboratory supplies

1. Plastic pots, trays, and lids for potting: thermoformed square pots with drainage 2.63” × 2.63” × 2.25”, no-hole trays 21” × 11” × 2.5”, and compatible plastic dome 21.50” × 11” × 2.10” (Teris) or thermoformed pots with drainage 95 × 78 × 75 mm, no-hole trays 54 × 28 × 6.5 cm, and compatible plastic domes 54 × 28 × 9 cm (Linyi Jiaxin Plastic Products Co., Ltd.)

2. All-purpose growing soil mixture (ASB Greenworld Grower Mix) or Jiffy FloraFleur potting soil M.70L/T002 (FarEastFlora, catalog number: 0000785)

3. 100 μL, 200 μL, and 1 mL pipettes with corresponding tips

4. 60 × 15 mm Petri dish (SARSTEDT, catalog number: 82.1194.500 or Corning^®, catalog number: 430196)

5. Laboratory film (Parafilm^TM)

6. Low lint tissue wipe (Kimwipes^TM, Kimberly-Clark)

7. Deionized H₂O

8. Precision tweezers with fine point (Dumont No. 5)

9. Syringe needles, 18G × 1 1/2 and 27G × 1 1/2 (BD^®, catalog number: 305196 and 301629)

10. Scalpel blades (FEATHER, #10)

11. Surgical tape (Micropore^TM tape 3M)

Equipment

1. Growth chamber, Conviron GEN1000 or Percival AR-66L3 with custom LED lighting (Grow Light C75 NS12, Valoya)

2. Autoclave

3. Laminar flow cabinet

4. Dissection stereomicroscope (Stemi 35, Zeiss or S8APO, Leica, equipped with KL 300 LED, Schott)

5. Upright confocal microscope equipped with long-working-distance water-dipping lenses (LSM800, Zeiss, with W Plan-Apochromat 40×/1.0 DIC M27 FWD = 2.5 mm or Stellaris 8 DM6 CFS, Leica, with HCX APO L 20×/1.0 W)

Software and datasets

1. Microscope software, Zeiss Zen 2.6 blue edition (Carl Zeiss Microscopy GmbH), or LAS X STELLARIS (Leica Microsystems)

2. MorphoGraphX 2.0.1 (https://morphographx.org/software/) (access date, 2026-02-26) [10–11]

3. All codes have been deposited to OSF: https://osf.io/uth78/ (access date, 2026-02-26)

Procedure