Confocal Live Imaging of Reproductive Organs Development in Arabidopsis

Materials and reagents

Biological materials

1. Reproductive organs of 4-week-old Arabidopsis thaliana Col-0 carrying a plasma membrane–localized fluorescent marker. Here, we used plants carrying pUBQ10::myr-YFP construct [14]; however, different plasma membrane marker lines can be used

Reagents

1. Murashige and Skoog basal salt mixture (Sigma, catalog number: M5524-50L)

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

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

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

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

6. 95% denatured alcohol (Fisher, catalog number: HC-1100-1GL)

7. Propidium iodide (PI) (Sigma, catalog number: P4170) (potential carcinogen)

Solutions

1. 1/2 MS medium (for 1 L) (see Recipes)

2. 0.1% PPM solution (see Recipes)

3. 70% ethanol solution (see Recipes)

4. 0.1% PI staining solution (see Recipes)

Recipes

1. 1/2 MS medium (for 1 L)

2.15 g of Murashige and Skoog basal salt mixture

10 g of sucrose

1 mL of Murashige and Skoog vitamin solution

Add deionized H₂O to 1 L

Adjust pH to 5.8

15 g of agar

2. 0.1% PPM solution

1 L of sterile deionized H₂O

1 mL of plant preservative mixture (PPM)

3. 70% ethanol solution

737 mL of 95% denatured alcohol

263 mL of sterile deionized H₂O

4. 0.1% PI staining solution (potential carcinogen)

1 mg of propidium iodide

1 mL of deionized H₂O

Laboratory supplies

1. 35 × 10 mm Petri dishes (SARSTEDT, catalog number: 82.1135.500)

2. Laboratory film (Parafilm^TM)

3. 200 μL and 1 mL pipette and corresponding tips

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

5. Syringe needles, 25 G × 1" (BD^®, catalog number: 305125)

6. Tungsten 1 µm probe tips (Lambda, catalog number: T20-10)

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

8. Deionized H₂O

9. Scalpel blades (FEATHER, # 15)

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

11. Plastic pots, trays, and lids for plant 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")

12. All-purpose growing soil mixture (ASB Greenworld Grower Mix)

Equipment

1. Autoclave

2. Laminar flow cabinet

3. Dissecting stereomicroscope with a minimum of 4× zoom magnification (Zeiss, model: Stemi 35)

4. Upright confocal microscope (Zeiss, model: LSM800) equipped with long working distance, water-dipping lenses with a good numerical aperture (W Plan-Apochromat 40×/1.0 DIC M27 FWD = 2.5 mm)

5. Growth chamber (Conviron GEN1000)

Software and datasets

1. Zeiss Zen 2.6 blue edition (Carl Zeiss Microscopy GmbH)

Procedure