Near-Infrared Autofluorescence Imaging of Nuclei in Living Plant Roots

Materials and reagents

Plant materials

1. Arabidopsis thaliana (L.) Heynh. ecotype: Col-0

2. Arabidopsis thaliana transgenic plant harboring BOR1pro:BOR1-GFP [7]

Reagents

1. Ethyl alcohol (Sigma-Aldrich, catalog number: 09-0770)

2. Gellan gum (Fujifilm Wako, catalog number: 073-03075)

3. 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Roche, catalog number: 10236276001)

4. NaH₂PO₄·2H₂O (Fujifilm Wako, catalog number: 192-02815)

5. NaHPO₄·12H₂O (Fujifilm Wako, catalog number: 196-02835)

6. MgSO₄·7H₂O (Fujifilm Wako, catalog number: 131-00405)

7. Ca(NO₃)₂·4H₂O (Fujifilm Wako, catalog number: 039-00735)

8. KNO₃ (Fujifilm Wako, catalog number: 160-04035)

9. MnSO₄·H₂O (Sigma, catalog number: M7634)

10. ZnSO₄·7H₂O (Fujifilm Wako, catalog number: 264-00402)

11. CuSO₄·5H₂O (Fujifilm Wako, catalog number: 039-04412)

12. CoCl₂·6H₂O (Fujifilm Wako, catalog number: 036-03682)

13. (NH₄)₆Mo₇O₂₄·4H₂O (Fujifilm Wako, catalog number: 016-06902)

14. FeSO₄·7H₂O (Fujifilm Wako, catalog number: 098-01085)

15. Na₂EDTA·2H₂O (Sigma, catalog number: 345-01865)

16. Boric acid (Fujifilm Wako, catalog number: 026-15715)

17. Sucrose (Fujifilm Wako, catalog number: 196-00015)

Solutions

1. 200× Pi stock solution (see Recipes)

2. 200× Mg stock solution (see Recipes)

3. 200× Ca-K-N stock solution (see Recipes)

4. 200× Micronutrient (-B, -Fe) stock solution (see Recipes)

5. 200× Fe stock solution (see Recipes)

6. DAPI stock and staining solution (see Recipes)

7. MGRL media (see Recipes)

8. MGRL liquid medium without sugar (see Recipes)

Recipes

1. 200× Pi stock solution (pH 5.8)

NaH₂PO₄·2H₂O 47.2 g/L

Na₂H PO₄·12H₂O 18.4 g/L

This stock solution should be prepared using Milli-Q ultrapure water. Store in plastic bottles at room temperature.

2. 200× Mg stock solution

MgSO₄·7H₂O 74.0 g/L

This stock solution should be prepared using Milli-Q ultrapure water. Store in plastic bottles at room temperature.

3. 200× Ca-K-N stock solution

Ca(NO₃)₂·4H₂O 94.4 g/L

KNO₃ 60.7 g/L

This stock solution should be prepared using Milli-Q ultrapure water. Store in plastic bottles at room temperature.

4. 200× Micronutrient (-B, -Fe) stock solution

MnSO₄·H₂O 348 mg/L

ZnSO₄·7H₂O 57.5 mg/L

CuSO₄·5H₂O 50 mg/L

CoCl₂·6H₂O 6.2 mg/L

(NH₄)₆Mo₇O₂₄·4H₂O 5.9 mg/L

This stock solution should be prepared using Milli-Q ultrapure water. Store in plastic bottles at room temperature.

5. 200× Fe stock solution

FeSO₄·7H₂O 2.78 g/L

Na₂EDTA·2H₂O 3.72 g/L

This stock solution should be prepared using Milli-Q ultrapure water. Store in plastic bottles at room temperature.

6. DAPI stock and staining solution

Dissolve DAPI in ethanol at a concentration of 5 mg/mL (stock solution). For the staining solution, dilute DAPI stock solution 1,000-fold using MGRL liquid medium without sugar (final concentration of DAPI: 5 μg/mL).

7. MGRL media

1. Add the following reagents into ~700 mL of Milli-Q ultrapure water:

a. 200× Pi stock solution 4 mL

b. 200× Mg stock solution 4 mL

c. 200× Ca-K-N stock solution 4 mL

d. 200× Micronutrient stock solution 4 mL

e. 200× Fe stock solution 4 mL

2. Add 1 mM or 500 mM boric acid solution: For low-B medium, add 400 μL of 1 mM boric acid solution (0.5 μM B, final concentration). For normal-B medium, add 48 μL of 500 mM boric acid solution (30 μM B, final concentration).

3. Adjust the volume to 800 mL with Milli-Q ultrapure water.

4. Transfer the solution into a 1,000 mL Nalgene^TM wide-mouth PPCO bottle.

5. Add 8 g of sucrose.

6. Add 12 g of gellan gum.

7. Autoclave at 121 °C for 15 min.

8. MGRL liquid medium without sugar

1. Add the following reagents into ~40 mL of Milli-Q ultrapure water in a 50 mL plastic conical tube.

a. 200× Pi stock solution 250 μL

b. 200× Mg stock solution 250 μL

c. 200× Ca-K-N stock solution 250 μL

d. 200× Micronutrient stock solution 250 μL

e. 200× Fe stock solution 250 μL

2. Add 1 mM or 500 mM boric acid solution: For low-B medium, add 25 μL of 1 mM boric acid solution (0.5 μM B, final concentration). For normal-B medium, add 3 μL of 500 mM boric acid solution (30 μM B, final concentration).

3. Adjust the volume to 50 mL with Milli-Q ultrapure water.

Laboratory supplies

1. Cover glass, 22 × 40 mm No. 1, 0.13–0.17 mm thickness (Matsunami Glass Ind., Ltd., catalog number: C022401)

2. 1.5 mL test tube

3. 1,000 μL pipette tip (used with 1,000 μL micropipette for washing seeds)

4. 200 μL pipette tip (used with 200 μL micropipette for sample mounting on slide glass)

5. 10 μL pipette tip (used with 20 μL micropipette for sowing)

6. 50 mL plastic conical tube

7. Sterile 25 mL serological pipette

8. Surgical tape 21N No. 12, 12 mm × 9 m (Nichiban Co., Ltd.)

9. Rectangle plastic dish, 140 mm × 100 mm × 14.5 mm (Eiken Chemical Co., Ltd., catalog number: AW2000)

10. Aluminum foil

11. Scalpel blade or razor blade

Equipment

1. 1,000 mL Nalgene^TM wide-mouth PPCO bottles with closure (Nalgene Nunc, Thermo Fisher Scientific, catalog number: 312105-0032)

2. Autoclave

3. Plant growth chamber (NK System, catalog number: LH-411PFPFDT-SP)

4. Confocal laser scanning microscope equipped with 640 nm laser (Zeiss, model: LSM800)

5. Microscope objective lens, 40× water-immersion (1.1 NA LD C-Apochromat, Zeiss, item number: 421867-9970-000)

6. Microscope objective lens, 63× oil-immersion (1.4 NA Plan-Apochromat, Zeiss, item number: 420782-9900-000)

7. Slide glass (Matsunami Glass Ind., Ltd., catalog number: S012260)

8. Clean bench

9. 1,000 μL micropipette

10. 200 μL micropipette

11. 20 μL micropipette

12. Electric automatic pipette

Software and datasets

1. Fiji/ImageJ (ImageJ2, ver2.9.0/1.53t) (see https://imagej.net/software/fiji/) [8]

Procedure