Measurement of root growth and cell death area in the root tip using Arabidopsis seedlings that grow under DNA stress

Measurement of root growth and cell death area in the root tip using Arabidopsis seedlings that grow under DNA stress

1. Sow sterilized Arabidopsis seeds linearly, 2 mm apart, on the surface of solidified MS medium.
2. Seal the rectangular petri dish with surgical tape and incubate at 4 °C for two days in the dark.
3. Place the petri dish at a 90° angle in a plant growth chamber and incubate at 23 °C for five days under continuous light conditions.

4. Transfer five-day-old seedlings having almost the same root length to the surface of new solidified MS medium containing DNA-damaging agents.

    

For root growth measurement:

1. Place the petri dish at a 90° angle in a plant growth chamber and incubate at 23 °C for an additional five days under continuous light conditions.
2. Mark the positions of the root tips on the petri dish every 24 h.
3. Scan the marked petri dish and save images.
4. Measure root length using Fiji image analysis software (https://fiji.sc) by calculating the distance between successive marks of root tips.

For propidium iodide (PI) staining:

5.   Place the petri dish at a 90° angle in a plant growth chamber and incubate at 23 °C for an additional 24 h under continuous light conditions.

6.   Transfer the seedlings onto a glass slide and apply 100 µL PI solution.

7.     Incubate the seedlings in PI solution for 1 min at room temperature.

8.   Gently apply a cover slip and observe root tips under a confocal laser scanning microscope (excitation wavelength, 488 nm).

9.   Measure cell death area in the root tip using Fiji image analysis software (https://fiji.sc) by defining the field in which PI infiltrates the cells.

Plant medium and PI solution

1. MS medium [1/2 Murashige and Skoog Plant Salt Mixture (Fujifilm Wako Co.), 1% (w/v) sucrose, 0.5 g/L MES, 1.2% (w/v) phytoagar, pH 5.7]
2. PI solution [0.02 mg/mL propidium iodide (Fujifilm Wako Co.)]

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