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Apertures are indicated in microns (pm); H2O2 concentrations in mm data are from five independent experiments. SEM = standard error of mean, n = 10

guard cells.

Apertures are indicated in microns (pm); H2O2 concentrations in mm data are from five independent experiments. SEM = standard error of mean, n = 10

guard cells.

1. First, prepare Murashige and Skoog medium full strength, with 0.5 g/L MES, pH 5.7, and with bacto-agar at a strength of 1.5% (w/v). Autoclave and cool until the medium bottle can be held in the hands.

2. In a laminar flow cabinet, pour the molten agar into Petri dishes (9 cm), either with or without H2O2 at various concentrations (see Note 13). When the agar has set, make sure the plates and lids are dry before sowing seeds onto them.

3. Surface sterilise a small amount of seeds (see Note 14) sufficient for 5-6 plates containing 15-20 seeds each. To surface sterilize, add 1 mL of 70% ethanol into each tube containing seeds and agitate gently at room temperature for 5 min.

4. Remove the ethanol by allowing the seeds to settle.

5. Add 1 mL of 10% bleach, 0.1% SDS solution and agitate gently for 5 min.

6. Remove the solution after 5 min and wash the seeds with sterile distilled water at least five times.

7. Resuspend the seeds in 1 mL of sterile water, ready to sow on the agar plates.

8. In a laminar flow cabinet, using a cut-off pipette tip and a 100-pL pipette, slowly pipet single seeds with water onto the surface of the agar plate, allowing approximately 1 cm between each seed. Sow the seeds in two horizontal rows, with a distance of approximately 3 cm between them.

9. Allow the plates to dry before sealing them with Parafilm.

10 Place the plates in the fridge for 2 d.

11. Subsequently incubate the plates for 1 week vertically on shelves in a controlled environment growth room (24°C, 16-h photoperiod with lights of intensity 100-150 pE/m2/s).

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