- Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
- Moisten the paper layers with deionized H2O.
- Spread the seeds on the filter paper surface.
- Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
- Select seedlings with similar length of their primary roots.
- Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
- Transfer the basket with seedlings to a second plastic tray containing 2 mM hydroxyurea in 0.1x Hoaglands nutrient solution and incubate for 18h.
- Wash the roots vigorously in several changes of deionized H2O.
- Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 0.1x Hoaglands nutrient solution and incubate for 4.5 h.
- Transfer the basket with seedlings to a tray filled with 10 ľM oryzalin in 0.1x Hoaglands nutrient solution and incubate for 2h.
- Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
- Place the container in a refrigerator and leave overnight
Notes
Approximately 30 seedlings are needed to prepare one sample (1 ml of chromosome suspension). Germinate the seeds at 25 ą 0.5°C in a biological incubator in the dark.
Adjust the temperature of all solutions to 25 ą 0.5°C prior their use. Perform all incubations in the dark in a biological incubator at 25 ą 0.5°C.
Aerate all solutions. The aeration stones and tubing must be kept clean to avoid extensive contamination by bacteria and fungi.
The degree of metaphase synchrony may be checked microscopically or after flow cytometric analysis of nuclear DNA content.
