- Imbibe the seeds for 8 h in deionized H2O with aeration.
- 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 1 day 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 2.5 mM hydroxyurea in 1x Hoagland's nutrient solution and incubate for 18.5 h.
- Wash the roots vigorously in several changes of deionized H2O.
- Incubate in hydroxyurea-free 1x Hoagland's nutrient solution for 3.5 h.
- Transfer the basket with seedlings to a tray filled with 5 ľM oryzalin in 1x Hoagland's nutrient solution and incubate for 2h.
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.
References
Kovarova et al. (in press)
