- Imbibe the seeds for 24 h in deionized H2O with aeration.
- Wet inert substrate (e.g., perlite) with 1x Hoagland's nutrient solution and put it into a plastic tray.
- Wash the seeds in deionized H2O, spread them over the surface of the substrate, cover them with 1-cm layer of wet substrate.
- Cover the tray with aluminium foil and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (approx. 4 cm).
- Remove the seedlings from the substrate and wash them in deionized H2O.
- 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 1.25 mM hydroxyurea in 1x Hoagland's nutrient solution and incubate for 18 h.
- Wash the roots vigorously in several changes of deionized H2O.
- Incubate in hydroxyurea-free 1x Hoagland's nutrient solution for 4.5 h.
- Transfer the basket with seedlings to a tray filled with 10 ľM amiprophos-methyl in 1x Hoagland's nutrient solution and incubate for 2h.
- 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.
References
Neumann P, Pozarkova D, Vrana J, Dolezel J, Macas J. Chromosome sorting and PCR-based physical mapping in pea (Pisum sativum L.). Chromosome Research 10: 63 - 71 (2002) Abstract
