Cell Cycle Synchronisation

These protocols are used to induce high degree of metaphase synchrony in meristem root-tip cells. The procedures use a combination of hydroxyurea, a DNA synthesis inhibitor, and the anti-microtubular drug amiprophos-methyl or oryzalin. The original version of the procedure has been published by Dolezel et al. (1992).

Cell Cycle Synchronisation in Barley

  1. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  2. Moisten the paper layers with deionized H2O.
  3. Spread the seeds on the filter paper surface.
  4. Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
  5. Select seedlings with similar length of their primary roots.
  6. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  7. Transfer the basket with seedlings to a second plastic tray containing 2 mM hydroxyurea in 0.1x Hoagland\s nutrient solution and incubate for 18h.
  8. Wash the roots vigorously in several changes of deionized H2O.
  9. Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 0.1x Hoaglands nutrient solution and incubate for 6.5 h.
  10. Transfer the basket with seedlings to a tray filled with 2.5 ľM amiprophos-methyl in 0.1x Hoagland\s nutrient solution and incubate for 2h.
  11. Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
  12. 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 

Lysak MA, Cihalikova J, Kubalakova M, Simkova H, Kunzel G, Dolezel J. Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chromosome Research 7: 431 - 444 (1999)

Cell Cycle Synchronisation in Chickpea

  1. Imbibe the seeds for 24 h in deionized H2O with aeration.
  2. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  3. Moisten the paper layers with deionized H2O.
  4. Spread the seeds on the filter paper surface.
  5. Cover the petri dish and leave the seeds to germinate for 1 - 2 days to achieve optimal root length (2 - 3 cm).
  6. Select seedlings with similar length of their primary roots.
  7. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  8. 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.
  9. Wash the roots vigorously in several changes of deionized H2O.
  10. Incubate in hydroxyurea-free 1x Hoagland's nutrient solution for 4 h.
  11. Transfer the basket with seedlings to a tray filled with 5 ľM oryzalin in 1x Hoagland's nutrient solution and incubate for 2h.
  12. 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 

Vlacilova K, Ohri D, Vrana J, Cihalikova J, Kubalakova M, Kahl G, Dolezel J. Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.). Chromosome Research 10: 695 - 706 (2002) Abstract

Cell Cycle Synchronisation in Durum Wheat

  1. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  2. Moisten the paper layers with deionized H2O.
  3. Spread the seeds on the filter paper surface.
  4. Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
  5. Select seedlings with similar length of their primary roots.
  6. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  7. Transfer the basket with seedlings to a second plastic tray containing the 1.25 mM hydroxyurea in 0.1x Hoaglands nutrient solution and incubate for 18h.
  8. Wash the roots vigorously in several changes of deionized H2O.
  9. Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 0.1x Hoaglands nutrient solution and incubate for 5 h.
  10. Transfer the basket with seedlings to a tray filled with 2.5 ľM amiprophos-methyl in 0.1x Hoaglands nutrient solution and incubate for 2h.
  11. Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
  12. 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 

Kubalakova M, Kovarova P, Suchankova P, Cihalikova J, Bartos J, Lucretti S, Watanabe N, Kianian SF, Dolezel J. Chromosome sorting in tetraploid wheat and its potential for genome analysis. Genetics 170: 823 - 829 (2005) Abstract PDF

Cell Cycle Synchronisation in Field Bean

  1. Imbibe the seeds for 24 h in deionized H2O with aeration.
  2. Wet inert substrate (e.g., perlite) with 1x Hoagland's nutrient solution and put it into a plastic tray.
  3. Wash the seeds in deionized H2O, spread them over the surface of the substrate, cover them with 1-cm layer of wet substrate.
  4. Cover the tray with aluminium foil and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (approx. 4 cm).
  5. Remove the seedlings from the substrate and wash them in deionized H2O.
  6. Select seedlings with similar length of their primary roots.
  7. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  8. Transfer the basket with seedlings to a second plastic tray containing 1.25 mM hydroxyurea in 1x Hoagland's nutrient solution and incubate for 18.5 h.
  9. Wash the roots vigorously in several changes of deionized H2O.
  10. Incubate in hydroxyurea-free 1x Hoagland's nutrient solution for 4.5h.
  11. Transfer the basket with seedlings to a tray filled with 2.5 ľM amiprophos-methyl 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 

Dolezel J, Cihalikova J, Lucretti S. A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba. L. Planta 188: 93 - 98 (1992)

Cell Cycle Synchronisation in Garden Pea

  1. Imbibe the seeds for 24 h in deionized H2O with aeration.
  2. Wet inert substrate (e.g., perlite) with 1x Hoagland's nutrient solution and put it into a plastic tray.
  3. Wash the seeds in deionized H2O, spread them over the surface of the substrate, cover them with 1-cm layer of wet substrate.
  4. Cover the tray with aluminium foil and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (approx. 4 cm).
  5. Remove the seedlings from the substrate and wash them in deionized H2O.
  6. Select seedlings with similar length of their primary roots.
  7. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  8. 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.
  9. Wash the roots vigorously in several changes of deionized H2O.
  10. Incubate in hydroxyurea-free 1x Hoagland's nutrient solution for 4.5 h.
  11. Transfer the basket with seedlings to a tray filled with 10 ľM amiprophos-methyl in 1x Hoagland's nutrient solution and incubate for 2h.
  12. 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

Cell Cycle Synchronisation in Oat

  1. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  2. Moisten the paper layers with deionized H2O.
  3. Spread the seeds on the filter paper surface.
  4. Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
  5. Select seedlings with similar length of their primary roots.
  6. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  7. Transfer the basket with seedlings to a second plastic tray containing 2 mM hydroxyurea in 0.1x Hoaglands nutrient solution and incubate for 18h.
  8. Wash the roots vigorously in several changes of deionized H2O.
  9. Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 0.1x Hoaglands nutrient solution and incubate for 4.5 h.
  10. Transfer the basket with seedlings to a tray filled with 10 ľM oryzalin in 0.1x Hoaglands nutrient solution and incubate for 2h.
  11. Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
  12. 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.

Cell Cycle Synchronisation in Rye

  1. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  2. Moisten the paper layers with deionized H2O.
  3. Spread the seeds on the filter paper surface.
  4. Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
  5. Select seedlings with similar length of their primary roots.
  6. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  7. Transfer the basket with seedlings to a second plastic tray containing 2.5 mM hydroxyurea in 1x Hoaglands nutrient solution and incubate for 18 h.
  8. Wash the roots vigorously in several changes of deionized H2O.
  9. Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 1x Hoaglands nutrient solution and incubate for 6.5 h.
  10. Transfer the basket with seedlings to a tray filled with 10 ľM oryzalin in 1x Hoaglands nutrient solution and incubate for 2h.
  11. Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
  12. 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 

Kubalakova M, Kovarova P, Suchankova P, Cihalikova J, Bartos J, Lucretti S, Watanabe N, Kianian SF, Dolezel J. Chromosome sorting in tetraploid wheat and its potential for genome analysis. Genetics 170: 823 - 829 (2005) Abstract PDF

Cell Cycle Synchronisation in Vicia sativa

  1. Imbibe the seeds for 8 h in deionized H2O with aeration.
  2. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  3. Moisten the paper layers with deionized H2O.
  4. Spread the seeds on the filter paper surface.
  5. Cover the petri dish and leave the seeds to germinate for 1 day to achieve optimal root length (2 - 3 cm).
  6. Select seedlings with similar length of their primary roots.
  7. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  8. 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.
  9. Wash the roots vigorously in several changes of deionized H2O.
  10. Incubate in hydroxyurea-free 1x Hoagland's nutrient solution for 3.5 h.
  11. 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)

Cell Cycle Synchronisation in Wheat

  1. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  2. Moisten the paper layers with deionized H2O.
  3. Spread the seeds on the filter paper surface.
  4. Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
  5. Select seedlings with similar length of their primary roots.
  6. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  7. Transfer the basket with seedlings to a second plastic tray containing the 2 mM hydroxyurea in 0.1x Hoaglands nutrient solution and incubate for 18h.
  8. Wash the roots vigorously in several changes of deionized H2O.
  9. Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 0.1x Hoaglands nutrient solution and incubate for 4.5 h.
  10. Transfer the basket with seedlings to a tray filled with 2.5 µM amiprophos-methyl in 0.1x Hoaglands nutrient solution and incubate for 2h.
  11. Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
  12. 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 

Vrana J, Kubalakova M, Simkova H, Cihalikova J, Lysak MA, Dolezel J. Flow-sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics 156: 2033 - 2041 (2000) Abstract PDF 
Kubalakova M, Vrana J, Cihalikova J, Simkova H, Dolezel J. Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.). Theoretical and Applied Genetics 104: 1362 - 1372 (2002) Abstract