Preparation of Chromosome Suspensions

In this procedure, chromosomes are released from synchronized root tips mechanically after a mild fixation with formaldehyde. Chromosomes are released into a polyamine lysis buffer (LB01) which stabilises their structure. The method has been originally developed for chromosome isolation in field bean (Dolezel et al. 1992). Chromosome suspensions prepared according to this procedure are suitable for flow cytometric analysis and sorting.

Chromosome Isolation in Barley

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 20 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 15000 rpm for 10 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation.

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)

Chromosome Isolation in Chickpea

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 10000 rpm for 15 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 15 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 

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

Chromosome Isolation in Durum Wheat

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 20 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 20000 rpm for 10 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 

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

Chromosome Isolation in Field Bean

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 4 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 7.5).
  5. Isolate chromosomes by homogenising at 15000 rpm for 15 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes 

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 
Root tips may be homogenised also using a razor blade. This method is more laborious. However, it results in higher yield of longer chromosomes in field bean:

  1. Add 1.25 ml of LB01 lysis buffer into a glass petri dish (6-cm diameter).
  2. Transfer fixed root tips into the petri dish.
  3. Chop meristem root-tips individually using a sharp razor blade, avoid dispersion or drying.
  4. Filter the suspension through a 50-µm nylon mesh into a polystyrene tube.
  5. Syringe the suspension once through a 22-gauge needle to disperse intact metaphases.
  6. Store the suspension on ice.

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)

Chromosome Isolation in Garden Pea

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 3 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 7.5).
  5. Isolate chromosomes by homogenising at 13000 rpm for 15 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 25 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation.

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

Chromosome Isolation in Oat

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 25 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 25000 rpm for 10 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 25 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation.

Chromosome Isolation in Rye

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 15000 rpm for 10 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 

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

Chromosome Isolation in Vicia sativa

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 10000 rpm for 15 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 15 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation.

References 

Kovarova et al. (in press)

Chromosome Isolation in Wheat

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 20 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 20000 rpm for 10 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 

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