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Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation

Abstract

Polyploid plants often have altered gene expression, biochemistry, and metabolism compared to their diploid predecessors. Therefore cultured diploid cells have distinct benefits over cultured polyploid cells for the study of gene regulation and metabolism of the parent plant. Here we report methods for establishing and maintaining a rapidly dividing diploid Arabidopsis thaliana cell suspension culture, and subsequent cell cycle synchronisation. Rapid growth of homogeneous cell populations was achieved after 3 months of initiation of cultures from leaf calluses. The cells were grown in the dark on an orbital shaker (110 rpm, 50 mm orbit) at 24 °C. Continued maintenance of the culture required the use of late-exponential stage cells for subculture at weekly intervals using careful subculturing techniques to achieve accurate biomass transfer. Cell cycle synchronisation was achieved following sucrose starvation, phosphate starvation, hydroxyurea treatment, aphidicolin treatment, and a combination of phosphate starvation and aphidicolin treatment. Inhibition of the cell cycle and accumulation of cells in specific phases was monitored by microscopy to determine the metaphase/anaphase index, and by flow cytometry. The cell cycle was partially and reversibly blocked by sucrose or phosphate starvation and by hydroxyurea (2.5 mM) treatment. A complete block at G1/S interphase was achieved after aphidicolin treatment or phosphate starvation combined with aphidicolin treatment. Release from the aphidicolin block achieved ca. 78% cell cycle synchronisation in the cell population. Endoreduplication was evident after release from the block in all treatments but after one cycle (24 h) the cells returned to the diploid state. This diploid culture is currently being used in our laboratory for the genetic analysis of cell death.

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Abbreviations

BAP:

6-benzylaminopurine

DAPI:

4′-6-diamidino-2-phenylindole

2,4-d :

2,4-dichlorophenoxyacetic acid

DAR:

6-(γ,γ-dimethylallylamino)-purine riboside

DMSO:

dimethyl sulfoxide

FDA:

fluorescein diacetate

B5:

Gamborg et al. (1968) medium

IAA:

indole-3-acetic acid

M/A index:

metaphase/anaphase index

MS medium:

Murashige and Skoog (1962) medium

NAA:

α-naphthaleneacetic acid

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Acknowledgements

We wish to thank Dr Ross Bicknell, Sylvia Erasmuson and Dianne Hall for the use of their equipment and technical expertise in flow cytometry. We thank Andrew Mullan, Margaret Young and Beverly Hoffmann for tissue culture media preparation, and Duncan Hedderley for statistical support. This research was funded by the Foundation for Research, Science and Technology.

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Pathirana, R., Eason, J.R. Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation. Plant Cell Tiss Organ Cult 85, 125 (2006). https://doi.org/10.1007/s11240-005-9057-z

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  • DOI: https://doi.org/10.1007/s11240-005-9057-z

Keywords

  • aphidicolin
  • cell cycle arrest
  • cell death
  • endoreduplication
  • flow cytometry
  • hydroxyurea
  • phosphate starvation
  • sucrose starvation