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Initiation, growth and cryopreservation of plant cell suspension cultures

Nature Protocols volume 6, pages 715–742 (2011)Cite this article

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Abstract

Methods described in this paper are confined to in vitro dedifferentiated plant cell suspension cultures, which are convenient for the large-scale production of fine chemicals in bioreactors and for the study of cellular and molecular processes, as they offer the advantages of a simplified model system for the study of plants when compared with plants themselves or differentiated plant tissue cultures. The commonly used methods of initiation of a callus from a plant and subsequent steps from callus to cell suspension culture are presented in the protocol. This is followed by three different techniques for subculturing (by weighing cells, pipetting and pouring cell suspension) and four methods for growth measurement (fresh- and dry-weight cells, dissimilation curve and cell volume after sedimentation). The advantages and disadvantages of the methods are discussed. Finally, we provide a two-step (controlled rate) freezing technique also known as the slow (equilibrium) freezing method for long-term storage, which has been applied successfully to a wide range of plant cell suspension cultures.

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Figure 1
Figure 2
Figure 3: Stabilization of cell suspension culture after change of 2,4-D (line 6) to NAA (line 6AA) as auxin in maintenance medium; the production of alkaloids in Tabernaemontana divaricata cell suspension cultures in 1 year.
Figure 4: Growth of plant cell suspension cultures determined by loss of weight by dissimilation.
Figure 5: Growth of a batch of Cinchona cell suspension cultures.
Figure 6: The effect of inoculum density on cell metabolism (different accumulation of metabolites) of Catharanthus roseus cell suspension cultures.
Figure 7: Comparison of different methods of growth characterization (time-course experiment).
Figure 8: Growth curves, alkaloid production and some enzyme activities of four alkaloid producing lines of Tabernaemontana divaricata5 cell suspension cultures (data are shown with the standard error for four determinations).
Figure 9: Device to measure cell volume after sedimentation (CVS) as a characteristic of the growth of a cell suspension culture without sacrificing cells.
Figure 10: Effect of biomass density and volume on the accuracy of the CVS growth measurement.
Figure 11: Regrowth and relative nuclear DNA content of Taxus × media Rehd.
Figure 12: Differences between control and cryopreserved cells of Tabernaemontana divaricata determined by NMR-based metabolomics.
Figure 13: The dimensions of the CVS device.

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Acknowledgements

We thank E.G. Wilson for correcting the English of the manuscript and Z. Saiman for his assistance in providing Figure 9. The financial support from SmartCell (EU Seventh Framework Programme) and ExPlant Technologies B.V. is gratefully acknowledged. During the revision of this protocol, unfortunately, Dr. Frank van Iren passed away. With him we lost a great friend who has made a very important contribution to the plant cell biotechnology research here in Leiden over the past three decades. The cryopreservation protocol is an important heritage of all his work.

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  1. Frank van Iren: Deceased.

Authors and Affiliations

  1. Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden University, Leiden, The Netherlands

    Natali R Mustafa & Robert Verpoorte

  2. Section of Plant Cell Physiology, Institute of Biology, Leiden University, Leiden, The Netherlands

    Ward de Winter & Frank van Iren

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  1. Natali R Mustafa
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Contributions

N.R.M., on the basis of her experience as the person responsible for the plant cell cultures in the Department of Pharmacognosy, made the draft proposals for the plant cell culture protocols and combined them for the manuscript. W.d.W. and F.v.I., who developed the methods for cryopreservation and have been applying them for the past 20 years, wrote the protocols for this method. R.V. coordinated and supervised the process of writing, and on the basis of his many years of experience in plant cell biotechnology was particularly involved in the process of identifying and describing critical steps in the protocols.

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Correspondence to Robert Verpoorte.

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Mustafa, N., de Winter, W., van Iren, F. et al. Initiation, growth and cryopreservation of plant cell suspension cultures. Nat Protoc 6, 715–742 (2011). https://doi.org/10.1038/nprot.2010.144

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