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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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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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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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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DOI: https://doi.org/10.1038/nprot.2010.144
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