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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. Access
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SIMILAR CONTENT BEING VIEWED BY OTHERS DEVELOPMENT OF A FAST AND USER-FRIENDLY CRYOPRESERVATION PROTOCOL FOR SWEET POTATO GENETIC RESOURCES Article Open access 07 September 2020 EFFICIENT
PLANT REGENERATION FROM EMBRYOGENIC CELL SUSPENSION CULTURES OF _EUONYMUS ALATUS_ Article Open access 23 July 2021 SUSPENSION CULTURE OF STEM CELLS ESTABLISHED OF _CALENDULA OFFICINALIS_ L.
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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. AUTHOR INFORMATION Author notes * Frank van Iren: Deceased. AUTHORS AND AFFILIATIONS * Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden
University, Leiden, The Netherlands Natali R Mustafa & Robert Verpoorte * Section of Plant Cell Physiology, Institute of Biology, Leiden University, Leiden, The Netherlands Ward de
Winter & Frank van Iren Authors * Natali R Mustafa View author publications You can also search for this author inPubMed Google Scholar * Ward de Winter View author publications You can
also search for this author inPubMed Google Scholar * Frank van Iren View author publications You can also search for this author inPubMed Google Scholar * Robert Verpoorte View author
publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to
Robert Verpoorte. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS
ARTICLE 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 Download citation * Published: 05 May 2011 * Issue Date: June 2011 * DOI: https://doi.org/10.1038/nprot.2010.144 SHARE THIS ARTICLE Anyone you share
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