Initiation, growth and cryopreservation of plant cell suspension cultures

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

through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Subscribe to this journal

Receive 12 print issues and online access $259.00 per year only $21.58 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices

may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support

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.

Article Open access 03 January 2024 REFERENCES * Endreß, R. _Plant Cell Biotechnology_ (Springer-Verlag, 1994). * Dixon, R.A. Isolation and maintenance of callus and cell suspension

cultures. In _Plant Cell Culture; a Practical Approach_ (ed. Dixon, R.A.) 1–20 (IRL Press, 1985). * Gamborg, O.L. & Phillips, G.C. Media preparation and handling. In _Plant Cell, Tissue

and Organ Culture; Fundamental Methods_ (eds. Gamborg, O.L. & Phillips, G.C.) 1–90 (Springer Lab Manual, Springer-Verlag, 1995). * Morris, P. Long term stability of alkaloid productivity

in cell suspension cultures of _Catharanthus roseus_. In _Secondary Metabolism in Plant Cell Cultures_ (eds. Morris, P., Scragg, A.H., Stafford, A. & Fowler, M.W.) 257–262 (Cambridge

University Press, 1986). * Sierra, M.I., van der Heijden, R., van der Leer, T. & Verpoorte, R. Stability of alkaloid production in cell suspension cultures of _Tabernaemontana

divaricata_ during long-term subculture. _Plant Cell Tiss. Org. Cult._ 28, 59–68 (1992). Article  CAS  Google Scholar  * Hulst, A.C., Meyer, M.M.T., Breteler, H. & Tramper, J. Effect of

aggregate size in cell-cultures of _Tagetes-patula_ on thiophene production and cell growth. _Appl. Microbiol. Biotechnol._ 30, 18–25 (1989). Article  CAS  Google Scholar  * Edahiro, J.-I.

& Seki, M. Phenylpropanoid metabolite supports cell aggregate formation in strawberry cell suspension culture. _J. Biosci. Bioeng._ 102, 8–13 (2006). Article  CAS  Google Scholar  * Su,

W.W. & Arias, R. Continuous plant cell perfusion culture: bioreactor characterization and secreted enzyme production. _J. Biosci. Bioeng._ 95, 13–20 (2003). Article  CAS  Google Scholar

  * Ten Hoopen, H.J.G., van Gulik, W.M. & Heijnen, J.J. Continuous culture of suspended plant cells. _In Vitro Cell Dev. Biol._ 28, 115–120 (1992). Article  Google Scholar  * Schripsema,

J., Meijer, A.H., van Iren, F., ten Hoopen, H.J.G. & Verpoorte, R. Dissimilation curves as a simple method for the characterization of growth of plant cell suspension cultures. _Plant

Cell Tiss. Org. Cult._ 22, 55–64 (1990). Article  Google Scholar  * Stafford, A., Smith, L. & Fowler, M.W. Regulation of product synthesis in cell cultures of _Catharanthus roseus_ (L)G.

Don. _Plant Cell Tiss. Org. Cult._ 4, 83–94 (1985). Article  CAS  Google Scholar  * Blom, T.J.M., Kreis, W., van Iren, F. & Libbenga, K.R. A non-invasive method for the

routine-estimation of fresh weight of cells grown in batch suspension cultures. _Plant Cell Rep._ 11, 146–149 (1992). Article  CAS  Google Scholar  * Contin, A., van der Heijden, R., ten

Hoopen, H.J.G. & Verpoorte, R. The inoculum size triggers tryptamine or secologanin biosynthesis in a _Catharanthus roseus_ cell culture. _Plant Sci._ 139, 205–211 (1998). Article  CAS 

Google Scholar  * Schripsema, J. & Verpoorte, R. Novel techniques for growth characterization and phytochemical analysis of plant cell suspension cultures. _J. Nat. Prod._ 58, 1305–1314

(1995). Article  CAS  Google Scholar  * David, H., Laigneau, C. & David, A. Growth and soluble proteins of cell cultures derived from explants and protoplasts of _Pinus pinaster_

cotyledons. _Tree Physiol._ 5, 497–506 (1989). Article  CAS  Google Scholar  * Hahlbrock, K. Further studies on the relationship between the rates of nitrate uptake, growth and conductivity

changes in the medium of plant cell suspension cultures. _Planta (Berl.)_ 124, 311–318 (1975). Article  CAS  Google Scholar  * Hahlbrock, K., Ebel, J. & Oaks, A. Determination of

specific growth stages of plant cell suspension cultures by monitoring conductivity changes in the medium. _Planta (Berl.)_ 118, 75–84 (1974). Article  CAS  Google Scholar  * Taya, M.,

Hegglin, M., Prenosil, J.E. & Bourne, J.R. On-line monitoring of cell growth in plant tissue cultures by conductometry. _Enzyme Microb. Technol._ 11, 170–176 (1989). Article  CAS  Google

Scholar  * Thom, M., Maretzki, A., Komor, E. & Sakai, W.S. Nutrient uptake and accumulation by sugarcane cell cultures in relation to the growth cycle. _Plant Cell Tiss. Org. Cult._ 1,

3–14 (1981). Article  CAS  Google Scholar  * Bradford, M.M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye

binding. _Anal. Biochem._ 72, 248–254 (1976). Article  CAS  Google Scholar  * Doležel, J., Greilhuber, J. & Suda, J. Estimation of nuclear DNA content in plants using flow cytometry.

_Nat. Protoc._ 2, 2233–2244 (2007). Article  Google Scholar  * Pfosser, M., Magyar, Z. & Bőgre, L. Cell cycle analysis in plants. In _Flow Cytometry with Plant Cells_ (eds. Doležel, J.,

Greilhuber, J. & Suda, J.) 323–347 (Wiley-VCH, 2007). * Widholm, J.M. The use of fluorescein diacetate and phenosafranin for determining viability of cultured plant cells. _Stain

Technol._ 47, 189–194 (1972). Article  CAS  Google Scholar  * Baker, J.C. & Mock, N.M. An improved method for monitoring cell death in cell suspension and leaf disc assays using evans

blue. _Plant Cell Tiss. Org. Cult._ 39, 7–12 (1994). Article  Google Scholar  * Verleysen, H., Samyn, G., Van Bockstaele, E. & Debergh, P. Evaluation of analytical techniques to predict

viability after cryopreservation. _Plant Cell Tiss. Org. Cult._ 77, 11–21 (2004). Article  CAS  Google Scholar  * Deus-Neumann, B. & Zenk, M.H. Instability of indole alkaloid production

in _Catharanthus roseus_ cell suspension cultures. _Planta Med._ 50, 427–431 (1984). Article  CAS  Google Scholar  * Kartha, K.K. Meristem culture and germplasm preservation. In

_Cryopreservation of Plant Cells and Organs_ (ed. Kartha, K.K.) 115–134 (CRC Press, 1985). * Benson, E.E. Cryopreservation of phytodiversity: a critical appraisal of theory & practice.

_Crit. Rev. Plant Sci._ 27, 141–219 (2008). Article  CAS  Google Scholar  * Schrijnemakers, E.W.M. & Van Iren, F. A two-step or equilibrium freezing procedure for the cryopreservation of

plant cell suspensions. In _Methods in Molecular Biology Vol. 38 Cryopreservation and Freeze-drying Protocols_ (eds. Day, J.G. & McLellan, M.R.) 103–111 (Humana Press, 1995). *

Reinhoud, P.J., Van Iren, F. & Kijne, J.W. Cryopreservation of undifferentiated plant cells. In _Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Application_

(eds. Engelmann, F. & Takagi, H.) 91–102 (International Plant Genetic Resources Institute, 2000). * Bachiri, Y.C., Gazeau, J., Hansz, J., Morisset, C. & Dereuddre, J. Successful

cryopreservation of suspension cells by encapsulation-dehydration. _Plant Cell Tiss. Org. Cult._ 43, 241–248 (1995). Google Scholar  * Yamazaki, H., Ayabe, K., Ishii, R. & Kuriyama, A.

Desiccation and cryopreservation of actively-growing cultured plant cells and protoplasts. _Plant Cell Tiss. Org. Cult._ 97, 151–158 (2009). Article  Google Scholar  * Steponkus, P.L.,

Langis, R. & Fujikawa, S. Cryopreservation of plant tissues by vitrification. In _Advances in Low Temperature Biology_ Vol. 2 (ed. Steponkus, P.L.) 1–62 (JAI Press, 1992). * Sakai, A.,

Hirai, D. & Niino, T. Development of PVS-based vitrification and encapsulation-vitrification protocols. In _Plant Cryopreservation: A Practical Guide_ Chapter 3 (ed. Reed, B.M.) 33–57

(Springer, 2008). * Langis, R., Schnabel, B., Earle, E.D. & Steponkus, P.L. Cryopreservation of _Brassica campestris_ L. cell suspensions by vitrification. _Cryo-Lett._ 10, 421–428

(1989). Google Scholar  * Uragami, A., Sakai, A., Nagai, M. & Takahashi, T. Survival of cultured cells and somatic embryos of _Asparagus officinalis_ cryopreserved by vitrification.

_Plant Cell Rep._ 8, 418–421 (1989). Article  CAS  Google Scholar  * Dereuddre, J., Scottez, C., Arnaud, Y. & Duron, M. Resistance of alginate-coated axillary shoot tips of pear tree

(_Pyrus communis_ L. cv. Beurré Hardy) _in vitro_ plantlets to dehydratation and subsequent freezing in liquid nitrogen: effects of previous cold hardening. _Comptes Rendus de

l'Académie des Sciences_ 310 Série III, 317–323 (1990). Google Scholar  * Withers, L.A. & King, P. A simple freezing unit and routine cryopreservation method for plant cell

cultures. _Cryo-Lett._ 1, 213–220 (1980). Google Scholar  * Škrlep, K. et al. Cryopreservation of cell suspension cultures of _Taxus x media_ and _Taxus floridana_. _Biol. Plant._ 52,

329–333 (2008). Article  Google Scholar  * Reed, B.M. Cryopreservation; practical consideration. In _Plant Cryopreservation: A Practical Guide_ Chapter 1 (ed. Reed, B.M.) 3–13 (Springer,

2008). * Heine-Dobbernack, E., Kiesecker, H. & Schumacher, H.M. Cryopreservation of dedifferentiated cell cultures. In _Plant Cryopreservation: A Practical Guide_ Chapter 8 (ed. Reed,

B.M.) 141–176 (Springer, 2008). * Reinhoud, P.J., Schrijnemakers, E.W.M., van Iren, F. & Kijne, J.W. Vitrification and a heat-shock treatment improve cryopreservation of tobacco cell

suspensions compared to two-step freezing. _Plant Cell Tiss. Org. Cult._ 42, 261–267 (1995). Article  Google Scholar  * Ishikawa, M. et al. Effect of growth phase on survival of Bromegrass

suspension cells following cryopreservation and abiotic stresses. _Ann. Bot._ 97, 453–459 (2006). Article  Google Scholar  * Lu, T.G. & Sun, C.S. Cryopreservation of millet (_Setaria

italica_ L.). _J. Plant Physiol._ 139, 295–298 (1992). Article  CAS  Google Scholar  * Sala, F., Cella, R. & Rollo, F. Freeze-preservation of rice cells grown in suspension culture.

_Physiol. Plant_ 45, 170–176 (1979). Article  CAS  Google Scholar  * Friesen, L.J. et al. Cryopreservation of _Papaver somniferum_ cell suspension cultures. _Planta Med._ 57, 53–55 (1991).

Article  CAS  Google Scholar  * Kim, S.I. et al. Cryopreservation of _Taxus chinensis_ suspension cell cultures. _CryoLetters_ 22, 43–50 (2001). PubMed  Google Scholar  * Suhartono, L. et

al. Metabolic comparison of cryopreserved and normal cells from _Tabernaemontana divaricata_ suspension cultures. _Plant Cell Tiss. Org. Cult._ 83, 59–66 (2005). Article  CAS  Google Scholar

  * Schripsema, J., Erkelens, C. & Verpoorte, R. Intra- and extracellular carbohydrates in plant cell cultures investigated by 1H-NMR. _Plant Cell Rep._ 9, 527–530 (1991). Article  CAS 

Google Scholar  * George, E.F., Puttock, D.J.M. & George, H.J. _Plant Culture Media Vol. 1; Formulations and Uses_ (Exegetics Limited, 1987). * Murashige, T. & Skoog, F. A revised

medium for rapid growth and bioassays with tobacco cultures. _Physiol. Plant_ 15, 473–497 (1962). Article  CAS  Google Scholar  * Gamborg, O.L., Miller, R.A. & Ojima, K. Nutrients

requirements of suspension cultures of soybean root cells. _Exp. Cell Res._ 50, 151–158 (1968). Article  CAS  Google Scholar  * Linsmaier, E.M. & Skoog, F. Organic growth requirements of

tobacco tissue cultures. _Physiol. Plant_ 18, 100–127 (1965). Article  CAS  Google Scholar  * Schenk, R.U. & Hildebrandt, A.C. Medium and techniques for induction and growth of

monocotyledonous and dicotyledonous plant cell cultures. _Can. J. Bot._ 50, 1999–2004 (1972). Article  Google Scholar  * Kors, F.T.M. _Biochemicals Plant Cell and Tissue Culture: Catalogue

2003–2005_ (Duchefa Biochemie, 2003). * Bhojwani, S.S., Dhawan, V. & Cocking, E.C. _Plant Tissue Culture: A Classified Bibliography_ 1–65 (Elsevier Science, 1986). * Bhojwani, S.S.,

Dhawan, V. & Arora, R. _Plant Tissue Culture: A Classified Bibliography 1985–1989_ 1–13 (Elsevier Science, 1990). * Knobloch, K.H., Bast, G. & Berlin, J. Medium- and light-induced

formation of serpentine and anthocyanins in cell suspension cultures of _Catharanthus roseus_. _Phytochemistry_ 21, 591–594 (1982). Article  CAS  Google Scholar  * Kadkade, P.G., Bare, C.B.,

Schnabel-Preiktas, B. & Yu, B. _Cryopreservation of Plant Cells_. US Patent no. 5,965,438 (1999). Google Scholar  * Lee, T.-J., Shultz, R.W., Hanley-Bowdoin, L. & Thompson, W.F.

Establishment of rapidly proliferating rice cell suspension culture and its characterization by fluorescence-activated cell sorting analysis. _Plant Mol. Biol. Rep._ 22, 259–267 (2004).

Article  CAS  Google Scholar  * Figueiredo, A.C.S. & Pais, M.S.S. _Achillea millefolium_ (Yarrow) cell suspension cultures: establishment and growth conditions. _Biotechnol. Lett._ 13,

63–68 (1991). Article  Google Scholar  * Flores-Sanchez, I.J. et al. Elicitation studies in cell suspension cultures of _Cannabis sativa_ L. _J. Biotechnol._ 143, 157–168 (2009). Article 

CAS  Google Scholar  * El-Sayed, M. & Verpoorte, R. Effect of phytohormones on growth and alkaloid accumulation by a _Catharanthus roseus_ cell suspension cultures fed with alkaloid

precursors tryptamine and loganin. _Plant Cell Tiss. Org. Cult._ 68, 265–270 (2002). Article  CAS  Google Scholar  * Mustafa, N.R., Kim, H.K., Choi, Y.H. & Verpoorte, R. Metabolic

changes of salicylic acid-elicited _Catharanthus roseus_ cell suspension cultures monitored by NMR-based metabolomics. _Biotechnol. Lett._ 31, 1967–1974 (2009). Article  CAS  Google Scholar

  * Moreno, P.R.H. et al. Induction of ajmalicine formation and related enzyme activities in _Catharanthus roseus_ cells: effect of inoculum density. _Appl. Microbiol. Biotechnol._ 39, 42–47

(1993). Article  CAS  Google Scholar  * Ramos-Valdivia, A.C., van der Heijden, R. & Verpoorte, R. Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl

diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of _Cinchona robusta_. _Planta_ 203, 155–161 (1997). Article  CAS  Google Scholar  *

Langezaal, C.R. & Scheffer, J.J.C. Initiation and growth characterization of some hop cell suspension cultures. _Plant Cell Tiss. Org. Cult._ 30, 159–164 (1992). Article  CAS  Google

Scholar  * Yuana Dignum, M.J.W. & Verpoorte, R. Glucosylation of exogenous vanillin by plant cell cultures. _Plant Cell Tiss. Org. Cult._ 69, 177–182 (2002). Article  Google Scholar  *

Han, Y., van der Heijden, R. & Verpoorte, R. Biosynthesis of anthraquinones in cell cultures of the Rubiaceae. _Plant Cell Tiss. Org. Cult._ 67, 201–220 (2001). Article  CAS  Google

Scholar  * Schripsema, J., Dagnino, D., Dos Santos, R.I. & Verpoorte, R. Breakdown of indole alkaloids in suspension cultures of _Tabernaemontana divaricata_ and _Catharanthus roseus_.

_Plant Cell Tiss. Org. Cult._ 38, 299–305 (1994). Article  CAS  Google Scholar  * Dagnino, D., Schripsema, J. & Verpoorte, R. Terpenoid indole alkaloid biosynthesis and enzyme activities

in two cell lines of _Tabernaemontana divaricata_. _Phytochemistry_ 39, 341–349 (1995). Article  CAS  Google Scholar  * Kraemer, K.H., Schenkel, E.P. & Verpoorte, R. _Ilex

paraguariensis_ cell suspension culture characterization and response against ethanol. _Plant Cell Tiss. Org. Cult._ 68, 257–263 (2002). Article  CAS  Google Scholar  Download references

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

the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer

Nature SharedIt content-sharing initiative