Cryopreservation of Dedifferentiated Cell Cultures

  • Elke Heine-Dobbernack
  • Heiko Kiesecker
  • Heinz Martin Schumacher

When Gottlieb Haberlandt made the first efforts to cultivate single isolated plant cells in salt solutions his goal was to prove the totipotency of single cells (Haberlandt 1902). The cultivation of isolated plant cells in a chemically defined culture medium became possible only after the discovery and application of auxins (Gautheret 1939). Today plant cells as well as tissues can be cultivated in vitro for many applications in plant breeding, plant propagation, germplasm preservation and molecular biology.

The idea to produce valuable plant metabolites by large-scale fermentation of cell cultures was soon born (Routien and Nickel 1952). Scientists were fascinated by the idea of using dedifferentiated cell cultures as a replacement for intact plants in research and biotechnology. Tulecke and Nickell (1959) wrote: “…in essence these cell cultures represent a new kind of microorganisms…” The formation of a number of secondary metabolites was detected in the new material and in some cases the concentration of these compounds in the cell cultures exceeded even that in intact plants (for review see Carew and Staba 1965). The concept to produce such valuable compounds by growing cell cultures in fermentation vessels offers many advantages: the production is independent from specific climatic conditions and it can be carried out under defined and sterile conditions. Furthermore, the production of extractable raw material from plant cell cultures can be quicker than with intact plants, especially for plants like ginseng where harvest starts many years after planting and destroys the plants. Other candidates for cell mass production by cell culture are plants growing extremely slowly or where the content of a certain compound is extremely low like for the drug paclitaxel in Taxus plants.

Although in many cases high concentrations of certain compounds were obtained in plant cell cultures, like rosmarinic acid in Coleus blumei cell lines (see Berlin 1997), ginsenosides in Panax ginseng cell cultures (Thanh et al. 2005) or Raucaffricine in cell lines of Rauvolfia serpentina (Schuebel and Stoeckigt 1984), examples for the economic application of large scale fermentation of dedifferentiated plant cell cultures remain rare. The first process was established by the Mitsui Petrochemical Company for the production of Shikonin, a red-colored antimicrobial compound traditionally used in Japan (Fujita et al. 1982). Presently the best known example is the production of paclitaxel by Phyton in a large scale fermentation facility specific for plant cell cultures close to the city of Hamburg (Venkat 1998).


Suspension Culture Rosmarinic Acid Evans Blue Plant Cell Culture Logarithmic Growth Phase 
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  1. Alfermann AW, Petersen M, Fuss E (2003) Production of natural products by plant cell biotechnology: Results problems and perspectives. In: Laimer M, Rücker W (eds.) Plant Tissue Culture - 100 years since Gottlieb Haberlandt. Springer, Wien, pp 153-166Google Scholar
  2. Alves E, Ballesteros I, Linacero R, Vazquez AM (2005) RYS1, a foldback transposon, is activated by tissue culture and shows preferential insertion points into the rye genome. Theor Appl Genet 111: 431-436CrossRefPubMedGoogle Scholar
  3. Bachiri Y, Gazeau C, Hansz J, Morisset C, Dereuddre J (1995) Successful cryopreservation of suspension cells by encapsulation-dehydration. Plant Cell Tiss Organ Cult 43: 241-248Google Scholar
  4. Baker CJ, Mock NM (1994) An improved method for monitoring cell death in cell suspension and disc assay using Evans blue. Plant Cell Tiss Organ Cult 39: 7-12CrossRefGoogle Scholar
  5. Bayliss MW (1980) Chromosomal variation in plant tissue culture. Ann Rev Cytol Suppl 11A: 113-144Google Scholar
  6. Berlin J (1997) Secondary products from plant cell cultures. In: Rehm HJ, Reed G (eds.) Biotechnology. VCH Verlagsgesellschaft, Weinheim, New York, Basel, Cambridge, pp 593-640Google Scholar
  7. Bode M, Stobe P, Thiede B, Schuphan I, Schmidt B (2004) Biotransformation of atrazine in transgenic tobacco cell culture expressing human P450. Pest Manag Sci 60: 49-58CrossRefPubMedGoogle Scholar
  8. Carew DP, Staba EJ (1965) Plant tissue culture, its fundamentals, application and relation to medicinal plant studies. Lloydia 28: 1-26Google Scholar
  9. Chandler SF, Vasil IK (1984) Selection and characterization of NaCl tolerant cells from embryogenic cultures of Pennisetum purpureum Schum. (Napier grass). Plant Sci Lett 37: 157-164CrossRefGoogle Scholar
  10. Chaprin N, Ellis BE (1984) Microspectrophotometric evaluation of rosmarinic acid accumulation in single cultured plant cells, Can J Bot 62: 2278-2282CrossRefGoogle Scholar
  11. Chen Y, Wang JH (2002) Cryopreservation of carrot (Daucus carota L.) cell suspensions and protoplasts by vitrification. CryoLetters 24: 57-64Google Scholar
  12. DeJong DW, Jansen EF, Olson AC (1967) Oxidoreductive and hydrolytic enzyme patterns in plant suspension cultured cells. Exper Cell Res 47: 139-156CrossRefGoogle Scholar
  13. Deus-Neumann B, Zenk MH (1984) Instability of indole alkaloid production in Catharanthus roseus cell suspension cultures. Planta Medica 50: 427-431CrossRefPubMedGoogle Scholar
  14. Dougall DK, Whitten GH (1980) A clonal analysis of anthocyanin accumulation by cell cultures of wild carrot. Planta 149: 292-297CrossRefGoogle Scholar
  15. Duncan DR, Widholm JM (2004) Osmotic induced stimulation of the reduction of the viability dye 2,3,5-triphenyltetrazolium chloride by maize roots and callus cultures. J Plant Physiol 161: 397-403CrossRefPubMedGoogle Scholar
  16. Fabre J, Dereuddre J (1990) Encapsulation-dehydration: a new approach to cryopreservation of Solanum shoot tips. CryoLetters 11: 413-426Google Scholar
  17. Fujita Y, Tabata M, Nishi A, Yamada Y (1982) New medium and production of secondary compounds with the two-staged culture method. In: Fujiwara A (ed.) Plant Tissue Culture. Maruzen, Tokyo, pp 399-400Google Scholar
  18. Gautheret RJ (1939) Sur la possibilité de réaliser la culture indefinie des tissus de tubercules de carotte. C R Acad Sci Paris 118-120Google Scholar
  19. Gazeau C, Elleuch H, David A, Morisset C (1998) Cryopreservation of transformed Papaver somniferum cells. CryoLetters 19: 147-159Google Scholar
  20. Grill E, Winnacker EL, Zenk MH (1991) Phytochelatins. Methods Enzymol 205: 333-341CrossRefGoogle Scholar
  21. Haberlandt G (1902) Kulturversuche mit isolierten Pflanzenzellen. Sitzungsberichte Akad Wiss Math Naturwiss Kl 11: 69-92Google Scholar
  22. Han Y-S, van der Heijden R, Verpoorte J (2001) Biosynthesis of anthraquinones in cell cultures of the Rubiaceae. Plant Cell Tiss Organ Cult 67: 201-220CrossRefGoogle Scholar
  23. Hao YJ, Cheng YJ, Deng XX (2003) GUS gene remains stable in transgenic citrus callus recovered from cryopreservation. CryoLetters 24: 375-380PubMedGoogle Scholar
  24. Huang C-H, Wang J-H, Yan Q-S, Zhang X-Q, Yan Q-F (1995) Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification. Plant Cell Rep 14: 730-734CrossRefGoogle Scholar
  25. Huang FC, Kutchan TM (2000) Distribution of morphinan and benzo [c] phenanthridine alkaloid gene transcript accumulation in Papaver somninferum. Phytochemistry 53: 555-564CrossRefPubMedGoogle Scholar
  26. Huang LF, Liu YK, Lu CA, Hsieh SL, Yu SM (2005) Production of human serum albumin by sugar starvation induced promoter and rice culture. Transgenic Res 14: 569-581CrossRefPubMedGoogle Scholar
  27. Ikegawa H, Yamamoto Y, Matsumoto H (1998) Cell death caused by a combination of aluminium and iron in cultured tobacco cells. Physiol Plant 104: 474-478CrossRefGoogle Scholar
  28. Ishikawa M, Robertson A J, Gusta LV (1995) Comparison of viability tests for assessing cross adaptation to freezing, heat and salt stresses induced by abscisic acid in bromegrass (Bromus inermis Leyss) suspension cultured cells. Plant Sci 107: 83-93CrossRefGoogle Scholar
  29. Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43: 179-188CrossRefPubMedGoogle Scholar
  30. Kutchan TM (1993) Strictosidine: from alkaloid to enzyme to gene. Phytochemistry 32: 493-506CrossRefPubMedGoogle Scholar
  31. Langis RA, Schnabel B, Earle ED, Steponkus P (1989) Cryopreservation of Brassica campetsris L. cell suspension by vitrification. CryoLetters 10: 421-428Google Scholar
  32. Larkin PJ, Scowcroft WR (1981) Somaclonal variation - a novel source of vari-ability from cell cultures for plant improvement. Theoret Appl Genet 60: 197-214CrossRefGoogle Scholar
  33. Lovelock JE, Bishop MWH (1959) Prevention of freezing damage to living cells by dimethyl sulfoxide. Nature 183: 1394-1395CrossRefPubMedGoogle Scholar
  34. Meijer EGM, van Iren F, Schrijnemakers E, Hensgen LAM, van Zijderveld M (1991) Retention of the capacity to produce plants from protoplasts in cryopreserved cell lines of rice (Oryza sativa L.) Plant Cell Rep 10: 171-174CrossRefGoogle Scholar
  35. Ogino T, Hiraoka N, Tabata M (1978) Selection of high nicotine-producing cell lines of tobacco callus by single-cell cloning. Phytochemistry 17: 1907-1910CrossRefGoogle Scholar
  36. Quatrano RS (1968) Freeze preservation of cultured flax cells utilizing dimethyl sulfoxide. Plant Physiol 43: 2057-2061CrossRefPubMedGoogle Scholar
  37. Reinhoud PJ, Uragami A, Sakai A, van Iren F (1995) Vitrification of plant cell suspensions. In: Day JG, McLellan MR (eds.) Cryopreservation and Freeze Drying Protocols. Humana Press, Totowa, NJ, pp 113-120CrossRefGoogle Scholar
  38. Routien JB, Nickel LG (1952) Cultivation of Plant Tissues. US Patent [2,747,334]Google Scholar
  39. Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep 9: 30-33CrossRefGoogle Scholar
  40. Sato F, Yamada Y (1984) High berberine-producing cultures of Coptis japonica cells. Phytochemistry 23: 281-285CrossRefGoogle Scholar
  41. Schinkel H, Schiermeyer A, Soeur R, Fischer R, Schillberg S (2005) Production of an active recombinant thrombomodulin derivative in transgenic tobacco plants and suspension cells. Transgenic Res 14: 251-259CrossRefPubMedGoogle Scholar
  42. Schuebel H, Stoeckigt J (1984) RLCC-Isolation of raucaffricine from its most efficient source—cell suspension cultures of Rauvolfia serpentina. Plant Cell Rep 3: 72-74CrossRefGoogle Scholar
  43. Schumacher HM (1999) Cryo-conservation of industrially important plant cell cultures. In: Benson EE (ed.) Plant Conservation Biotechnology. Taylor & Francis, London, pp 125-137Google Scholar
  44. Schumacher HM, Gundlach H, Fiedler F, Zenk MH (1987) Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures. Plant Cell Rep 6: 410-413Google Scholar
  45. Shibli RA, Al-Juboory KH (2000) Cryopreservation of ‘Nabali’ olive (Olea europaea L.) somatic embryos by encapsulation-dehydration and encapsulationvitrification. CryoLetters 21: 357-366PubMedGoogle Scholar
  46. Shibli RA, Smith MA, Shatnawi MA (1999) Pigment recovery from encapsulateddehydrated Vaccinium pahalae (ohelo) cryopreserved cells. Plant Cell Tiss Organ Cult 55: 119-123CrossRefGoogle Scholar
  47. Stafford AM (2002) Plant cell cultures as a source of bioactive small molecules. Curr Opin Drug Discov Devel 5: 296-303PubMedGoogle Scholar
  48. Steponkus PL, Lanphear FO (1967) Refinement of the triphenyl tetrazolium chloride method of determining cold injury. Plant Physiol 42: 1423-1426CrossRefPubMedGoogle Scholar
  49. Sunil Kumar GB, Ganapathi TR, Srinivas L, Bapat VA (2005) Secretion of hepatitis B surface antigen in transformed tobacco cell suspension cultures. Biotech Letters 27: 927-932CrossRefGoogle Scholar
  50. Swan TW, Deakin EA, Hunjan G, Souch GR, Spencer ME, Stafford AM, Lynch PT (1998) Cryopreservation of cell suspension of Polygonum aviculare using traditional controlled rate freezing and encapsulation-dehydration protocols, a comparison of post-thaw recovery. CryoLetters 19: 237-248Google Scholar
  51. Swan TW, O’Hare D, Gill RA, Lynch PT (1999) Influence of preculture conditions on the post-thaw recovery of suspension cultures of Jerusalem Artichoke (Helianthus tuberosus L.). CryoLetters 20: 325-336Google Scholar
  52. Thanh NT, Murthy HN, Yu KW, Hahn EJ, Paek KY (2005) Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors. Appl Microbiol Biotech 67: 197-201CrossRefGoogle Scholar
  53. Tsukatsaki H, Mii M, Tokuhara K, Ishikawa K (2000) Cryopreservation of Doritaenopsis suspension culture by vitrification. Plant Cell Rep 19: 1160-1164CrossRefGoogle Scholar
  54. Tulecke W, Nickell LG (1959) Production of large amounts of plant tissue by submerged culture. Science 130: 863-864CrossRefPubMedGoogle Scholar
  55. Venkat K (1998) Paclitaxel production through plant cell cultures: An exciting approach to harnessing biodiversity. Pure Appl Chem 70: 2177CrossRefGoogle Scholar
  56. Verpoorte R, van der Heijden R, ten Hoopen HJG, Memelink J (1999) Metabolic engineering of secondary metabolite pathways for the production of fine chemicals. Biotech Lett 21: 467-479CrossRefGoogle Scholar
  57. Wang Q, Gafny R, Sahar N, Sela I, Mawassi M, Tanne E, Perl A (2002) Cryopreservation of grapevine (Vitis vinifera L.) embryogenic cell suspensions by encapsulation-dehydration and subsequent plant regeneration. Plant Sci 162: 551-558CrossRefGoogle Scholar
  58. Watanabe K, Mitsuda H, Yamada H (1983) Retention of metabolic capacity of green Lavandula vera callus after freeze-preservation. Plant Cell Physiol 24: 119-122Google Scholar
  59. Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol 47: 189-194PubMedGoogle Scholar
  60. Withers LA (1985) Cryopreservation of cultured plant cells and protoplasts. In: Kartha KK (ed.) Cryopreservation of Plant Cells and Organs. CRC, Boca Raton, FL, pp 243-267Google Scholar
  61. Withers LA, King PJ (1980) A simple freezing unit and routine cryopreservation method for plant cell cultures. CryoLetters 1: 213-220Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Elke Heine-Dobbernack
    • 1
  • Heiko Kiesecker
    • 1
  • Heinz Martin Schumacher
    • 1
  1. 1.Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbHGermany

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