Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 112, Issue 3, pp 303–310 | Cite as

Cellular aggregation is a key parameter associated with long term variability in paclitaxel accumulation in Taxus suspension cultures

  • Rohan A. Patil
  • Martin E. Kolewe
  • Susan C. Roberts
Original Paper


Plant cell cultures provide a renewable source for synthesis and supply of commercially valuable plant-derived products, particularly for secondary metabolites. However, instability in product yields over multiple passages has hampered the efficient and sustainable use of this technology. Paclitaxel accumulation in Taxus cell suspension culture was quantified over multiple passages and correlated to mean aggregate size, extracellular sugar level, ploidy, and cell cycle distribution. Paclitaxel levels varied approximately 6.9-fold over the 6-month timeframe investigated. Of all of the parameters examined, only mean aggregate size correlated with paclitaxel accumulation, where a significant negative correlation (r = −0.75, p < 0.01) was observed. These results demonstrate the relevance of measuring, and potentially controlling, aggregate size during long term culture passages, particularly for plant suspensions where industrially relevant secondary metabolites are not pigmented to enable rapid culture selection.


Plant cell culture Bioprocess Cellular aggregation Paclitaxel Taxus Ploidy DNA content 


  1. Baebler S, Hren M, Camloh M, Ravnikar M, Bohanec B, Plaper I, Ucman R, Zel J (2005) Establishment of cell suspension cultures of yew (Taxus x media rehd.) and assessment of their genomic stability. In Vitro Cell Dev Biol Plant 41:338–343CrossRefGoogle Scholar
  2. Bergounioux C, Perennes C, Brown SC, Gadal P (1988) Nuclear-rna quantification in protoplast cell-cycle phases. Cytometry 9:84–87PubMedCrossRefGoogle Scholar
  3. Bergounioux C, Brown SC, Petit PX (1992) Flow-cytometry and plant protoplast cell biology. Physiol Plantarum 85:374–386CrossRefGoogle Scholar
  4. Boisson AM, Gout E, Bligny R, Rivasseau C (2012) A simple and efficient method for the long-term preservation of plant cell suspension cultures. Plant Methods 8:4PubMedCrossRefGoogle Scholar
  5. Callebaut A, Terahara N, Haan M, Decleire M (1997) Stability of anthocyanin composition in Ajuga reptans callus and cell suspension cultures. Plant Cell Tiss Org 50:195–201CrossRefGoogle Scholar
  6. Capataz-Tafur J, Trejo-Tapia G, Rodríguez-Monroy M, Sepúlveda-Jimenez G (2011) Arabinogalactan proteins are involved in cell aggregation of cell suspension cultures of Beta vulgaris L. Plant Cell Tiss Org 106:169–177CrossRefGoogle Scholar
  7. Citterio S, Sgorbati S, Levi M, Colombo BM, Sparvoli E (1992) Pcna and total nuclear-protein content as markers of cell-proliferation in pea tissue. J Cell Sci 102:71–78Google Scholar
  8. Cragg GM, Grothaus PG, Newman DJ (2009) Impact of natural products on developing new anti-cancer agents. Chem Rev 109:3012–3043PubMedCrossRefGoogle Scholar
  9. Creemers-Molenaar J, Loeffen JPM, van Rossum M, Colijn-Hooymans CM (1992) The effect of genotype, cold storage and ploidy level on the morphogenic response of perennial ryegrass (Lolium perenne L.) suspension cultures. Plant Sci 83:87–94CrossRefGoogle Scholar
  10. Dehghan E, Hakkinen ST, Oksman-Caldentey KM, Ahmadi FS (2012) Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.). Plant Cell Tiss Org 110:35–44CrossRefGoogle Scholar
  11. De Jesus-Gonzalez L, Weathers PJ (2003) Tetraploid Artemisia annua hairy roots produce more artemisinin than diploids. Plant Cell Rep 21:809–813Google Scholar
  12. Deusneumann B, Zenk MH (1984) Instability of indole alkaloid production in Catharanthus roseus cell suspension cultures. Planta Med 50:427–431CrossRefGoogle Scholar
  13. Dolezel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat Protocols 2:2233–2244CrossRefGoogle Scholar
  14. Eibl R, Eibl D (2002) Bioreactors for plant cell and tissue cultures. In: Oksman-Caldentey KM, Barz W (eds) Plant biotechnology and transgenic plants. Marcel Dekker, New York, pp 163–199Google Scholar
  15. Gaurav V, Kolewe ME, Roberts SC (2010) Flow cytometric methods to investigate culture heterogeneities for plant metabolic engineering. In: Fett-Neto A (ed) Plant secondary metabolism engineering: methods and applications, methods in molecular biology. Springer, New York, pp 243–262CrossRefGoogle Scholar
  16. Gibson D, Ketchum R, Vance N, Christen A (1993) Initiation and growth of cell lines of Taxus brevifolia (Pacific yew). Plant Cell Rep 12:479–482CrossRefGoogle Scholar
  17. Gundlach H, Muller MJ, Kutchan TM, Zenk MH (1992) Jasmonic acid is a signal transducer in elicitor induced plant cell cultures. Proc Natl Acad Sci USA 89:2389–2393PubMedCrossRefGoogle Scholar
  18. Hall RD, Yeoman MM (1986) Factors determining anthocyanin yield in cell cultures of Catharanthus roseus (L.) G. Don. New Phytol 103:33–43CrossRefGoogle Scholar
  19. Hall RD, Yeoman MM (1987) Intercellular and intercultural heterogeneity in secondary metabolite accumulation in cultures of Catharanthus roseus following cell line selection. J Exp Bot 38:1391–1398CrossRefGoogle Scholar
  20. Hanagata N, Ito A, Uehara H, Asari F, Takeuchi T, Karube I (1993) Behavior of cell aggregate of Carthamus tinctorius L cultured cells and correlation with red pigment formation. J Biotechnol 30:259–269CrossRefGoogle Scholar
  21. Harvey AL (2008) Natural products in drug discovery. Drug Discov Today 13(19–20):894–901PubMedCrossRefGoogle Scholar
  22. Hirasuna TJ, Shuler ML, Lackney VK, Spanswick RM (1991) Enhanced anthocyanin production in grape cell cultures. Plant Sci 78:107–120CrossRefGoogle Scholar
  23. Ishikawa M, Suzuki M, Nakamura T, Kishimoto T, Robertson AJ, Gusta LV (2006) Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses. Ann Bot-London 97:453–459CrossRefGoogle Scholar
  24. Kim BJ, Gibson DM, Shuler ML (2004) Effect of subculture and elicitation on instability of Taxol production in Taxus sp suspension cultures. Biotechnol Progr 20:1666–1673CrossRefGoogle Scholar
  25. Kinnersley AM, Dougall DK (1980) Increase in anthocyanin yield from wild carrot cell cultures by a selection system based on cell aggregate size. Planta 149:200–204CrossRefGoogle Scholar
  26. Kolewe ME, Gaurav V, Roberts SC (2008) Pharmaceutically active natural product synthesis and supply via plant cell culture technology. Mol Pharm 5:243–256PubMedCrossRefGoogle Scholar
  27. Kolewe ME, Henson MA, Roberts SC (2010) Characterization of aggregate size in Taxus suspension cell culture. Plant Cell Rep 29:485–494PubMedCrossRefGoogle Scholar
  28. Kolewe ME, Henson MA, Roberts SC (2011) Analysis of aggregate size as a process variable affecting paclitaxel accumulation in Taxus suspension cultures. Biotechnol Progr 27:1365–1372CrossRefGoogle Scholar
  29. Kolewe ME, Roberts SC, Henson MA (2012) A population balance equation model of aggregation dynamics in Taxus suspension cell cultures. Biotechnol Bioeng 109:472–482PubMedCrossRefGoogle Scholar
  30. Kosuth J, Koperdakova J, Tolonen A, Hohtola A, Cellarova E (2003) The content of hypericins and phloroglucinols in Hypericum perforatum L. seedlings at early stage of development. Plant Sci 165:515–521Google Scholar
  31. Kotogany E, Dudits D, Horvath GV, Ayaydin F (2010) A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine. Plant Methods 6:5PubMedCrossRefGoogle Scholar
  32. Krzyzanowska J, Czubacka A, Pecio L, Przybys M, Doroszewska T, Stochmal A, Oleszek W (2012) The effects of jasmonic acid and methyl jasmonate on rosmarinic acid production in Mentha × piperita cell suspension cultures. Plant Cell Tiss Org 108:73–81CrossRefGoogle Scholar
  33. Lee EK, Jin YW, Park JH, Yoo YM, Hong SM, Amir R, Yan Z, Kwon E, Elfick A, Tomlinson S, Halbritter F, Waibel T, Yun BW, Loake GJ (2010) Cultured cambial meristematic cells as a source of plant natural products. Nat Biotech 28:1213–1217CrossRefGoogle Scholar
  34. Maciejewska U, Skierski JS, Szczerbakowa A (1999) Nuclear DNA content of Solanum species grown in vitro, as determined by flow cytometry. Acta Physiol Plantarum 21:37–43Google Scholar
  35. McChesney JD, Venkataraman SK, Henri JT (2007) Plant natural products: back to the future or into extinction? Phytochemistry 68:2015–2022PubMedCrossRefGoogle Scholar
  36. Mishiba KI, Okamoto T, Mii M (2001) Increasing ploidy level in cell suspension cultures of Doritaenopsis by exogenous application of 2,4-dichlorophenoxyacetic acid. Physiol Plantarum 112:142–148CrossRefGoogle Scholar
  37. Morris P (1986) Long term stability of alkaloid productivity in cell suspension cultures of Catharanthus roseus. In: Morris P, Scragg AH, Stafford A, Fowler MW (eds) Secondary metabolism in plant cell cultures. University Press, Cambridge, pp 257–262Google Scholar
  38. Morris P, Rudge K, Cresswell R, Fowler MW (1989) Regulation of product synthesis in cell cultures of Catharanthus roseus. 5. Long term maintenance of cells on a production medium. Plant Cell Tiss Org 17:79–90CrossRefGoogle Scholar
  39. Mustafa NR, de Winter W, van Iren F, Verpoorte R (2011) Initiation, growth and cryopreservation of plant cell suspension cultures. Nat Protocols 6:715–742CrossRefGoogle Scholar
  40. Naill MC, Roberts SC (2004) Preparation of single cells from aggregated Taxus suspension cultures for population analysis. Biotechnol Bioeng 86:817–826PubMedCrossRefGoogle Scholar
  41. Naill MC, Roberts SC (2005) Cell cycle analysis of Taxus suspension cultures at the single cell level as an indicator of culture heterogeneity. Biotechnol Bioeng 90:491–500PubMedCrossRefGoogle Scholar
  42. Neumann KH, Kumar A, Imani J (2009) Cell division, cell growth, cell differentiation. In: Neumann KH, Kumar A, Imani J (eds) Plant cell and tissue culture—a tool in biotechnology. Basics and application. Springer, Berlin, pp 235–247CrossRefGoogle Scholar
  43. Patil RA, Kolewe ME, Normanly J, Walker EL, Roberts SC (2012) Contribution of taxane biosynthetic pathway gene expression to observed variability in paclitaxel accumulation in Taxus suspension cultures. Biotechnol J 7:418–427PubMedCrossRefGoogle Scholar
  44. Pauwels L, Morreel K, Witte ED, Lammertyn F, Montagu MV, Boerjan W, Inzé D, Goossens A (2008) Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells. Proc Natl Acad Sci USA 105:1380–1385PubMedCrossRefGoogle Scholar
  45. Qu JG, Zhang W, Yu XJ, Jin MF (2005) Instability of anthocyanin accumulation in Vitis vinifera L. var. Gamay Freaux suspension cultures. Biotechnol Bioproc E 10:155–161CrossRefGoogle Scholar
  46. Qu JG, Zhang W, Hu QL, Jin MF (2006) Impact of subculture cycles and inoculum sizes on suspension cultures of Vitis vinifera. Chin J Biotechnol 22:984–989CrossRefGoogle Scholar
  47. Reinhoud PJ, Schrijnemakers EWM, Iren F, Kijne JW (1995) Vitrification and a heat-shock treatment improve cryopreservation of tobacco cell suspensions compared to two-step freezing. Plant Cell Tiss Org 42:261–267CrossRefGoogle Scholar
  48. Roberts SC (2007) Production and engineering of terpenoids in plant cell culture. Nat Chem Biol 3:387–395PubMedCrossRefGoogle Scholar
  49. Saito K, Mizukami H (2002) Plant cell cultures as producers of secondary compounds. In: Oksman-Caldentey KM, Barz W (eds) Plant biotechnology and transgenic plants. Marcel Dekker, New York, pp 66–90Google Scholar
  50. Suzuki H, Reddy MSS, Naoumkina M, Aziz N, May GD, Huhman DV, Sumner LW, Blount JW, Mendes P, Dixon RA (2005) Methyl jasmonate and yeast elicitor induce differential transcriptional and metabolic re-programming in cell suspension cultures of the model legume Medicago truncatula. Planta 220:696–707PubMedCrossRefGoogle Scholar
  51. Yamada Y, Hashimoto T (1990) Possibilities for improving yield of secondary metabolites in plant cell cultures. In: Nijkamp HJ, Van der Plas L, Van Aartrijk J (eds) Current plant science and biotechnology in agriculture. progress in plant cellular and molecular biology. Kluwer, Dordrecht, pp 547–556CrossRefGoogle Scholar
  52. Yanpaisan W, King NJC, Doran PM (1998) Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry. Biotechnol Bioeng 58:515–528PubMedCrossRefGoogle Scholar
  53. Yanpaisan W, King NJC, Doran PM (1999) Flow cytometry of plant cells with applications in large-scale bioprocessing. Biotechnol Adv 17:3–27PubMedCrossRefGoogle Scholar
  54. Yeoman MM, Yeoman CL (1996) Manipulating secondary metabolism in cultured plant cells. New Phytol 134:553–569CrossRefGoogle Scholar
  55. Zeliang PK, Pattanayak A, Iangrai B, Khongwir EA, Sarma BK (2010) Fertile plant regeneration from cryopreserved calli of Oryza rufipogon Griff. and assessment of variation in the progeny of regenerated plants. Plant Cell Rep 29:1423–1433PubMedCrossRefGoogle Scholar
  56. Zhao J, Verpoorte R (2007) Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors: from biochemical processing to metabolic engineering. Phytochem Rev 6:435–457CrossRefGoogle Scholar
  57. Zhong J, Yoshida M, Yoshida T (1995) Effects of biological factors on cell growth and anthocyanin formation by suspended cultures of Perilla frutescens cells. Chin J Biotechnol 11:143–147PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Rohan A. Patil
    • 1
  • Martin E. Kolewe
    • 1
  • Susan C. Roberts
    • 1
  1. 1.Department of Chemical Engineering, Institute for Cellular EngineeringUniversity of MassachusettsAmherstUSA

Personalised recommendations