Skip to main content
SpringerLink
Log in

Assessing factors that affect the growth of Corylus avellana cell suspension cultures: a statistical approach

  • Plant Tissue Culture
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

The detection of taxol and related taxanes in Corylus avellana has generated considerable interest, particularly for in vitro cell cultures. Cell suspensions are a sustainable and rational option for obtaining a continuous and reliable source of secondary metabolites in large-scale processes. We therefore focused our study on the main factors that affect the growth of C. avellana cell suspensions as a key approach to improving culture productivity. In this work, calli were successfully induced from C. avellana seeds, leaves, and stems, and the efficiency of different sterilization methods was analyzed. The effects of the basal medium, carbon source, and the type and quantity of plant growth regulators on culture growth were studied. A fractional factorial design allowed us to reduce the number of experiments and analyze all the combinations in one run, thereby reducing time, variability, and costs. Statistical analysis (ANOVA) revealed that 1-naphthaleneacetic acid (NAA) and sucrose are mandatory for the growth of C. avellana cell suspension cultures, with no interactions detected between the parameters analyzed, while growth did not depend on the addition of cytokinins. The secondary metabolism was not inhibited, detecting 1175.45 ng/L of baccatin III and traces of taxol, deacetyltaxol, and cephalomannine. Additionally, prompted by the high growth rate of the C. avellana calli, we assayed a new cold-temperature-based method to maintain a stock of calli using half-strength Murashige and Skoog solid medium, concluding that up to 5 mo at 4°C is optimal to ensure white friable calli upon regrowth at 25°C.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.

Similar content being viewed by others

References

  • Asano S, Ohtsubo S, Nakajima M, Kusunoki M, Keneko K, Katayama H, Nawa Y (2002) Production of anthocyanins by habituated cultured cells of Nyoho strawberry (Fragaria ananassa Duch.). Food Sci Technol Res 8:64–69

    Article  CAS  Google Scholar 

  • Bacchetta L, Aramini M, Bernardini C (2008) In vitro propagation of traditional Italian hazelnut cultivars as a tool for the valorization and conservation of local genetic resources. HortSci 43:562–566

    Google Scholar 

  • Bemani E, Ghanati RA, Jamshidi M (2013) Effect of phenylalanine on taxol production and antioxidant activity of extracts of suspension-cultured hazel (Corylus avellana L) cells. J Nat Med 67:446–451

    Article  CAS  PubMed  Google Scholar 

  • Bentebibel S, Moyano E, Palazón J, Cusidó RM, Bonfill M, Eibl R, Piñol MT (2005) Effects of immobilization by entrapment in alginate and scale-up on paclitaxel and baccatin III production in cell suspension cultures of Taxus baccata. Biotechnol Bioeng 89:647–655

    Article  CAS  PubMed  Google Scholar 

  • Bestoso F, Ottaggio L, Armirotti A, Balbi A, Damonte G, Degan P, Mazzei M, Cavalli F, Ledda B, Miele M (2006) In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol and taxanes. BMC Biotechnol 6:45

    Article  PubMed Central  PubMed  Google Scholar 

  • Binns A, Meins F (1973) Habituation of tobacco pith cells for factors promoting cell division is heritable and potentially reversible. Proc Natl Acad Sci U S A 70:2660–2662

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Charlwood BV, Rhodes MJC (1990) Secondary products from plant tissue culture. Clarendon, Oxford

    Google Scholar 

  • Contessa C, Valentini N, Caviglione M, Botta R (2011) Propagation of Corylus avellana L. by means of semi-hardwood cuttings: rooting and bud retention in four Italian cultivars. Eur J Hortic Sci 76:170–175

    Google Scholar 

  • Cusidó RM, Palazón J, Bonfill M, Navia-Osorio A, Morales C, Piñol MT (2002) Improved paclitaxel and baccatin III production in suspension cultures of Taxus media. Biotechnol Prog 18:418–423

  • Cusidó RM, Palazón J, Navia-Osorio A, Mallol A, Bonfill M, Morales C, Piñol MT (1999) Production of Taxol® and baccatin III by a selected Taxus baccata callus line and its derived cell suspension culture. Plant Sci 146:101–107

    Article  Google Scholar 

  • Diaz-Sala C, Rey M, Rodríguez R (1990) In vitro establishment of a cyclonal chain from nodal segments and apical buds on adult hazel (Corylus avellana L.). Plant Cell Tissue Organ Cult 23:151–157

    CAS  Google Scholar 

  • Dixon RA (2001) Natural products and plant disease resistance. Nature 411:843–847

    Article  CAS  PubMed  Google Scholar 

  • Eisenhauer EA, Vermorken JB (1998) The taxoids: comparative clinical pharmacology and therapeutic potential. Drugs 55:5–30

    Article  CAS  PubMed  Google Scholar 

  • Farjaminezhad R, Zare N, Asgahari-Zakaria R, Farjaminezhad M (2013) Establishment and optimization of cell growth in suspension culture of Papaver bracteatum: a biotechnology approach for thebaine production. Turk J Biol 37:689–697

    Article  CAS  Google Scholar 

  • Fornale S, Esposti DD, Navia-Osorio A, Cusido RM, Piñol MT, Bagni N (2002) Taxol transport in Taxus baccata cell suspension cultures. Plant Physiol Biochem 40:81–88

    Article  CAS  Google Scholar 

  • GuiZhi F, YaGuang Z, Bo W, YuTing Y (2009) Effects of light on growth and triterpenes content in callus of Betula platyphylla. J Northeast For Univ 37:1–3

    Google Scholar 

  • Hartig K, Beck E (2005) Endogenous cytokinin oscillations control cell cycle progression of tobacco BY-2 cells. Plant Biol 7:33–40

    Article  CAS  PubMed  Google Scholar 

  • Hoffman A, Khan W, Worapong J, Strobel G, Griffin D, Arbogast B, Barofsky B, Boone D, Ning RB, Zheng P, Daley L (1998) Bioprospecting for Taxol in angiosperm plant extracts: using high performance liquid chromatography–thermospray mass spectrometry to detect the anticancer agent and its related metabolites in filbert trees. Spectroscopy 13:22–32

    CAS  Google Scholar 

  • Holmes FA, Walters RS, Theriault RL, Buzdar AU, Fry DK, Hortobagyi GN, Forman AD, Neston LK, Raben MN (1991) Phase II trial taxol, an active drug in the treatment of metastatic breast cancer. J Natl Cancer Inst 83:1797–1805

    Article  CAS  PubMed  Google Scholar 

  • Hong S, Xiuxia L, Zhijian X, Dan M, Yinhui L (2010) Callus induction and protoplast isolation from hybrid hazel. J Northeast For Univ 38:23–26

    Google Scholar 

  • Kajani AA, Moghim S, Mofid MR (2012) Optimization of the basal medium for improving production and secretion of taxanes from suspension cell culture of Taxus baccata L. Daru 20:54

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Karwasara VS, Dixit VK (2012) Culture medium optimization for improved puerarin production by cell suspension cultures of Pueraria tuberosa (Roxb. ex Willd.) DC. In Vitro Cell Dev Biol Plant 48:189–199

    Article  Google Scholar 

  • Karwasara VS, Dixit VK (2013) Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley. Plant Biotechnol Rep 7:357–369

    Article  Google Scholar 

  • Kevers C, Coumans M, De Greef W, Hofinger M, Gaspar TH (1981) Habituation in sugarbeet callus: auxin content, auxin protectors, peroxidase pattern and inhibitors. Physiol Plant 51:281–286

    Article  CAS  Google Scholar 

  • McGuire WP, Rowinsky EK, Rosenshein HB, Grumbine FC, Ettiger DS, Amstrong DK, Donehower RC (1989) Taxol, a unique antineoplastic agent significant activity in advanced ovarian epithelial neoplasms. Ann Intern Med 111:273–279

    Article  CAS  PubMed  Google Scholar 

  • Mirjalili HM, Fakhr-Tabatabaei SM, Bonfill M, Alizadeh H, Cusido RM, Ghassempour A, Palazon J (2009) Morphology and withanolide production of Withania coagulans hairy root cultures. Eng Life Sci 9:197–204

    Article  CAS  Google Scholar 

  • Miura GA, Miller CO (1969) Cytokinins from a variant strain of cultured soybean cells. Plant Physiol 44:1035–1039

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Mustafa NR, de Winter W, van Iren F, Verpoorte R (2011) Initiation, growth and cryopreservation of plant cell suspension cultures. Nat Protoc 6:715–742

    Article  CAS  PubMed  Google Scholar 

  • Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. Int Rev Cytol 132:1–30

    Article  CAS  Google Scholar 

  • Nagella P, Murthy HN (2010) Establishment of cell suspension cultures of Withania somnifera for the production of withanolide A. Bioresour Technol 101:6735–6739

    Article  CAS  PubMed  Google Scholar 

  • Nas MN (2004) Inclusion of polyamines in the medium improves shoot elongation in hazelnut (Corylus avellana L.) micropropagation. Turk J Agric For 28:189–194

    CAS  Google Scholar 

  • Nas MN, Read PE (2004) A hypothesis for the development of a defined tissue culture medium of higher plants and micropropagation of hazelnuts. Sci Hortic 101:189–200

    Article  CAS  Google Scholar 

  • Navia-Osorio A, Garden H, Cusidó RM, Palazón J, Alfermann AW, Piñol MT (2002) Production of paclitaxel and baccatin III in a 20-L airlift bioreactor by a cell suspension of Taxus wallichiana. Planta Med 68:336–340

    Article  CAS  PubMed  Google Scholar 

  • Nishinari N, Syono K (1980) Changes in endogenous cytokinin levels in partially synchronized cultured tobacco cells. Plant Physiol 65:437–441

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ottaggio L, Betoso F, Armirotti A, Balbi A, Damonte G, Mazzei M, Sancandi M, Miele M (2008) Taxanes from shells and leaves of Corylus avellana. J Nat Prod 71:58–60

    Article  CAS  PubMed  Google Scholar 

  • Pollard JW, Walker JM (1990) Methods in molecular biology, vol 6. Humana Press, Clifton New Jersey

    Google Scholar 

  • Rao SR, Ravishankar GA (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 20:101–153

    Article  CAS  PubMed  Google Scholar 

  • Redig P, Shaul O, Inze D, Van Montagu M, Van Onckelen H (1996) Levels of endogenous cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY-2 cells. FEBS Lett 391:175–180

    Article  CAS  PubMed  Google Scholar 

  • Reed B, Mentzer J, Trampraset P, Yu X (1998) Internal bacterial contamination of micropropagated hazelnut: identification and antibiotic treatment. Plant Cell Tissue Organ Cult 52:67–70

    Article  CAS  Google Scholar 

  • Rezaei A, Ghanati F, Behmanesh M, Mokhtari-Dizaji M (2011) Ultrasound-potentiated salicylic acid-induced physiological effects and production of taxol in hazelnut (Corylus avellana L.) cell culture. Ultrasound Med Biol 37:1938–1947

    Article  PubMed  Google Scholar 

  • Rezaei A, Ghanati F, Behmanesh M, Safari M, Sharafi Y (2013) Synergistic accumulative effect of salicylic acid and dibutyl phthalate on paclitaxel production in Corylus avellana cell culture. J Stress Physiol Biochem 9:157–168

    Google Scholar 

  • Richheimer SL, Tinnermeier DM, Timmons DW (1992) High-performance liquid chromatographic assay of taxol. Anal Chem 64:2323–2326

    Article  CAS  PubMed  Google Scholar 

  • Rolland F, Moore B, Sheen J (2002) Sugar sensing and signaling in plants. Plant Cell 14:S185–S205

    PubMed Central  CAS  PubMed  Google Scholar 

  • Safari M, Ghanati F, Hajnoruzi A, Rezaei A, Abdolmaleki P, Mokhtari-Dizaji M (2012) Maintenance of membrane integrity and increase of taxanes production in hazel (Corylus avellana L.) cells induced by low-intensity ultrasounds. Biotechnol Lett 34:1137–1141

    Article  CAS  PubMed  Google Scholar 

  • Sanchez-Olate M, Rios D, Rodriguez R, Materan ME, Pereira G (2004) Duration of the reinvigorating effect of severe pruning of mature European hazelnut plants (Corylus avellana L.) cv. Negretta with in vitro cultivation. Agric Technol 64:338–346

    Google Scholar 

  • Saville M, Lietzau J, Pluda J, Feuerstein I, Odom J, Wilson W, Humphrey R, Feigal E, Steinberg S, Broder S (1995) Treatment of HIV-associated Kaposi’s sarcoma with paclitaxel. Lancet 346:26–28

    Article  CAS  PubMed  Google Scholar 

  • Singh M, Chaturvedi R (2012) Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthes acmella Murr. Bioprocess Biosyst Eng 35:943–951

    Article  CAS  PubMed  Google Scholar 

  • Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Muthuselvam M, Selvaraj N, Manickavasagam M, Ganapathi A (2013) A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal. Protoplasma 250:885–898

    Article  CAS  PubMed  Google Scholar 

  • Suehara K, Kameoka T, Hashimoto A (2012) Sugar uptake analysis of suspension Arabidopsis, tobacco, and rice cells in various media using an FT-IR/ATR method. Bioprocess Biosyst Eng 35:1259–1268

    Article  CAS  PubMed  Google Scholar 

  • Sung LS, Huang SY (2000) Medium optimization of transformed root cultures of Stizolobium hassjoo producing L-DOPA with response surface methodology. Biotechnol Prog 16:1135–1140

    Article  CAS  PubMed  Google Scholar 

  • Vasilev N, Grömping U, Lipperts A, Raven N, Fischer R, Schillberg S (2013) Optimization of BY-2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method. Plant Biotechnol J 11:867–874

    Article  CAS  PubMed  Google Scholar 

  • Verpoorte R (2000) Secondary metabolisms. In: Verpoorte R, Alfermann AW (eds) Metabolic engineering of plant secondary metabolism. Kluwer Academic Publishers, London, pp 1–29

    Chapter  Google Scholar 

  • Wyndaele R, Christiansen J, Horseele R, Rüdelsheim P, Van Onckelen H (1988) Functional correlation between endogenous phytohormone levels and hormone autotrophy of transformed and habituated soybean cell lines. Plant Cell Physiol 29:1095–1101

    CAS  Google Scholar 

  • Yu X, Reed BM (1995) A micropropagation system for hazelnuts (Corylus species). HortSci 30:120–123

    Google Scholar 

Download references

Acknowledgments

Work in the Plant Physiology Laboratory (University of Barcelona) was financially supported by the Spanish MEC (BIO2011-29856-C02-1) and the Generalitat de Catalunya (2014SGR215). A. Gallego held a grant from the Universitat Pompeu Fabra.

Author information

Authors and Affiliations

  1. Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Avda. Dr. Aiguader 80, E-08003, Barcelona, Spain

    Ana Gallego, Manuel Pastor & Elisabeth Moyano

  2. Laboratori de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII sn., E-08028, Barcelona, Spain

    Mercedes Bonfill, Rosa M. Cusido & Javier Palazon

Authors
  1. Ana Gallego
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mercedes Bonfill
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rosa M. Cusido
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Manuel Pastor
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Javier Palazon
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Elisabeth Moyano
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elisabeth Moyano.

Additional information

Editor: J. Forster

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gallego, A., Bonfill, M., Cusido, R.M. et al. Assessing factors that affect the growth of Corylus avellana cell suspension cultures: a statistical approach. In Vitro Cell.Dev.Biol.-Plant 51, 530–538 (2015). https://doi.org/10.1007/s11627-015-9693-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-015-9693-x

Keywords

Search

Navigation