Document Type : Original Article


1 Department of Biology, Faculty of Sciences, Razi University, Kermanshah, Iran

2 Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran


Taxus baccata is one of the native forest species in Iran. It is one of the most important sources of taxol, an anti-cancer compound. This plant is an endangered species due to its low growth and regeneration and also over-harvesting. Taxol production by tissue culture technique can protect this plant from the danger of extinction. In this study, the improvement of the growth of callus and taxol production in Taxus baccata were assessed using chemical absorbents and antioxidant compounds.
Material and methods:
Activated charcoal (1 g/l), Polyvinylpyrrolidone (PVP; 250 mg/l) and an antioxidant compound (0.2 g/l glutamine, 0.05 g/l ascorbic acid and 0.05 g/l citric acid) were used for treating in a callus growth medium. Four grams of 60 day-old calli were transferred to a callus growth medium containing each of the treatments, where the untreated culture medium was considered as the control group. After 21 days, the amount of callus browning, wet weight, dry weight and viability as growth and morphological factors and also hydrogen peroxide (H2O2), malondialdehyde, total phenol, polyphenol oxidase activity and taxol amount as phytochemical factors were studied and evaluated.
Results and discussion:
The results of morphological and phytochemical changes of calli under treatments showed that the antioxidant compound reduced the H2O2 and lipid peroxidation amount and with the reduction of oxidative stress, browning of tissues decreased. Also, under this treatment, with decreasing total phenol and activity of phenoloxidase, the amount of phenol oxidation decreased significantly. So, cell viability and wet and dry weight were increased and the amount of tissue browning was decreased. By absorbing the phenolic compound, activated charcoal and PVP decreased the polyphenol oxidase activity. The decrease in the polyphenol oxidase activity increased cell viability and prevented tissue browning. Under these treatments, no significant changes in H2O2 and MDA content, and wet and dry weight was observed. No changes were observed in the callus growth, which may be due to the absorption of hormones and vitamins along with the absorption of toxic compounds from the medium.
All treatments were effective in decreasing the amount of tissue browning and increasing the viability of calli, but antioxidant compound treatment was the most effective in improving growth and increasing wet and dry weight with increasing taxol. Therefore, it is suggested as the best treatment for optimizing the callus culture of yew.


  1. Abdelwahd, R., Hakam, N., Labhilili, M. and Udupa, S.M., 2008. Use of an adsorbent and antioxidants to reduce the effects of leached phenolics in in vitro plantlet regeneration of faba bean. African Journal of Biotechnology. 7, 997- 1002.
  2. Ainsworth, E.A. and Gillespie, K.M., 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nature Protocols. 2, 875-877.
  3. Alemi, A., Eslami, A. and Shataee joibari, S., 2013. Investigation on development potential of endangered species of Taxus baccata at Golestan Province, based on GIS technology (Case study: Pooneh Aram reserve). Iranian journal of Forests and Poplar Research. 21, 678- 689 (In Persian with English abstract(.
  4. Alexieva, V., Sergiev, I., Mapelli, S. and Karanov, E., 2001. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant, Cell & Environment. 24, 1337-1344.
  5. Behera, M.D., Srivastava, S., Kushwaha, S. and Roy, P., 2000. Stratification and mapping of Taxus baccata L. bearing forests in Talle Valley using remote sensing and GIS. Current Science. 1008-1013.
  6. Bruňakova, K., Babincova, Z. and Čellarova, E., 2004. Selection of callus cultures of Taxus baccata L. as a potential source of paclitaxel production. Engineering in Life Sciences. 4, 465-469.
  7. Delevoy, D., Jackson, W. and Harrison, S., 1967. A handbook of Coniferae and Ginkgoaceae, New York: St. Martin's Press.
  8. Exposito, O., Bonfill, M., Onrubia, M., Jane, A., Moyano, E., Cusido, R.M., Palazon, J. and Piñol, M.T., 2009. Effect of taxol feeding on taxol and related taxane production in Taxus baccata suspension cultures. New biotechnology. 25, 252-259.
  9. Fett-Neto, A.G., Melanson, S.J., Sakata, K. and DiCosmo, F., 1993. Improved growth and taxol yield in developing calli of Taxus cuspidata by medium composition modification. Nature Biotechnology. 11, 731.
  10. FILOVÁ, A., 2014. Production of secondary metabolities in plant tissue cultures. Research Journal of Agricultural Science. 46, 236- 245.
  11. Gapińska, M., Skłodowska, M. and Gabara, B., 2008. Effect of short-and long-term salinity on the activities of antioxidative enzymes and lipid peroxidation in tomato roots. Acta Physiologiae Plantarum. 30, 11- 18.
  12. Gill, R. and Gill, S., 1994. In-vitro Exudation of Phenols in Eucalyptus. Indian Forester. 120, 504-509.
  13. Golalizadeh, D., 2010. Phytosociological study on natural T. baccata in forests of Gorgan, Qaemshahr and Noor. MSc thesis, Department of Forestry, Faculty of Natural Resources, Gorgan University of Agricultural Sciences and Natural Resources, Iran.
  14. Heath, R.L. and Packer, L., 1968. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics. 125, 189-198.
  15. Hoffman, A. and Shahidi, F., 2009. Paclitaxel and other taxanes in hazelnut. Journal of Functional Foods. 1, 33-37.
  16. Jaziri, M., Zhiri, A., Guo, Y.-W., Dupont, J.-P., Shimomura, K., Hamada, H., Vanhaelen, M. and Homès, J., 1996. Taxus sp. cell, tissue and organ cultures as alternative sources for taxoids production: a literature survey. Plant Cell, Tissue and Organ Culture. 46, 59-75.
  17. Jones, A.M.P. and Saxena, P.K., 2013. Inhibition of phenylpropanoid biosynthesis in Artemisia annua L.: a novel approach to reduce oxidative browning in plant tissue culture. PloS One. 8, 1- 13.
  18. Khosroushahi, A.Y., Naderi-Manesh, H. and Simonsen, H.T., 2011. Effect of antioxidants and carbohydrates in callus cultures of Taxus brevifolia: evaluation of browning, callus growth, total phenolics and paclitaxel production. BioImpacts: BI. 1, 37.
  19. Krishna, H., Sairam, R., Singh, S., Patel, V., Sharma, R., Grover, M., Nain, L. and Sachdev, A., 2008. Mango explant browning: Effect of ontogenic age, mycorrhization and pre-treatments. Scientia Horticulturae. 118, 132-138.
  20. Lan, W., Yu, L., Li, M. and Qin, W., 2003. Cell death unlikely contributes to taxol production in fungal elicitor-induced cell suspension cultures of Taxus chinensis. Biotechnology Letters. 25, 47-49.
  21. Liu, X. and Huang, B., 2000. Heat stress injury in relation to membrane lipid peroxidation in creeping bentgrass. Crop Science. 40, 503-510.
  22. Madhusudhanan, K. and Rahiman, B., 2000. The effect of activated charcoal supplemented media to browning of in vitro cultures of Piper species. Biologia Plantarum. 43, 297-299.
  23. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science. 7, 405-410.
  24. Mousavi, B., Parsaeimehr, A. and Irvani, N., 2011. Influences of growth regulators on callus induction, ephedrine and pseudoephedrine contents and chemical analysis of mature embryo of Ephedra strobilacea Bunge.
  25. Neill, S.J., Desikan, R., Clarke, A., Hurst, R.D. and Hancock, J.T., 2002. Hydrogen peroxide and nitric oxide as signalling molecules in plants. Journal of Experimental Botany. 53, 1237-1247.
  26. Palma, M., Piñeiro, Z. and Barroso, C.G., 2001. Stability of phenolic compounds during extraction with superheated solvents. Journal of Chromatography A. 921, 169-174.
  27. Price, R.A., 1990. The genera of Taxaceae in the southeastern United States. Journal of the Arnold Arboretum. 71, 69-91.
  28. Rajewski, M., Lange, S. and Hattemer, H., 2000. Problems of reproduction in the genetic conservation of rare tree species: the example of common yew (Taxus baccata L.). Forest Snow and Landscape Research. 75, 251-266.
  29. Sairam, R. and Saxena, D., 2000. Oxidative stress and antioxidants in wheat genotypes: possible mechanism of water stress tolerance. Journal of Agronomy and Crop Science. 184, 55- 61.
  30. Soliva, R.C., Elez, P., Sebastian, M. and Martı́n, O., 2000. Evaluation of browning effect on avocado puree preserved by combined methods. Innovative Food Science and Emerging Technologies. 1, 261-268.
  31. Svenning, J.-C. and Magård, E., 1999. Population ecology and conservation status of the last natural population of English yew Taxus baccata in Denmark. Biological Conservation. 88, 173-182.
  32. Titov, S., Bhowmik, S.K., Mandal, A., Alam, M.S. and Uddin, S.N., 2006. Control of phenolic compound secretion and effect of growth regulators for organ formation from Musa spp. cv. Kanthali floral bud explants. Am. J. Biochem. Biotechnol. 2, 97-104.
  33. Toulabi, S.B., Moieni, A., Ghanati, F. and Emami, F., 2015. Investigation of the Effects of the Basal Medium, Auxin and Antioxidants on the Induction and Maintenance of Callus and Taxol Production in Yew (Taxus baccata). Journal of Advances in Biology & Biotechnology. 3, 2394- 1081.
  34. Vahdatpour, F., Mashayekhi, K. and Piri Zirkouhi, M., 2009. Investigation of ontioxidant effect turmeric in comparing with active coal and ascorbic acid in cultural medium of Ulmas pavrifolia Jasq. Callus. plant production researches. 16, 1- 14 (In Persian with English abstract(.
  35. Witherup, K.M., Look, S.A., Stasko, M.W., Ghiorzi, T.J., Muschik, G.M. and Cragg, G.M., 1990. Taxus spp. needles contain amounts of taxol comparable to the bark of Taxus brevifolia: analysis and isolation. Journal of Natural Products. 53, 1249-1255.
  36. Woo, D.D., Miao, S.Y., Pelayo, J.C. and Woolf, A.S., 1994. Taxol inhibits progression of congenital polycystic kidney disease. Nature. 368, 750- 753.
  37. Zare, H., 2001. Introduced and Native Conifers in Iran. Agricultural Research, Educational and Extention Organization.
  38. Zhang, C.-H. and Wu, J.-Y., 2003. Ethylene inhibitors enhance elicitor-induced paclitaxel production in suspension cultures of Taxus spp. cells. Enzyme and Microbial Technology. 32, 71-77.
  39. Zhang, C.H., Fevereiro, P.S., He, G. and Chen, Z., 2007. Enhanced paclitaxel productivity and release capacity of Taxus chinensis cell suspension cultures adapted to chitosan. Plant Science. 172, 158-163.
  40. Zhao, J., Davis, L.C. and Verpoorte, R., 2005. Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances. 23, 283-333.