Development of an Agrobacterium-mediated transformation method for Taxus suspension cultures

  • Sarah A. Wilson
  • Patricia Keen
  • Michelle C. McKee
  • Nicole Raia
  • Joyce Van Eck
  • Susan C. Roberts
Genetic Transformation


The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.


Agrobacterium Stable transformation Taxus β-glucuronidase 



Wilson acknowledges the support of the National Science Foundation-sponsored Institute for Cellular Engineering IGERT program (DGE-0654128). Roberts also acknowledges support from the National Institutes of Health (RO1 GM070852).


  1. Akcay UC, Mahmoudian M, Kamci H, Yucel M, Oktem HA (2009) Agrobacterium tumefaciens-mediated genetic transformation of a recalcitrant grain legume, lentil (Lens culinaris Medik). Plant Cell Rep 28(3):407–417. CrossRefPubMedGoogle Scholar
  2. Altunkaya A, Gökmen V (2008) Effect of various inhibitors on enzymatic browning, antioxidant activity and total phenol content of fresh lettuce (Lactuca sativa). Food Chem 107(3):1173–1179. CrossRefGoogle Scholar
  3. Cragg GM, Newman DJ (2009) Nature: a vital source of leads for anticancer drug development. Phytochem Rev 8(2):313–331. CrossRefGoogle Scholar
  4. Cusido RM, Onrubia M, Sabater-Jara AB, Moyano E, Bonfill M, Goossens A, Pedreño MA, Palazon J (2014) A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp. Biotechnol Adv 32(6):1157–1167. CrossRefPubMedGoogle Scholar
  5. Dalton S, Bettany A, Timms E, Morris P (1995) The effect of selection pressure on transformation frequency and copy number in transgenic plants of tall fescue (Festuca arundinacea Schreb.) Plant Sci 108(1):63–70. CrossRefGoogle Scholar
  6. Dong Y, Duan W, He H, Su P, Zhang M, Song G, Fu C, Yu L (2015) Enhancing taxane biosynthesis in cell suspension culture of Taxus chinensis by overexpressing the neutral/alkaline invertase gene. Process Biochem 50(4):651–660. CrossRefGoogle Scholar
  7. Enríquez-Obregón GA, Prieto-Samsónov DL, de la Riva GA, Pérez M, Selman-Housein G, Vázquez-Padrón RI (1999) Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an antinecrotic treatment. Plant Cell Tissue Organ Cult 59(3):159–168. CrossRefGoogle Scholar
  8. Fettneto AG, Zhang WY, Dicosmo F (1994) Kinetics of taxol production, growth, and nutrient uptake in cell suspenesions of Taxus cuspidata. Biotechnol Bioeng 44(2):205–210. CrossRefGoogle Scholar
  9. Jones AMP, Saxena PK (2013) Inhibition of phenylpropanoid biosynthesis in Artemisia annua L.: a novel approach to reduce oxidative browning in plant tissue culture. PLoS One 8(10):e76802. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Ketchum REB, Wherland L, Croteau RB (2007) Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures. Plant Cell Rep 26(7):1025–1033. CrossRefPubMedGoogle Scholar
  11. Kolewe ME, Henson MA, Roberts SC (2010) Characterization of aggregate size in Taxus suspension cell culture. Plant Cell Rep 29(5):485–494. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Kolewe ME, Henson MA, Roberts SC (2011) Analysis of aggregate size as a process variable affecting paclitaxel accumulation in Taxus suspension cultures. Biotechnol Prog 27(5):1365–1372. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Krügel T, Lim M, Gase K, Halitschke R, Baldwin IT (2002) Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. Chemoecology 12(4):177–183. CrossRefGoogle Scholar
  14. Li FL, Ma XJ, XL H, Hoffman A, Dai JG, Qiu DY (2011) Antisense-induced suppression of taxoid 14 beta-hydroxylase gene expression in transgenic Taxus x media cells. Afr J Biotechnol 10:8720–8728Google Scholar
  15. Li S, Zhang P, Zhang M, Fu C, Yu L (2013) Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis. Plant Biol 15(1):19–26. CrossRefPubMedGoogle Scholar
  16. Li ST, CH F, Zhang M, Zhang Y, Xie S, LJ Y (2012) Enhancing taxol biosynthesis by overexpressing a 9-cis-epoxycarotenoid dioxygenase gene in transgenic cell lines of Taxus chinensis. Plant Mol Biol Rep 30(5):1125–1130. CrossRefGoogle Scholar
  17. Opabode JT (2006) Agrobacterium-mediated transformation of plants: emerging factors that influence efficiency. Biotechnol Mol Biol Rev 1:12–20Google Scholar
  18. 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(3):418–427. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Patil RA, Kolewe ME, Roberts SC (2013) Cellular aggregation is a key parameter associated with long term variability in paclitaxel accumulation in Taxus suspension cultures. Plant Cell Tissue Organ Cult 112(3):303–310. CrossRefPubMedGoogle Scholar
  20. Shekhawat UKS, Ganapathi TR, Srinivas L, Bapat VA, Rathore TS (2008) Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. Plant Cell Tissue Organ Cult 92(3):261–271. CrossRefGoogle Scholar
  21. Song GQ, Sink KC (2004) Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.) Plant Cell Rep 23(7):475–484. CrossRefPubMedGoogle Scholar
  22. Vaucheret H, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, Mourrain P, Palauqui JC, Vernhettes S (1998) Transgene-induced gene silencing in plants. Plant J 16(6):651–659. CrossRefPubMedGoogle Scholar
  23. Vongpaseuth K, Roberts SC (2007) Advancements in the understanding of paclitaxel metabolism in tissue culture. Curr Pharm Biotechnol 8(4):219–236. CrossRefPubMedGoogle Scholar
  24. Wilson SA, Cummings EM, Roberts SC (2014) Multi-scale engineering of plant cell cultures for promotion of specialized metabolism. Curr Opin Biotechnol 29:163–170. CrossRefPubMedGoogle Scholar
  25. Wilson SA, Roberts SC (2012) Recent advances towards development and commercialization of plant cell culture processes for the synthesis of biomolecules. Plant Biotechnol J 10(3):249–268. CrossRefPubMedGoogle Scholar
  26. Zhang D, Yang R, Wang S, Dong Z (2014) Paclitaxel: new uses for an old drug. Drug Des Devel Ther 8:279–284. PubMedPubMedCentralGoogle Scholar
  27. Zhang P, Li ST, Liu TT, CH F, Zhou PP, Zhao CF, LJ Y (2011) Overexpression of a 10-deacetylbaccatin III-10 beta-O-acetyltransferase gene leads to increased taxol yield in cells of Taxus chinensis. Plant Cell Tissue Organ Cult 106(1):63–70. CrossRefGoogle Scholar
  28. Zhang X, Henriques R, Lin S-S, Niu Q-W, Chua N-H (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2):641–646. CrossRefPubMedGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  • Sarah A. Wilson
    • 1
  • Patricia Keen
    • 2
  • Michelle C. McKee
    • 3
  • Nicole Raia
    • 1
  • Joyce Van Eck
    • 2
  • Susan C. Roberts
    • 4
  1. 1.Chemical EngineeringUniversity of MassachusettsAmherstUSA
  2. 2.Boyce Thompson InstituteIthacaUSA
  3. 3.Biology and BiotechnologyWorcester Polytechnic InstituteWorcesterUSA
  4. 4.Chemical EngineeringWorcester Polytechnic InstituteWorcesterUSA

Personalised recommendations