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

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)36-44
Number of pages9
JournalIn Vitro Cellular and Developmental Biology - Plant
Volume54
Issue number1
DOIs
StatePublished - Feb 1 2018

Bibliographical note

Funding Information:
Acknowledgements 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).

Publisher Copyright:
© 2018, The Society for In Vitro Biology.

Keywords

  • Agrobacterium
  • Stable transformation
  • Taxus
  • β-glucuronidase

ASJC Scopus subject areas

  • Biotechnology
  • Plant Science

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