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Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures

  • Genetic Transformation and Hybridization
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Abstract

A cell line of Taxus cuspidata has been transformed with wild-type Agrobacterium rhizogenes ATCC strain 15834 containing binary vector pCAMBIA1301 and, separately, with A. tumefaciens strain EHA105 containing binary vector pCAMBIA1305.2. Additionally, a cell line of T. chinensis has been transformed with wild-type A. rhizogenes ATCC strain 25818 containing binary vector pCAMBIA1301. The two transgenic T. cuspidata cell lines have been maintained in culture for more than 20 months, and the transgenic T. chinensis cell line for more than 9 months, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernable effect on growth or Taxol production in the transgenic cell lines when compared to the parent control. The methods for transforming non-embryogenic Taxus suspension cultures are described.

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Abbreviations

X-gluc:

5-Bromo-4-chloro-3-indolyl-β-d-glucuronide

GUS:

β-Glucuronidase

CaMV35S:

Cauliflower mosaic virus 35S promoter

HPLC-MS:

High performance liquid chromatography coupled mass spectroscopy

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Acknowledgments

This investigation was supported by U.S. National Institutes of Health grant CA-55254, and by McIntire-Stennis Project 0967 from the Washington State University Agricultural Research Center. The authors wish to thank James Green and Carina Ng for technical assistance and Mark Wildung for insightful discussions.

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Authors and Affiliations

  1. Institute of Biological Chemistry, Washington State University, Pullman, WA, 99164-6340, USA

    Raymond E. B. Ketchum, Lea Wherland & Rodney B. Croteau

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  1. Raymond E. B. Ketchum
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  2. Lea Wherland
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  3. Rodney B. Croteau
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Correspondence to Raymond E. B. Ketchum.

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Communicated by G.C. Phillips.

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Ketchum, R.E.B., Wherland, L. & Croteau, R.B. Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures. Plant Cell Rep 26, 1025–1033 (2007). https://doi.org/10.1007/s00299-007-0323-x

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