Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures
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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.
KeywordsTaxus Taxol Paclitaxel Agrobacterium Transformation Cell suspension culture
Cauliflower mosaic virus 35S promoter
High performance liquid chromatography coupled mass spectroscopy
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.
- Castle LA, Morris RO (1994) Transient expression assays using GUS constructs and fluorometric detection for analysis of T-DNA transfers. In: Gelvin S, Schilperoot R (eds) Plant molecular biology manual, 2nd edn. Kluwer, Dordrecht, B5, pp 1–16Google Scholar
- Hefner J, Rubenstein SM, Ketchum REB, Gibson DM, Williams RM, Croteau R (1996) Cytochrome P450-catalyzed hydroxylation of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5α-ol: the first oxygenation step in Taxol biosynthesis. Chem Biol 3:479–489. doi:10.1016/S1074-5521(96)90096-4PubMedCrossRefGoogle Scholar
- Ketchum REB, Croteau RB (2006) The taxoid metabolome and the elucidation of the paclitaxel biosynthetic pathway in cell suspension cultures of Taxus. In: Saito K, Dixon R, Willmetzer L (eds) Biotechnology in agriculture and forestry, Springer, Berlin Heidelberg New York, 57:291–309Google Scholar
- Kim CH, Kim KI, Chung IS (2000) Expression of modified green fluorescent protein in suspension culture of Taxus cuspidata. J Micro Biotech 10:91–94Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning : a laboratory manual, 2nd edn. Cold Spring Harbor, New YorkGoogle Scholar
- Tabata H (2004) Paclitaxel production by plant-cell-culture technology. Adv Biochem Eng Biotechnol 7:1–23. doi:10.1007/b95986Google Scholar
- Tian LN, Brown DCW, Webb J (2000) Transient expression of a reporter gene changes significantly during somatic embryogenesis in alfalfa. Can J Plant Sci 80:765–771Google Scholar
- Vincent JM (1970) A manual for the practical study of root-nodule bacteria. Blackwell, OxfordGoogle Scholar