Date of Award

5-13-2011

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Plant Biology

First Advisor

Susan C. Roberts

Second Advisor

Elsbeth L. Walker

Third Advisor

Magdalena Bezanilla

Subject Categories

Plant Biology

Abstract

Human society has benefited greatly from plant secondary metabolites, often utilizing a variety of compounds as dyes, food additives, and drugs. In particular, pharmaceutical development has benefited greatly from plant secondary metabolites. One example of this utility is paclitaxel, a highly substituted diterpene approved in the treatment of breast cancer, ovarian cancer, non-small cell lung cancer, and the AIDSrelated Kaposi’s sarcoma. Demand of paclitaxel is likely to increase, due to the current examination of paclitaxel in numerous clinical trials against a variety of other cancers.

Taxus cell culture represents a production source of paclitaxel to meet future demand. However, paclitaxel production through Taxus cell culture is often variable and low. Targeted metabolic engineering of Taxus to produce superior paclitaxelaccumulating lines is a viable strategy to address variable and low yields. To facilitate the production of genetically engineered Taxus cell lines, stable transformation is required to examine the long-term effect of gene expression in vitro. Additionally, suitable transient transformation systems are necessary to characterize novel Taxus genes related to paclitaxel accumulation.

A transient particle bombardment-mediated transformation protocol was developed to introduce transgenes into Taxus cells in vitro. Additionally, agroinfiltration in Nicotiana benthamiana was examined as a system to express genes related to paclitaxel biosynthesis and lead to the accumulation of the first dedicated taxane, taxa- 4(5), 11(12)-diene. In regard to stable transformation, an Agrobacterium-mediated transformation protocol was developed, though this method requires further optimization for reliability and increased transformation efficiency. These transformation technologies will aid in the creation of elite paclitaxel-accumulating Taxus cell lines.

Included in

Plant Biology Commons

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