The overall goal of this study is to develop procedure for the production of taxol and/or its derivatives in vitro. One of the most critical strategies in developing alternative taxol production source via in vitro culture is to start the culture with the explants which have the highest possible taxol production gene combinations. The heart of this proposal is to obtain such explants and use them to develop tissue clones in vitro. The goal is to use the investigator's expertise in natural product chemistry, in embryo culture of woody plants and in Agrobacterium transformation to carry out the following in vitro culture projects of T. brevifolia: Project I: Using in vitro embryo culture technique to by- pass the complex seed dormancy and to produce 1,024 seedlings. The taxol/taxane derivatives production capacity of these seedlings will be screened as soon as the seedlings reach the proper size.The ten top taxol producing seedlings will be used as the starting materials for Project III. Project II: To develop techniques on Agrobacterium transformation of Taxus embryonic and young seedling tissues in order to produce (1) fast grown tumorous callus tissue for the production of semi-organized """"""""nodule cultures,"""""""" (2) genetically stable fast-grown """"""""hairy roots,"""""""" and/or (3) genetically stable, fast-grown """"""""teratoma."""""""" After nodule culture, hairy root and/or teratoma tissues established, their taxol production capacity will be compared. Project III: Applying the transformation procedure, developed in Project II for the type of tissue (nodule, hairy root, or teratoma) with the highest taxol production capacity, to the tissues of the ten top taxol producing seedlings from Project I.The resultant transformed tissues will be cloned to be used in the future projects such as maximizing taxol production in vitro. Reverse phase column HPLC will be used to screen the cultured tissue clones to identify the high taxol/taxane derivatives producing clones. Tandem mass spectrophotometric procedure will be used to confirm the structure of the compounds produced by cultured tissues. Taxol production of the above clones will be maximized by optimizing culture medium composition, addition of elicitors, providing artificial sink to remove product from culture medium, and permeating membranes to assist the nondestructive product recovering.
