(599bc) An Optimized Transformation Method for Estrogen-Inducible Transgene Expression in Catharanthus Roseus Hairy Roots

The medicinal plant, Catharanthus roseus, is the source of two powerful anticancer alkaloids, vincristine (VCR) and vinblastine (VBL). These alkaloids are produced only in C. roseusand at extremely low levels (0.0002 wt%). Due to low levels, the isolation of these compounds is both laborious and costly, ranging from $4 to $60 million/kg. Despite these barriers, these alkaloids have been effectively used to treat cancer for over 50 years.

   To improve alkaloid production in C. roseus, the expression of several enzymes and transcription factors involved in alkaloid biosynthesis have been genetically engineered using a constitutive expression system (cauliflower mosaic virus 35S promoter). An inducible system, however, provides many benefits over a constitutive system. In an inducible system, the timing of transgene expression can be controlled, clonal variation in negative control lines is avoided, and deleterious effects such as growth retardation can be circumvented. A glucocorticoid-inducible system has been established and successfully used to express transgenes in C. roseushairy roots. However, growth defects in Arabidopsis and rice have been attributed to one or more components of the glucocorticoid-inducible system. Therefore the estrogen-inducible (XVE) system is an alternative inducible system.

   The process of obtaining stable transgenic hairy roots through Agrobacterium-mediated transformations can be timely and inefficient. Here, we report an optimized method of establishing transgenic C. roseus hairy roots (33% efficiency) under the estrogen-inducible XVE system driving GFP expression.