Optimization of the Fast Agro-Mediated Seedling Transformation Method in Catharanthus roseus

Abstract

Catharanthus roseus produces numerous pharmaceutically valuable Terpenoid Indole Alkaloids (TIAs), including the anticancer drugs vincristine and vinblastine. Because of their low concentrations in the plant, these drugs are very expensive. Several methods for stable and transient plant transformation have been developed to engineer transgenic lines. A naturally occurring plant pathogen, Agrobacterium rhizogenes, has been utilized for both stable and transient transformation. A. rhizogenes integrates DNA from its plasmids into its hostÍs genome and causes transgenic roots to grow at the site of infection. This bacterium provides a unique tool for genetically modifying plants and can be engineered to transfer our DNA of interest into the C. roseus genome. However, the process of obtaining a stable transgenic line can take several months, while transient expression methods take only weeks. Some methods used for transient expression analysis are particle bombardment, protoplast transformation, and Agrobacterium-mediated transformation. While particle bombardment and protoplast transformation require expensive, specialized equipment and extensive method development, Agrobacterium-mediated transformation relies on the bacteriumÍs natural ability to insert DNA into the plant genome. Here, we adapted and optimized the Fast Agro-mediated Seedling Transformation (FAST) method in C. roseus, where the Gus reporter gene was used to evaluate transformation efficiency.