Highly Efficient Method of Transformation Using Domestic Strain A13

, a medicinal plant growing in Asian countries, produces shikonin derivatives that are lipophilic secondary metabolites. These red naphthoquinone pigments are traditionally used as a natural drug and a dye in East Asia. In intact plants, shikonin derivatives are produced in the root epidermal cells and secreted into extracellular spaces. The biosynthetic pathway for shikonin derivatives remains incompletely understood and the secretion mechanisms are largely unknown. Understanding the molecular mechanisms underlying shikonin biosynthesis and transport in cells requires functional analysis of candidate genes using transgenic plants. To date, however, standard transformation methods have not yet been established. This study describes an efficient method for transformation using hairy roots by strain A13, present domestically in Japan. Hairy roots of were generated from explants of the axenic shoots that were infected with strain A13. Integration into the genome was assessed by PCR amplifying a transgene encoding green fluorescent protein (GFP) and by monitoring GFP expression. This method enhanced transformation efficiency 50-70%. Although methods for the systematic stable transformation of plants have not yet been reported, the method described in this study resulted in highly efficient stable transformation using hairy roots. This method enables the functional analysis of genes.