In vitro regeneration, Agrobacterium-mediated transformation, and genetic assay of chalcone synthase in the medicinal plant Echinacea pallida

Abstract

In vitro plant regeneration was established in Echinacea pallida, a plant that is commonly used as a folk medicine to treat the common cold, fevers, inflammation and so on. Conditions for callus induction, lateral root and shoot regeneration were determined. Subsequently, two vectors pCHS and pOSAG78, carrying different selection marker genes resistant to kanamycin and hygromycin, respectively, were independently used to transform leaf explants of E. pallida using an Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of the transgene and selection marker gene in obtained transgenic lines. Southern hybridization indicated that the T-DNA insertion in some transgenic E. pallida was single copy. Among them, transformants carrying Petunia chalcone synthase (CHS) were selected for further study. CHS is a key enzyme in the biosynthesis of diverse flavonoids including anthocyanin pigmentation. Here, we analyzed the roles and compared the gene expression of two clusters of CHSs, EpaCHS-A and EpaCHS-B (EpaCHS-B1 and EpaCHS-B2), isolated from E. pallida. Two of the genes, EpaCHS-A and EpaCHS-B1, were abundantly expressed in petals, whereas EpaCHS-B2 was expressed at high levels in leaves. The expression of EpaCHSs remained constant in leaves and roots of Petunia CHS transformants, while EpaCHS-B2 expression was changed in flowers of transgenic plants. The biosynthesis of caffeic acid derivatives, cichoric acid and caftaric acid, was increased in leaves and roots of CHS transformants, respectively, while the amount of echinacoside in roots of transgenic plants was decreased. This is the first report on genetic engineering of E. pallida. The information contained herein can be used as a tool for further study of the biological pathways and secondary metabolism of specific compounds from medicinal Echinacea species.