Production of transgenic Hypericum perforatum plants via particle bombardment-mediated transformation of novel organogenic cell suspension cultures

Abstract

We have developed particle bombardment-mediated transformation procedure for Hypericum perforatum L. (St. John’s wort), an important medicinal species that remains highly recalcitrant towards Agrobacterium-mediated transformation. Among the major transformation techniques evaluated in the present study (Agrobacterium tumefaciens-, A. rhizogenes- and biolistics-mediated), particle-bombardment-mediated gene transfer was found to be the most successful one. GUS positive cells were obtained from organogenic nodules bombarded with the plasmid vector pCAMBIA1301 encoding an intron-containing b-glucuronidase (gusA) and hygromycin phosphotransferase (hpt) genes. After 3 months of continuous selection of bombarded nodules with 20 mg l 1 hygromycin, transgenic hygromycin-resistant callus cultures and subsequently transgenic plants were produced. PCR analysis of DNA isolated from GUS positive plants showed the presence of both gusA and hpt genes. Southern blot analysis confirmed the transgene integration and revealed diverse copy numbers and insertion sites. The data presented here demonstrate for the first time H. perforatum can be efficiently transformed via particle bombardment of organogenic cell suspension. Our results open the possibility of using particle bombardment-mediated transformation to elucidate biosynthetic pathways and to improve secondary metabolite production in H. perforatum.