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

摘要

We have developed particle bombardment-mediated transformation procedure for Hypericum perforatum L. (St. John’s wort), an importantmedicinal species that remains highly recalcitrant towards Agrobacterium-mediated transformation. Among the major transformation techniquesevaluated in the present study (Agrobacterium tumefaciens-, A. rhizogenes- and biolistics-mediated), particle-bombardment-mediated genetransfer was found to be the most successful one. GUS positive cells were obtained from organogenic nodules bombarded with the plasmid vectorpCAMBIA1301 encoding an intron-containing b-glucuronidase (gusA) and hygromycin phosphotransferase (hpt) genes. After 3 months ofcontinuous selection of bombarded nodules with 20 mg l 1 hygromycin, transgenic hygromycin-resistant callus cultures and subsequentlytransgenic 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 heredemonstrate for the first time H. perforatum can be efficiently transformed via particle bombardment of organogenic cell suspension. Our resultsopen the possibility of using particle bombardment-mediated transformation to elucidate biosynthetic pathways and to improve secondarymetabolite production in H. perforatum.