Transgenic patchouli plants produced by Agrobacterium-mediated transformation

A successful transformation procedure using Agrobacterium was established for the most important aromatic crop, patchouli (Pogostemon cablin Benth.). To avoid inhibition of Agrobacterium infection by patchouli oil accumulated in leaf tissues, complete plants regenerated in vitro which possessed no or trace amounts of patchouli oil in leaf tissues were used as an explant source. Conditions for transformation were examined using two A. tumefaciens strains containing a different chimeric plasmid. Leaf explants were infected with A. tumefaciens strain EHA101/pIG121-Hm carrying beta-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes. Following co-cultivation for 3 days and selection by 50 mg l(-1) hygromycin B, greenish calli with adventitious shoots were selected, from which putative transformants with roots were regenerated. Histochemical assay showed that GUS expression is detected in every organs of transformants, which was confirmed by the detection of high activity of GUS. Using another strain LBA4404/pBI 121-PaCP1 encoding the coat protein precursor gene of patchouli mild mosaic virus (CP-P) and neomycin phosphotransferase (NPTII) gene, putative transformants were also obtained after co-cultivation for 7 days and selection by 100 mg l(-1) kanamycin. Using total DNAs from the transformants, the full length of CP-P was detected by PCR reaction. Comparing between two strains examined, it was noted that prolonged co-cultivation period and higher dose of a selection drug were indispensable for successful infection with LBA4404/pBI 121 - PaCP1.