Background: Papaver bracteatum Lindl. Commonly known as Iranian poppy is an important medicinal plant due to the presence of benzylisoquinoline alkaloids. Objective: To evaluate the hairy root culture as a novel method for thebaine production. Methods: To optimize the hairy root culture of P. bracteatum, five strains of Agrobacterium rhyzogenes (ATCC15834, C318, A13, 9453 and A4) were used by means of sonication-assisted Agrobacterium-mediated transformation (SAAT). Results: Strain C318 proved to be more efficient than the other strains to hairy root induction ability. Among different experimental conditions, the highest transformation rate (28.5 %) was observed 4 weeks after inoculation, once the explants were subjected to sonication (45 s) following a heat treatment of 40°C for 5 min. Transgenic status of the hairy roots was confirmed by PCR using rolB-specific primers. Conclusion: HPLC analysis showed the ability of hairy roots to synthesize of thebaine as a valuable medicinal compound.

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