Efficient Agrobacterium-mediated transformation of Artemisia annua L. using young inflorescence

Abstract

Artemisia annua L. is currently the only practical source of artemisinin, an effective antimalarial agent. Development of an efficient gene delivery system is crucial for genetic manipulation of this medicinal plant to enhance artemisinin production. Here, we report a protocol for efficient Agrobacterium-mediated transformation and plant regeneration of A. annua using young inflorescence as explants. We evaluated the influence of explant types and Agrobacterium strains on transformation and regeneration efficiency. Young inflorescence and leaf explants were co-cultivated with Agrobacterium tumefaciens strains, EHA105 or LBA4404, carrying the pCAMBIA2301 binary vector containing a s-glucuronidase reporter. Shoot regeneration was observed when explants were incubated on modified Murashige and Skoog (MS) medium supplemented with 0.5 mg L−1 6-benzylaminopurine and 0.3 mg L−1 α-naphthalene acetic acid. Shoot bud development from young inflorescence explants was observed within 7 wk of culture whereas those from the leaf explants regenerated after 9 wk. PCR and Southern blot analyses showed that 95% of the putative transgenic lines were positive for the GUS reporter gene. The maximum transformation frequency of 17% was achieved by co-cultivation of A. tumefaciens strain EHA105 with young inflorescence, representing a twofold improvement compared to using leaf explants. Molecular and genetic analysis of T1 progeny further confirmed the inheritance of the introduced gene. The transformation protocol using young inflorescence of A. annua reduces the transformation time and increases the efficiency; hence, this represents a practical platform for metabolic engineering of this important medicinal plant.