In vitro regeneration and optimization of factors affecting Agrobacterium mediated transformation in Artemisia Pallens, an important medicinal plant.

Abstract

Artemisia pallens is an important medicinal plant. In-vitro regeneration and multiplication of A. pallens have been established using attached cotyledons. Different growth regulators were considered for regeneration of multiple shoots. An average of 36 shoots per explants were obtained by culturing attached cotyledons on Murashige and Skoog's medium containing 2mg/L BAP and 0.1mg/L NAA, after 45days. The shoots were rooted best on half Murashige and Skoog's medium with respect to media containing 1mg/L IBA or 1mg/L NAA. Different parameters such as type of bacterial strains, OD600 of bacterial culture, co-cultivation duration, concentration of acetosyringone and explants type were optimized for transient expression of the reporter gene. Agrobacterium tumefaciens harbouring pCambia1301 plasmid carrying β-glucuronidase as a reporter gene and hygromycin phosphotransferase as plant selectable marker genes were used for genetic transformation of A. pallens. Hygromycin lethality test showed concentration of 15mg/L were sufficient to inhibit the growth of attached cotyledons and multiple shoot buds of nontransgenics in selection media. Up to 83% transient transformation was found when attached cotyledons were co-cultivated with Agrobacterium strain AGL1 for 2days at 22°C on shoot induction medium. The bacterial growth was eliminated by addition of cefotaxime (200mg/L) in selection media. T0 transgenic plants were confirmed by GUS histochemical assay and further by polymerase chain reaction (PCR) using uidA and hpt gene specific primers. The study is useful in establishing technological improvement in A. pallens by genetic engineering.