title: 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 2 mg/L BAP and 0.1 mg/L NAA, after 45 days. The shoots were rooted best on half Murashige and Skoog's medium with respect to media containing 1 mg/L IBA or 1 mg/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 15 mg/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 2 days at 22 °C on shoot induction medium. The bacterial growth was eliminated by addition of cefotaxime (200 mg/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.
authors.affiliation: School of Biotechnology and Bioinformatics, D. Y. Patil University, Navi Mumbai, India ; National Agri-Food Biotechnology Institute, Govt. of India, Mohali, Punjab India.
authors.forename: Anshu
authors.initials: A
authors.lastname: Alok
authors.affiliation: School of Biotechnology and Bioinformatics, D. Y. Patil University, Navi Mumbai, India ; National Agri-Food Biotechnology Institute, Govt. of India, Mohali, Punjab India.
authors.forename: Vishnu
authors.initials: V
authors.lastname: Shukla
authors.affiliation: School of Biotechnology and Bioinformatics, D. Y. Patil University, Navi Mumbai, India ; Department of Biological Sciences, BITS Pilani, Pilani Campus, Pilani, Rajasthan India.
authors.forename: Zarna
authors.initials: Z
authors.lastname: Pala
authors.affiliation: School of Biotechnology and Bioinformatics, D. Y. Patil University, Navi Mumbai, India ; National Agri-Food Biotechnology Institute, Govt. of India, Mohali, Punjab India.
authors.forename: Jitesh
authors.initials: J
authors.lastname: Kumar
authors.affiliation: School of Biotechnology and Bioinformatics, D. Y. Patil University, Navi Mumbai, India.
authors.forename: Subhash
authors.initials: S
authors.lastname: Kudale
authors.affiliation: School of Biotechnology and Bioinformatics, D. Y. Patil University, Navi Mumbai, India ; Amity Institute of Biotechnology, Amity University, Mumbai, India.
authors.forename: Neetin
authors.initials: N
authors.lastname: Desai
keywords: A. pallens; Agrobacterium tumefaciens; Attached cotyledons; GUS; Transient transformation