Agrobacterium-Mediated Genetic Transformation and Cloning of Reference Genes in Suspension Cells of Artemisia Pallens

Abstract A reliable and stable Agrobacterium-mediated genetic transformation system has been developed using cell suspension cultures derived from Artemisia pallens cotyledon explants. Cotyledon, attached cotyledon, and compound leaf were found to be suitable for the induction of callus among five different types of explants tested. Yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2.4-dichlorophenoxyacetic acid (2,4-D) and in combination with different concentrations of Zeatin (ZEA). Among the two different shock treatments, cold shock (at 4 oC) for 20 minutes and heat shock (at 45oC) treatment for 5 minutes, heat shock treatment increased the transformation efficiency. Supplementation of chemical additives such as Silwet L-77 (0.05%) and Pluronic F-68 (0.05%) significantly enhanced suspension cultures' transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity was measured as 879.4±113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant callus derived from micro-calli showed intense blue colour in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The cloned and mRNA expression levels of three candidate reference genes ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) along with transgene (uidA) were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understand the metabolic pathways of this medicinally important plant and its genetic improvement.