Induction of hairy roots and over production of anthraquinones in Oldenlandia umbellata L.: a dye yielding medicinal plant by using wild type Agrobacterium rhizogenes strain

Abstract

Hairy root cultures of Oldenlandia umbellata L. was established by infecting leaf explants with wild type Agrobacterium rhizogenes strain (MTCC 532). Leaf explants were co-cultured with activated bacteria in half strength Murashige and Skoog (MS) basal medium containing 500 mg l⁻¹ ampicillin for the induction of hairy roots. To facilitate effective transfer of T-DNA of Ri plasmid, duration of co-cultivation and concentration of acetosyringone were standardized as 48 h and 200 µM, respectively. Roots were produced from the infected leaves within 14 days. Fast growing roots (2–3 cm) were excised and sub-cultured in half strength basal MS liquid medium for the development of transgenic lines. Thus ten hairy root lines were raised and superior line L3 was selected on the basis of fresh weight (1.5 g), dry weight (0.26 g) and anthraquinone (AQ) content (10.374 mg g⁻¹ dry wt). Superior line (L3) was further cultured to test stability of AQ production and found that stable production of AQ during five consecutive sub-cultures. High performance liquid chromatographic method was adopted for quantification of purpurin from samples. Hairy roots produced 21.83 mg g⁻¹ purpurin, while roots from wild growing plants produced 6.08 mg g⁻¹ purpurin. Thus purpurin content in hairy roots was about 3.6 times higher than wild growing roots. Further, polymerase chain reaction based amplification using primers specific for genes rolA, rolB, and rolC confirmed the transformation.