Development of transgenic hairy roots and augmentation of secondary metabolites by precursor feeding in Sphagneticola calendulacea (L.) Pruski

Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] is an important medicinal plant that possesses a variety of bioactive secondary metabolites. A stable transgenic hairy root culture has been developed using engineered Agrobacterium rhizogenes strain LBA1334 harboring pCAM:2 x 35S:gusA binary vector, to achieve high yield of secondary metabolites. Putative transgenic hairy roots appeared with highest transformation frequency (66.67%) when leaf explants were immersed in A. rhizogenes suspension for 10 min and co-cultivated for three days. Incorporation of foreign genes was confirmed by initial GUS staining assay followed by polymerase chain reaction analysis of rotA, rolB, rolC and gusA genes. Time course study revealed that the transgenic hairy roots grew rapidly in 1/2 Murashige and Skoog liquid medium with highest biomass production of 0.61 g per 50 ml dry weight after 28 days of culture. In the precursor feeding experiment, the biomass of hairy roots was further increased to 0.698 g per 50 ml dry weight when 0.5 mM phenylalanine was added to the 10 days old growing culture. High Performance Thin Layer Chromatography showed that the transgenic hairy roots accumulated enhanced level of wedelolactone (422.01 mu g g(-1) dry weight), which was 1.43 and 1.37 times higher than those of in vivo (294.44 mu g g(-1) dry weight) and in vitro-grown (308.28 mu g g-(1) dry weight) plantlets, respectively. Precursor feeding with phenylalanine further augmented the accumulation of wedelolactone to 440.33 mu g g(-1) dry weight in the transgenic hairy roots. In addition, the content of several important phenolic and flavonoid compounds were increased in the transgenic hairy roots as analyzed by High Performance Liquid Chromatography. The protocol offers a scope to introduce foreign genes and further regulate the production of secondary metabolites.