Question

Agrobacterium rhizogenes mediated genetic transformation in rauwolfia serpentina thesis

Answer 1

Root extracts of Rauwolfia serpentina have been used for centuries in Ayurvedic medicine as a panacea for a wide variety of physical as well as mental disorders. The potential of Agrobacterium rhizogenes-mediated genetic transformation for the synthesis of phytomolecules of high pharmaceutical value is now well established and documented. Transgenic roots were induced from R. serpentina leaf explants in response to A. rhizogenes A4 strain on semi-solid 1⁄2-strength MS medium. Amongst 200 hairy root clones developed, 27 showing persistent and incessant growth over several generations were selected. Transformed roots grew vigorously and branched profusely on hormone-free liquid B5 medium with 3% sucrose with higher biomass yields compared to the control and showed two stable and distinct morphotypes. Medium devoid of any carbon source served as the control. The transformed nature of the roots was confirmed by PCR amplification with rolA primers. Growth kinetic studies exhibited the highest growth index (58.57 ± 1.92) at the 10 week followed by slow growth in the subsequent period up to 14 weeks. Reserpine content increased with root growth and was highest in 10-weeks-old cultures. Hairy root clones showed a wide array of variation in relative reserpine content, varying from 0.0064 to 0.0858% dry weight (DW). On the basis of relative reserpine content, these hairy root clones were classified into 5 different groups. SM12 clone had the highest reserpine level (0.0858% DW) producing 2-3 times more than the content of field-grown roots harvested after 18-24 months. A distinct relationship between root morphology and reserpine content was observed. The present study is the first report of reserpine production in quantifiable amounts from the hairy roots of any Rauwolfia species.