Scale-Up of Agrobacterium rhizogenes-Mediated Hairy Root Cultures of Rauwolfia serpentina: A Persuasive Approach for Stable Reserpine Production.
Roots of Rauwolfia serpentina, also known as "Sarpagandha" possess high pharmaceutical value due to the presence of reserpine and other medicinally important terpene indole alkaloids. Ever increasing commercial demand of R. serpentina roots is the major reason behind the unsystematic harvesting and fast decline of the species from its natural environment. Considering Agrobacterium rhizogenes-mediated hairy root cultures as an alternative source for the production of plant-based secondary metabolites, the present optimized protocol offers a commercially feasible method for the production of reserpine, the most potent alkaloid from R. serpentina roots. This end-to-end protocol presents the establishment of hairy root culture from the leaf explants of R. serpentina through the infection of A. rhizogenes strain A4 in liquid B5 culture medium and its up-scaling in a 5 L bench top, mechanically agitated bioreactor. The transformed nature of roots was confirmed through PCR-based rol A gene amplification in genomic DNA of putative hairy roots. The extraction and quantification of reserpine in bioreactor grown roots has been done using monolithic reverse phase high-performance liquid chromatography (HPLC).
Plant Biotechnology Division, Central Institute of Medicinal & Aromatic Plants, P.O. CIMAP; Picnic Spot Road, Lucknow, 2256015, India. shaktimehrotra22@gmail.com.
Shakti
S
Mehrotra
National Institute of Plant Genome Research, Aruna Asaf Ali Road, 10531, New Delhi, 110 067, India.
Vikas
V
Srivastava
BioSeed Research India Limited, ICRISAT, Patancheru, 502324, Hyderabad, India.
Manoj K
MK
Goel
Plant Biotechnology Division, Central Institute of Medicinal & Aromatic Plants, P.O. CIMAP; Picnic Spot Road, Lucknow, 2256015, India.
Arun K
AK
Kukreja
D060054:Agrobacterium / Q000235:genetics / Q000502:physiology*; D019149:Bioreactors; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D018920:Coculture Techniques / Q000379:methods*; D003470:Culture Media / Q000378:metabolism; D018744:DNA, Plant / Q000235:genetics; D005784:Gene Amplification; D018517:Plant Roots / Q000235:genetics / Q000254:growth & development / Q000382:microbiology* / Q000502:physiology; D011926:Rauwolfia / Q000235:genetics / Q000254:growth & development / Q000382:microbiology* / Q000502:physiology; D012110:Reserpine / Q000032:analysis / Q000378:metabolism*; D014170:Transformation, Genetic
D003470:Culture Media; D018744:DNA, Plant; D012110:Reserpine
Bioreactor; HPLC; Hairy roots; Rauwolfia serpentina; Reserpine; Up-scaling