REJUVENATION RESEARCH Volume 8, Number 1, 2005 © Mary Ann Liebert, Inc. Genetically Modified Hairy Roots of Withania somnifera Dunal: A Potent Source of Rejuvenating Principles VINOD KUMAR, 1 KOTAMBALLI N. CHIDAMBARA MURTHY, 1 SURESH BHAMIDI, 1 C.G. SUDHA, 2 and GOKARE A. RAVISHANKAR 1 ABSTRACT Transgenic hairy roots were induced from Withania somnifera Dunal, by infecting leaf ex- plants with Agrobacterium rhizogenes. Polymerase chain reaction for rol A gene and South- ern blot confirmed the integration of T-DNA in the genome. Cultures were grown in Murashige and Skoog solid as well as in liquid medium. The antioxidant activity was assayed in roots grown in solid media and liquid media. Hairy roots grown in liquid media found to possess highly significant activity in 1,1-diphenyl-2-pecryl-hydrazyl radical, -carotene linoleic acid model system. The activity was 57.34%, 75.64%, and 93.41% in case DPPH model and 55.3%, 76.3%, and 90.5% in case of b-CLAMS in 25, 50, and 100 mg L 1 concentration, re- spectively. In case of hydroxyl radical trapping and brain lipid peroxidation assay, the activ- ity was more significant in hairy roots grown on solid medium in comparison with commer- cial formulation prepared using normal roots and standard withanaloids. Root extract grown in solid medium has shown 93.2% hydroxyl radical trapping activity at 100 mg L 1 concen- tration, and 500 mg L 1 has shown 83.6% in case of brain lipid peroxidation assay. High-per- formance liquid chromatography analysis demonstrated the presence of withanaloids in the hairy root extracts. The results of the study clearly indicate that there is enhancement of sec- ondary metabolites in hairy roots, which is indicated through significant enhancement of the antioxidant activity, since these are the major constituents responsible for the activity. This is the first report on the presence of antioxidant principles in genetically modified roots of W. somnifera. These results of the present study may aid in utilization of the W. somnifera hairy roots for its rejuvenating principles. 37 INTRODUCTION W ITHANIA SOMNIFERA (WS) Dunal (family, Solanaceae) is a herb known for its med- icinal application since more than 2,500 years in the treatment of diseases and human ail- ments. 1 This is popularly known as “Indian Ginseng” and used in Ayurvedic medicines to attenuate cerebral function defects. Numerous studies show Withania somnifera to be superior to ginseng (Panax ginseng) as a rejevenative, an- tistress, and adaptogen. 2,3 It is also known to improve learning and memory retention in normal and deficient individuals, and to pro- vide non-specific host defense. 4 The root of this plant contains various steroidal and other al- kaloids generally known as “withania alka- loids.” The important ones are 5,20--dihydroxy, 1 Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore, Karnataka, India. 2 Plant Biotechnology Division, Tropical Botanic Garden and Research Institute, Polode, Tiruvananthapuram, India.