Indian Journal of Experimental Biology Vol. 53, March 2015, pp. 177-183 Effect of carbon and nitrogen sources on in vitro flower and fruit formation and withanolides production in Withania somnifera (L.) Dunal Ganeshan Sivanandhan 1 *, Jeevaraj Theboral 1 , Gnanajothi Kapil Dev 1 , Natesan Selvaraj 2 , Markandan Manickavasagam 1 & Andy Ganapathi 1 * 1 Department of Biotechnology and Genetic Engineering, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India 2 Government Arts College, Kulithalai 639 120, Tamil Nadu, India Received 06 November 2013; revised 21 February 2014 We studied the influence of sucrose and nitrogen concentration on in vitro flowering and fruit setting in elongated shoots of Withania somnifera. BA (1.5 mg/l) and IAA (0.3 mg/l) on MS medium supplemented with 4% sucrose showed 67% of in vitro flower induction frequency, 9 flowers/shoot, 4 fruits/shoot and 11 seeds/fruit in elongated-shoots. Different concentrations of nitrogen sources (L-glutamine, adenine sulphate, ammonium nitrate, potassium nitrate and sodium nitrate 5-25 mg/l) were tested in combination with 4% sucrose and BA at 1.5 mg/l and IAA at 0.3 mg/l. Highest number of flowers (20 flowers/shoot; 2.2-fold) and fruits (16 fruits/shoot; 3.39-fold), fruit setting (12 seeds/fruit; 1.08-fold) at a higher frequency (88 %) were achieved on MS medium augmented with 15 mg/l adenine sulphate with same PGRs and sucrose concentration. The maximum production of withanolide A (0.68 mg/g DW) and withanolide B (0.77 mg/g DW) was recorded in in vitro fruits. Highest accumulation of withaferin A (2 mg/g DW) was quantified from in vitro flowers, whereas, it was low in in vitro fruits (0.49 mg/g DW withaferin A). However, withanone (0.23 mg/g DW) was found accumulated uniformly in both in vitro flowers and fruits compared to control. Keywords:Ashwagandha, Indian ginseng, Withaferin A, Withanone Withania somnifera (L.) Dunal (“Indian ginseng”) is an important medicinal plant in the Indian traditional medicinal system. Withanolide A, withanolide B, withaferin A and withanone, the major bioactive constituents of this plant, have remarkable activities in physiological and metabolic restoration, anti-arthritic, anti-aging, anti-cancer, cognitive function improvement in geriatric states and recovery from neurodegenerative disorders 1 .These major withanolides are biosynthesized in leaves, roots, flowers and fruits 2 . Since W. somnifera flowers and fruits possess pharmacological activities, in vitro culture of flowers and fruits is considered as a viable option for uniform bioactive component synthesis. In addition, knowledge on in vitro flowering, development of fruits and seeds is precious in understanding the floral biology and prompt breeding of W. somnifera 3 . Although, Saritha and Naidu 4 reported in vitro flowering in W. somnifera with a low frequency of flowering and fruit setting, they did not account seed germination ability and the ploidy level in the progenies. Also, they did not record withanolides content in in vitro flowers and fruits. Although, many reports are available on withanolides production in shoot culture, adventitious root culture, hairy root culture and cell suspension culture 5-9 , there are no reports so far on withanolides production in in vitro flowers and fruits. The formation of flowers in vitro depends on numerous factors, which comprise the environment and stage of the explants, composition of the medium (PGRs, nitrogen and carbon sources) and environmental conditions 10 . The type and concentration of carbohydrate source are important in in vitro flowering and fruit setting, and its effect may differ depending up on the plant species 10 . Sucrose is the most favoured carbon source for flower induction and fruit setting in Solanaceae members 11 . Nitrogen bearing compounds at optimal level have played a desicive part in influencing the flower induction and fruit setting 12-14 . In the present study, we explored potential carbon and nitrogen sources to improve in vitro flower formation, fruit setting and withanolides production in W. somnifera. —————— *Correspondence: Tel.: 91 0431 2407086; Fax: 0431-2407045 aganapathi2003@rediffmail.com (AG); gsivabdu@gmail.com (GS)