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Not AvailableJawahar Ashwagandha-20 (JA-20) and Arka Ashwagandha (AA) seeds were raised in in vitro conditions and phenotypic differences between the plants was recorded. The adventitious roots were derived from leaves of the two varieties in in vitro conditions. The effects of the strength of media and concentration and the combination of auxins (IAA and IBA) for adventitious roots multiplication using suspension culture were studied. After 30 days of suspension culture, root biomass was measured and HPLC analysis of major withanolides in leaves and adventitious roots was conducted. Arka Ashwagandha variety had higher total withanolide content of 1.621 mg/g, Withaferin A content of 1.362 mg/g and root yield of 4.066 g from 0.1g inoculum in 30 days compared to JA-20 which had total withanolide content of 1.156 mg/g, Withaferin A content of 0.930 mg/g on dry weight basis and root mass of 3.71g from 0.1g of inoculum in 30 days. The present study thus helps in the identification of an elite cultivar of Ashwagandha and development of a standard protocol for mass multiplication of adventitious root in hormone-free media. This is beneficial in the preparation of health supplements in terms of human health issues due to negligible residual effects of hormones in the final product.Not Availabl