Mature seeds of Helicteres isora L. were collected from seven geographical locations of Maharashtra and Goa (India) and evaluated for diosgenin (a bioactive steroidal sapogenin of prime importance) extraction and quantification. Chemotypic variations were evidenced with diosgenin quantity ranging from 33 μg g−1 seeds (Osmanabad forests) to 138 μg g−1 (Khopoli region). Nodal and leaf explants from in vitro-raised seedlings were used for callus and Agrobacterium-mediated transformation, respectively. Compact, hard, whitish-green callus (2.65 g explant−1) was obtained on MS + 13.32 μM BAP + 2.32 μM Kin after 30 days of inoculation. Various parameters including types of explant and Agrobacterium strain, culture density, duration of infection and various medium compositions were optimized for hairy root production. A. rhizogenes strain ATCC-15834 successfully induced hairy roots from leaf explants (1 cm2) with 42 % efficiency. Transgenic status of the roots was confirmed by PCR using rolB and VirD specific primers. Hairy roots showed an ability to synthesize diosgenin. Diosgenin yield was increased ~8 times in hairy roots and ~5 times in callus than the seeds of wild plants. Enhanced diosgenin content was associated with proline accumulation in hairy roots. This is the first report on induction of hairy roots in H. isora.