Asian Journal of Agriculture and Allied Sciences

Black henbane (Hyoscyamus niger) is a medicinal plant that can be propagated under controlled conditions using plant tissue culture. This study evaluated callus induction, spontaneous shoot differentiation, rooting and acclimatisation from subcotyledonous stem segments. Seeds were surface-sterilised, germinated on solid Murashige and Skoog (MS) medium without growth regulators and maintained under controlled culture-room conditions. Subcotyledonous stem segments from 15-day-old seedlings were cultured on solid MS medium supplemented with 0.5 mg L⁻¹ 2,4-D and 1.0 mg L⁻¹ BA. The sterilisation procedure produced sterile seedlings with a 90% germination rate after six days. Callus induction occurred in 100% of the cultured stem segments. Ten days after the first callus subculture on the same medium, green structures appeared spontaneously and subsequently developed into shoots. After 30 days, the cultures produced an average of 15 shoots per callus piece, with a mean shoot length of approximately 3 cm and a 90% differentiation rate based on 60 recultured callus pieces. Excised shoots cultured on growth-regulator-free MS medium rooted within 8–10 days, with a 100% rooting rate. The regenerated plantlets were acclimatised in a sterilised soil and peat mixture and subsequently transferred to greenhouse conditions. Under the conditions tested, subcotyledonous stem callus supported spontaneous shoot regeneration, rooting and plant establishment in soil.