Plant regeneration of Ferula assa-foetida (asafoetida) via root-derived callus from seed- embryo plantlets

Ferula assa-foetida L. (asafoetida), a monocarpic and endangered medicinal-industrial plant native to the cold steppes of Iran and Afghanistan, faces extinction due to overexploitation for its oleo-gum-resin and complex seed dormancy. To develop an efficient micropropagation protocol using root-derived callus from seed-embryo plantlets and to determine optimal plant growth regulator concentrations for callus induction and plant regeneration. Mature embryos excised from surface-sterilized seeds were cultured on half-strength MS medium without hormones. Root segments (1 cm) from 21-day-old plantlets were cultured on half-strength MS medium supplemented with BAP (0, 0.5, 1.0, 1.5 mg/L) and TDZ (0, 0.5, 1.0, 1.5 mg/L). Callus growth parameters (length, width, height, fresh weight) were measured after four weeks. Analyzing the data, embryo culture achieved > 95% germination within 5–7 days. Callus formation from root explants occurred only in four hormone combinations. Treatment T1 (0.5 mg/L BAP + 0.5 mg/L TDZ) produced the highest callus length (4.73 cm) and fresh weight (5.94 g), significantly outperforming all other treatments (p < 0.01). The results revealed that TDZ had a stronger effect on callus growth than BAP. Only T1-derived calli regenerated whole plantlets. Regenerated plantlets exhibited severe vitrification on ½ MS medium but recovered completely after transfer to ½ QL medium (lower nitrogen content), followed by successful rooting and > 85% survival under greenhouse conditions. Using only 1 cm of root, approximately 30 healthy plantlets were produced. The optimized protocol provides an efficient, reproducible method for large-scale asexual propagation, conservation, and sustainable cultivation of this endangered medicinal species.