Prediction of the concentration of plant growth regulators for somatic embryogenesis and regeneration of Hyoscyamus niger using Box–Behnken design of response surface methodology

Optimizing of the in vitro culture condition, somatic embryogenesis, and plant regeneration are the first step in transformation. In this study, response surface methodology was used to optimize and predict the concentrations of TDZ, NAA, BAP, and kinetin for callus formation, and regeneration by indirect somatic embryogenesis of Hyoscyamus niger . The results showed that TDZ was more efficient than NAA for the regeneration of H. niger . By using different concentrations of TDZ, BAP, and kinetin, the petiole explants produced the highest percentage of callusing (100%) on MS medium containing 0.25 mg/L TDZ and 1 mg/L BAP, percentage of somatic embryogenesis (92.66%) with addition 0.5 mg/L TDZ, and the number of the shoots (7.75 per callus) with addition 0.25 mg/L TDZ and 1 mg/L BAP. The leaf explants produced the highest callus fresh weight (1207.75 mg/explant) by using 0.5 mg/L TDZ and 1 mg/L kinetin, and percentage of shooting (93.30%) by using 0.50 mg/L TDZ and 1 mg/L kinetin. Also, the highest percentage of callusing (100%), callus fresh weight (3273.64 mg/explant), percentage of somatic embryogenesis (100%), shooting (100%), and the number of the shoots (8.61 per callus) were predicted by culturing of petiole explant on the MS medium containing 0.11 mg/L TDZ and 1.253 mg/L BAP, leaf explant by using 0.52 mg/L TDZ, petiole explant by using 0.533 mg/L TDZ and 1 mg/L BAP, leaf explant with addition 0.18 mg/L TDZ and 2.89 mg/L BAP and petiole explant by using 0.1 mg/L TDZ and 3 mg/L BAP.