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

Abstract

Optimizing of the in vitro culture condition, somatic embryogenesis, and plant regeneration is the first step in transformation. In this study, the 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 proved that TDZ is more efficient than NAA for the regeneration of H. niger. By using different concentrations of TDZ, BAP, and kinetin, the highest percentage of callusing (100.00%), callus fresh weight (1207.75 mg/explant), percentage of somatic embryogenesis (92.66%) and shooting (93.30%), and the number of the shoot (7.75 per callus) was recorded from petiole explants on MS medium containing 0.25 mg/L TDZ and 1 mg/L BAP, leaf explants by using 0.5 mg/L TDZ and 1 mg/L kinetin, petiole explants with addition 0.5 mg/L TDZ, leaf explants by using 0.50 mg/L TDZ and 1 mg/L kinetin, and petiole explants with addition 0.25 mg/L TDZ and 1 mg/L BAP, respectively. Also, the highest percentage of callusing (100.00%), callus fresh weight (3273.64 mg/explant), percentage of somatic embryogenesis (100%), shooting (100.00%), and the number of the shoot (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.