Antioxidant enzymes metabolism and cellular differentiation during the developmental stages of somatic embryogenesis in Torilis japonica (Houtt.) DC

Abstract

In the present study, an efficient protocol for high frequency somatic embryogenesis from stem explants of Torilis japonica was established. Explants were cultured in Murashige and Skoog (MS) medium containing 0-2.0 mg·L -1 2,4- dichlorophenoxy acetic acid (2,4-D) with or without the combination of indole-3-butyric acid (IBA). The embryogenic callus induction and somatic embryogenesis frequency were measured at four week intervals. The highest embryogenic callus induction (100±0.0%) was achieved in 2.0 mg·L-1 2,4-D and 1.0 mg·L-1 IBA. Maximum somatic embryo induction (100±0.0%) was noted on the MS medium augmented with 2.0 mg·L-1 2, 4-D, 1.0 mg·L-1 IBA, and 1.0 mg·L-1 gibberellic acid (GA3 ) with an average of 75.8±3.7 somatic embryos per explant. Scanning electron microscopic investigation and histological analysis demonstrated the systemic development of somatic embryos. Comparatively , the embryogenic callus induction and plantlet conversion stages possessed more total protein and hydrogen peroxide content than other stages. As a consequence, the activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), and catalase (CAT) were also increased during the induction of somatic embryos and bipolar shoot-root axis differentiation stages. Clonal fidelity assessment by random amplified polymorphic DNA (RAPD) fingerprinting and inter simple sequence specific repeats (ISSR) markers displayed the monomorphic banding pattern across the micropropagated plantlets. Thus, the RAPD and ISSR markers validated the genetic homogeneity or the true-to-type nature of the in vitro plants.