In Vitro Regeneration via Somatic Embryogenesis of Schizozygia Coffeoides Baill (Mpelepele)

摘要

Schizozygia coffeoides (Mpelepele) is an endangered medicinal shrub with substantial antifungal and antibacterial properties. The sustainable utilization of this shrub is hindered by its limited regeneration potential since there exists no protocol for In vitro regeneration of not only this plant but the entire Apocynaceae family. The aim of this study was to develop an appropriate sterilization and regeneration protocol for In vitro regeneration of S. coffeoides using leaf discs, with the specific aims of determining the best NaOCL concentrations for explant sterilization as well as the appropriate plant growth hormone combinations for development of embryogenic calli, shoot development and rooting. Sterilization was optimized using NaOCl at the concentrations of 5.2mM, 7.8mM, 10.4mM and 13mM at exposure times of 10, 15, 20 and 25 minutes. The different concentrations of NaOCl had a significant effect on the survival of explants to sterilization (P=<0.0001), with the lower concentrations (5.2mM and 7.8mM) showing high levels of explant contamination and high concentration (13mM) leading to explant scotching. Similarly, the time explants were subjected to the sterilization substances was also found to significantly affect their ability to survive the sterilization process (P=0.0027). The best time and NaOCl concentration interaction for the sterilization of leaf explants was found out to be 10.4mM for 20 minutes. This time-NaOCl concentration interaction was found out to significantly produce a higher number of clean surviving explants than all other interactions (P=<0.0001). The best callus formation frequency of 68% was observed in MS media supplemented with 2.0mg/lBAP+0.8mg/lKin+0.4mg/l NAA+0.5mg/lTDZ, compared to a 39% callus formation frequency observed in media supplemented with 1.5mg/lBAP+0.8mg/lKin+0.4mg/lNAA+0.5mg/lTDZ and a 13% callus formation frequency observed in media supplemented with 1mg/lBAP+0.4mg/lKin+0.2mg/lNAA+0.05mg/lTDZ. Although somatic embryos formed in all media types, only media supplemented with 2.0mg/lBAP+0.8mg/lKin+0.4mg/lNAA+0.5mg/lTDZ formed most somatic embryos which survived to maturity and formed shoots, as most calli and embryos forming in other PGR supplementations died with subsequent subcultures. Roots only formed in shoots cultured in media supplemented with 1.0 BAP+0.5 IBA. The successful development of this regeneration protocol is expected to greatly contribute to mass production and conservation of this important shrub.