Effective Decontamination And Multiplication Of Croton Membranaceus Müll. Arg. In Vitro

ABSTRACT Croton membranaceus is a herb with useful medicinal properties. The leaves, bark and roots are used for the treatment of diverse ailments. The harvesting of the plant by many traditional medical practioners from the wild for the treatment of diseases in the country without replacement exposes the plant to possible extinction in the near future. The only means of propagating the plant is by use of seeds which is relatively slow. Thus, an alternative mode of propagation is needed to develop planting materials for nursery and field establishment. Therefore this study aims at determining an effective sterilization regime and subsequent in vitro regeneration using different explants. Intact seeds, coatless seeds, isolated embryos and nodal cutting explants were used to initiate cultures for multiplication of C. membranaceus. The explants were decontaminated using double sterilization. Generally, the best sterilization for seed explants was achieved by pre-treatment with 70% ethanol for 3 minutes prior to immersion in sodium hypochlorite (NaOCl). However, intact seeds were effectively decontaminated by immersion in 15% NaOCl solution for 20 minutes followed by 10% NaOCl solution for 15 minutes, whilst coatless seeds were effectively decontaminated when isolated from intact seeds immersed in 20% NaOCl solution for 20 minutes followed by 15% NaOCl solution for 15 minutes. Further, embryos isolated from intact seeds were effectively decontaminated in 20% NaOCl solution for 15 minutes followed by 15% NaOCl for 10 minutes sequentially. With these sterilization regimes, 86% intact seeds, 80% coatless seeds and 100% isolated embryos were successfully decontaminated. Nodal cutting explants were best decontaminated by immersion in 20% NaOCl solution for 15 minutes followed by 15% NaOCl for 10 minutes sequentially without ethanol pre-treatment. With this sterilization regime, 100% of the nodal cutting explants were successfully xi decontaminated. The development of shoots from explants in response to sterilization varied. Intact seeds did not develop into shoots while coatless seeds, isolated embryos and nodal cutting explants developed into shoots independent of sterilization regime. Shoot development was highest with shoot tip explants and when BAP, NAA and GA3 were added to the medium. Shoot multiplication was best achieved on an MS basal medium amended with 5.0μM BAP, 0.5μM NAA and 5.0μM GA3.