Effective decontamination and multiplication of Croton membranaceus in vitro

Abstract. Adukonu IA, Ayeh KO, Acheampong E. 2022. Effective decontamination and multiplication of Croton membranaceus in vitro. Cell Biol Dev 6: 68-81. Croton membranaceus Müll. Arg. is a useful herb with medicinal properties, and its leaves, roots, and bark are used to treat diverse ailments. However, the harvesting method by traditional medical practitioners without replacement exposes it to extinction. Therefore, the only means of propagating is by relatively slow use of seeds, and an alternative propagation method is needed for field establishment and nursery. Therefore, this study aims at determining an effective propagation by sterilization regime and subsequent in vitro regeneration using different explants; intact seeds, isolated embryos, coatless seeds, and nodal-cutting. The explants were decontaminated using double sterilization; the best was achieved by pre-treatment with 70% ethanol for 3 min before immersion in sodium hypochlorite (NaOCl). Intact seeds were effectively decontaminated by immersion in 15% NaOCl solution for 20 min, then by 10% NaOCl for 15 min. Conversely, coatless seeds were effectively decontaminated when isolated from intact seeds immersed in 20% NaOCl solution for 20 min and 15% NaOCl for 15 min. Further, embryos isolated from intact seeds were effectively decontaminated in 20% NaOCl for 15 min, then by 15% NaOCl for 10 min sequentially. These sterilization regimes successfully decontaminated 86% intact seeds, 100% isolated embryos, and 80% coatless seeds. Nodal-cutting explants were best decontaminated by immersion in 20% NaOCl solution for 15 min, followed by 15% NaOCl for 10 min sequentially without ethanol pre-treatment. This sterilization regime successfully decontaminated 100% of the nodal-cutting explants. However, the development of shoot explants varied in response to sterilization. Intact seeds did not develop into shoots, while isolated embryos, coatless seeds, and nodal-cutting explants developed into shoots independent of the sterilization regime. Shoot development was highest with the medium’s shoot-tip explants added with BAP, NAA, and GA3. Shoot multiplication was best achieved on an MS basal medium with 5.0 ?M BAP, 0.5 ?M NAA, and 5.0 ?M GA3 amendment.