Production of the first transgenic cassava in Africa via direct shoot organogenesis from friable embryogenic calli and germination of maturing somatic embryos

The impact of cassava transformation technologies for agricultural development in Africa will depend largely on how successfully these capabilities are transferred and adapted to the African environmentand local needs. Here we report on the first successful establishment of cassava regeneration and transformation capacity in Africa via organogenesis, somatic embryogenesis and friable embryogeniccallus (FEC). As a prerequisite for genetic engineering, we evaluated six African cassava genotypes for the ability of a) induction of FEC b) hygromycin sensitivity and c) T-DNA integration potential bydifferent Agrobacterium strains. FEC was induced in genotypes TMS 60444, TME 1 and TMS 91/02327. Potential tissues for FEC formation were induced in TMS 91/02324, TME 12 and TME 13. Pure andproliferating FEC was obtained and maintained only in TMS 60444. FEC growth and shoot organogenesis were completely suppressed when hygromycin was used at a concentration of 20 mg/l in all tissue types and genotypes. With somatic cotyledons, statistically significant differences (p0.05) were observed between Agrobacterium strains and genotypes with respect to T-DNA transfer efficiency.Using somatic cotyledons, TME 8 was found to be the most amenable to transformation with maximum blue spots per GUS-positive explants, and Agrobacterium GV3101 proved to be superior to EHA105,LBA4404, and AGl-1 for T-DNA transfer based on transient assays with a reporter gene (GUS). With FEC, Agrobacterium LBA4404 was superior to other strains. This study also identified EHA105 as a newvir helper strain to recover transgenic cassava plants. PCR and Southern hybridization of genomic DNA of the hygromycin-resistant cassava plants to a hpt probe confirmed the integration of hpt withintegration events varying between 1 and 2 insertions. The benefit of combining the FEC and shoot organogenesis systems for recovering transgenic cassava plants is described. The contributions ofthis report to enhancing the development and deployment of genetic engineering of cassava for agricultural biotechnology development in Africa are discussed

CALLUS INDUCTION AND MORPHOGENESIS IN DIOSCOREA DUMETORUM FOR STEROID PRODUCTION

Diosgenin is characteristic of the genus Dioscorea (Dioscoreaceae), which is used in partial synthesis of steroid drugs. It was not detected in the crude sapogenin extract of Dioscorea dumetorum and in the calluses. Callus was induced from the meristem of the two genotypes and maintained successfully on Murashige and Skoog's medium (MS) supplemented with 0.1mg/l of 2, 4-dichlorophenoxyacetic acid (2,4-D). Shoots induced from nodal cuttings of the white and yellow genotypes regenerated plantlets, which were subsequently maintained on MS medium, and Linsmaier and Skoog's medium (LS) supplemented with 0.5mg/l kinetin. Control of microbial contamination was achieved with tetracycline. Microtubers were also obtained from the yellow genotype plantlet growing on the MS medium. Steroids, especially the Δ5, 3-hydroxy steroids were detected only in the yellow genotype callus. Keywords: Dioscorea dumetorum; diosgenin; 3-hydroxy steroids; callus; shoot culture; meristem culture; yellow genotype Nigerian Journal of Natural Products and Medicine Vol.7 2003: 5-