Browsing by Author "Apio, Hellen B."
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Item Efficient conditions for in vitro establishment and regeneration of disease-free Ugandan farmer-preferred cassava genotypes(African Journal of Biotechnology, 2021) Apio, Hellen B.; Alicai, Titus; Ogwok, EmmanuelCassava (Manihot esculenta Crantz) is majorly devastated by two viral diseases, cassava brown streak disease (CBSD) and cassava mosaic disease (CMD), resulting in 100% yield loss. Being a clonal plant, nodal cuttings (NC) and shoot apical meristems (SAMs) are the best explants for production of disease free planting materials. In this study, NCs and SAMs were used to determine reliable indicators for successful in vitro establishment of cassava. Eight cassava genotypes were used for the study. Leaf samples were collected from 30 stakes of each of the eight genotypes planted in the screen house. The leaf samples were pooled and screened for presence and/or absence of CBSD and CMD by PCR using virus specific primers. Nodal cuttings were excised from screen house grown plants, surface sterilized to rid-off contaminants and established on Murashige and Skoog (MS) Medium. Using the sprouted stakes, 5-mm sized SAMs were excised, surface sterilized and reduced to 0.5-1 and 2-3 mm sizes. The SAMs were established on MS medium with varying concentrations of plant growth regulators (0.5, 1, 2) ml/L Benzylaminopurine (BAP) and (2, 4) ml/L Naphthalene acetic acid (NAA), Kinetin (K) and BAP respectively. PCR results revealed the pooled leaf samples were free of both CBSD and CMD for all genotypes. Establishment and regeneration of NCs was possible with MS medium for all genotypes. For the SAMs, the concentrations of (2, 4) ml/LBAP followed by 2 ml/LNAA facilitated their establishment and regeneration in comparison to KIN.SAMs of 2-3 mm sizes regenerated better than 0.5 - 1 mm size. Both NCs and SAMs of the different genotypes produced leaves, nodes, roots and there was an increase in plant length. These parameters are critical indicators for in vitro establishment and regeneration of cassava.Item Production of friable embryogenic callus and regeneration of Ugandan farmer-preferred cassava genotypes(African Journal of Biotechnology, 2015) Apio, Hellen B.; Alicai, Titus; Baguma, Yona; Mukasa, Settumba B.; Bua, Anton; Taylor, NigelGeneration of embryogenic callus is a key step in genetic engineering of many crop species, including cassava. Protocols for generation of friable embryogenic callus (FEC) have been lacking for Ugandan cassava genotypes, thereby delaying their genetic engineering for agronomic and other desirable traits. The objective of this study was to determine conditions suitable for production and regeneration of FEC in the Ugandan cassava genotypes; Aladu, Bukalasa and Ebwanateraka, and control cultivar 60444. Immature leaf lobe explants were established on Murashige and Skoog (MS) based media for initiation of organized embryogenic callus (OES). To produce FEC, resulting OES were established on Gresshoff and Doy based callus induction media with varying levels of sucrose, maltose, tyrosine, tryptophan, naphthalene acetic acid (NAA) under light and dark conditions. Subsequently, FEC was subcultured to MS-based embryo maturation and embryo regeneration media. All genotypes produced OES. All genotypes produced FEC except Bukalasa. The amino acid tyrosine favoured production of FEC in Aladu and Ebwanatereka, but not in 60444, while 20 g/L of sucrose trigged production of FEC in Aladu and 60444, but 40 g/L of sucrose was superior for Ebwanatereka. Media supplemented with 1 ml/L naphthalene acetic acid NAA facilitated embryo regeneration in Ebwanatereka and 60444, while Aladu responded better to 5 ml/L NAA. Light, tyrosine and sucrose were essential for FEC production in Uganda cultivars while NAA was required for regeneration of somatic embryos. Ability to produce FEC in these genotypes lays a foundation for their improvement through genetic transformation for the desired and agronomic traits.Item Tree Lab: Portable Genomics for Early Detection of Plant Viruses and Pests in Sub-Saharan Africa(Genes, 2019) Boykin, Laura M.; Sseruwagi, Peter; Alicai, Titus; Ateka, Elijah; Umar Mohammed, Ibrahim; Stanton, Jo-Ann L.; Kayuki, Charles; Mark, Deogratius; Fute, Tarcisius; Erasto, Joel; Bachwenkizi, Hilda; Muga, Brenda; Mumo, Naomi; Mwangi, Jenniffer; Abidrabo, Phillip; Okao-Okuja, Geofrey; Omuut, Geresemu; Akol, Jacinta; Apio, Hellen B.; Osingada, Francis; Kehoe, Monica A.; Eccles, David; Savill, Anders; Lamb, Stephen; Kinene, Tonny; Rawle, Christopher B.; Muralidhar, Abishek; Mayall, Kirsty; Tairo, Fred; Ndunguru, JosephIn this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an e ective point-of-need field diagnostic system. The PDQeX extractsDNAusing a cocktail of thermophilic proteinases and cell wall-degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This closed system delivers purified DNA with no cross-contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into nucleotide base called data locally in real-time, removing the need for high-specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and setup. To evaluate and validate capability of the system for unbiased pathogen identification by real-time sequencing in a farmer’s field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or destroy cassava crops. Eight hundred (800) million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint to its production. Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus-resistant or replacement planting. This case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, Internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making e ective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis, including sample collection, processing and provisional sequencing results was complete in under 3 h. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation.