Browsing by Author "Runo, Steven"
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Item Application and Evaluation of the Loop-Mediated Isothermal Amplification assay for the Detection of Aflatoxigenic Fungi Contaminants of Rice Grains in Kenya(Longdom Publishing, 2020) Douksouna, Youmma; Kwallah, Allan Ole; Nyerere, Andrew; Runo, Steven; Ambang, ZachéeAflatoxigenic fungi are most common filamentous fungi that synthesis aflatoxins and represent the major fungal pathogens to agricultural products. Aflatoxins remain a major threat to global food security, these molecules could be resisted into food during processing and in additional may remain within the food chain. Aflatoxins are carcinogenic, hepatotoxic, mutagenic, teratogenic, can inhibit numerous metabolic systems and immunosuppressive properties. Studies of aflatoxigenic strains can help to enhance strategies control and prevent aflatoxigenic fungi contamination and aflatoxins production in foodstuffs. In this study, isolation of Aspergillus species was based on morphological characteristics including the mycelium growth pattern, color, and properties of fruiting bodies of the fungi. The innovated technique loop-mediated isothermal amplification assay was applied to amplify Norsolorinic Acid gene. The loop-mediated isothermal amplification have been optimized by combination of the rapidity, simplicity and specificity to detect the target genomic DNA in the reactions. The amplification curves monitored by Loopamp realtime Turbidi meter were analyzed in order to distinguish aflatoxigenic and non-aflatoxigenic strains. Overall, the results showed that the loop-mediated isothermal amplification method was effective in detecting aflatoxigenic strains with high specificity of 71.5 % and sensitivity under lower concentrations of DNA. In additional, it was faster than the conventional polymerase chain reaction. The loop-mediated isothermal amplification assay described in this study might be a promising tool for prediction potential threats by aflatoxigenic fungi and aflatoxins risk in food and commoditiesItem Effects of Auxin and Source of Explants on Callus Induction of Tropical Maize(Biotechnology, 2012) Runo, Steven; Omer, Rasha Adam; Matheka, J. M.; Abdelbagi M. Ali, Abdelbagi M.; Kuria, Eric; Masiga, Clet; Mugoya, Charles; Machuka, JesseInduction of callus from explants is a critical process in regeneration, micropropagation and transformation of plants. Formation of callus from plant tissues on culture is affected by different factors. This study sought to establish the effect of genotype, source of explants and auxin concentration on callus induction from different Sudanese maize genotypes (222F, Hudiba-1, 441, Giza-2, PR5655 and Mojtamma-45). Callus induction of the six maize varieties was investigated using mature embryos, leaf disks and shoot tips as explants and different concentrations of the auxin; 2,4-dichlorophenoxyacetic acid (2,4-D), ranging from 0 to 10 mg L-1. The highest callus, induction frequency was observed in shoot tips while the lowest was observed in mature embryos. Leaf disks gave a higher callus induction frequency than mature embryos and lower than shoot tips. Concentrations of 2,4-D of 2 mg L-1 gave the highest callus induction for most genotypes while 0 and 10 mg L-1 gave the lowest callus induction for all the genotypes.Item Engineering host derived resistance against plant parasites through RNA interference - Challenges and opportunities(Bioengineered Bugs., 2011) Runo, StevenRNA interference (RNAi) has rapidly advanced to become a powerful genetic tool and holds promise to revolutionizing agriculture by providing a strategy for controlling a wide array of crop pests. Numerous studies document RNAi efficacy in achieving silencing in viruses, insects, nematodes and weeds parasitizing crops. In general, host derived pest resistance through RNAi is achieved by genetically transforming host plants with double stranded RNA constructs targeted at essential parasite genes leading to generation of small interfering RNAs (siRNAs). Small interfering RNAs formed in the host are then delivered to the parasite and transported to target cells. Delivery can be oral - worms and insects, viral infections, viruses - or through a vascular connections - parasitic plants, while delivery to target cells is by cell to cell systemic movement of the silencing signal. Despite the overall optimism in generating pest resistant crops through RNAi-mediated silencing, some hurdles have recently begun to emerge. Presently, the main challenge is delivery of sufficient siRNAs, in the right cells, and at the right time to mount; a strong, durable, and broad-spectrum posttranscriptional gene silencing (PTGS) signal. This review highlights the novel strategies available for improving host derived RNAi resistance in downstream applied agriculture.Item Enhanced Utilization of Biotechnology Research and Development Innovations in Eastern and Central Africa for Agro-ecological Intensification(Springer International Publishing, 2014) Matheka, J. M.; Masiga, C.W.; Mugoya, C.; Ali, R.; Mohamed, A.; Osama, S.; Ngugi, A.; Kiambi, D.; De Villiers, S.; Ngugi, K.; Niyibigira, T.; Tesfamichel, A.; Machuka, J.; Oduor, R.; Runo, Steven; Adam, R.; Bedada, L.; Seth, M.; Kuria, E.; Ndirigwe, J.; Ndolo, P.; Muthamia, Z.; Nasona, B.; Ntimpirangeza, M.The Association for Strengthening Agricultural Research in Eastern and Central Africa (ASARECA) through its Agrobiodiversity and Biotechnology Programme is enhancing the utilization of biotechnology research and development innovations in Eastern and Central Africa (ECA). We present successes in the application of biotechnology to enhance the productivity of cassava, sweet potato, banana, maize and sorghum in ECA. These products—drought tolerant maize, sorghum resistant to striga, as well as the technology for producing and distributing disease free planting materials of cassava, sweet potato and banana to farmers—are central for the agro-ecological intensification of farming systems in the central African highlandsItem Enhancement of Drought Tolerance in Tropical Maize Through Silencing of Poly (ADP-Ribose) Polymerase-1 Gene(2014-08-19) Matheka, Jonathan Mutie; Machuka, J.; Runo, Steven; Gethi, J.; Anami, S.In most of the production areas under maize, mild drought can significantly reduce yields while severe drought can sometimes completely destroy an entire plantation. Consequently, the livelihood of farmers is affected due to reduction in their incomes leading to poverty. Currently, very few highly transformable maize genotypes such as B73, W22, A188 and CM216 are used in genomic studies and genetic engineering of traits such as drought tolerance. Following trait enhancement through genetic engineering, consumer uptake of the transgenic product may be hindered due to presence of undesirable genetic elements such as selectable marker genes (SMG). Therefore generation of transgenic plants free of SMG is important to make them biosafe. The objective of this study was to generate SMG-free maize plants having an artificial microRNA (amiRNA) targeting the poly (ADP-ribose) polymerase 1 (PARP1) gene and assess transgenic plants for tolerance to severe oxidative and drought stress. In earlier studies using arabidopsis and rapeseed, the PARP1 gene silencing approach was demonstrated to enhance drought tolerance through preservation of cellular energy and reduced damage by reactive oxygen species. In this study, maize PARP1 gene silencing constructs (amiRNA-PARP1) were cloned in the same T-DNA region as the pmi or the bar SMG or placed in a separate T-DNA region for cotransformation of plants. The cotransformation vectors were first validated in tobacco before use in transformation of different maize genotypes. Maize transformation was achieved by co-cultivation of immature embryos with Agrobacterium tumefaciens harboring the amiRNA-PARP1 construct. Transgenic plants were assessed for downregulation of the PARP1 gene using qRT-PCR. The effect of PARP1 gene downregulation on drought tolerance was also assessed. Out of 13 genotypes evaluated, two (TL03B6754A-20 and TL03B6757-68) were found to be highly regenerable and were chosen for recovery of transgenic plants using either the PMI/mannose or bar/PPT selection system. Using the PMI/mannose selection system, the two maize genotypes were found to be highly transformable, averaging transformation frequencies (TF) of 48% and 34.16%, respectively. The TF for the control genotype CML216 was 32.19%. The TF under PPT selection` for the inbred lines CML216, TL03B 6757-68 and TL03B 6754A-20 was 26.16%, 14.81%, and 21.69% for pMarkfree3.1 and 27.22%, 32.10% and 36.32% for pMarkfree3.2, respectively. A qRT- PCR analysis conducted on six amiRNA-PARP1 transgenic lines revealed that the expression of the PARP1 gene was reduced in plants exposed to methyl viologen-induced oxidative stress. However, the level of PARP1 gene expression was higher in non- transformed plants. Plants with reduced expression of the PARP1 gene were tolerant to oxidative and drought stress indicated by higher chlorophyll content, relative water content, growth and biomass as well as reduced anthocyanin accumulation, compared to non-transformed plants. In conclusion TL03B6754A-20 and TL03B6757-68 genotypes are highly responsive to transformation. Therefore these inbred lines extend the pool of highly transformable genotypes available for genomic and trait improvement studies. In addition the cotransformation binary vectors developed here (pMarkfree3.0, pMarkfree2.1 or pMarkfree2.2) are applicable in any plant to produce SMG-free plants containing one or more transgenes of interest. In the long term, the drought tolerant transgenic maize plants produced in this study can help stabilize maize yields and increase production during water stressed conditions. This will impact targeted farmers and their households by increasing their incomes thereby improving their livelihoods.Item Genetic structure and diversity of East African taro [Colocasia esculenta (L.) Schott](Academic Journals, 2014) Macharia, M. W.; Runo, Steven; Muchugi, A. N.; Palapala, V.Taro [Colocasia esculenta (L) Schott] is mainly produced in Africa by small holder farmers and plays an important role in the livelihood of millions of poor people in less developed countries. The genetic diversity of East African taro has not been determined. This study utilizes six microsatellite primers to analyze five populations of taro from three different regions of East Africa. Plant material consisted of 98 taro cultivars obtained from East Africa (Kenya, Tanzania and Uganda). Principal component analysis of microsatellite data indicated variation but did not show any distinct structure. Population diversity estimate was relatively low with the highest being 0.27, for accessions sourced from Lake Victoria basin. Analysis of molecular variance (AMOVA) revealed most variation among individuals within population at 79%. Nei’s genetic distance showed that relatedness is not based on geographical proximity. Based on these findings, this study proposes establishment of a regional collection that will be conserved and ensure a broad genetic base for available varieties and enable development of improved varieties through breeding programmes.Item Genetic variability of sorghum landraces from lower Eastern Kenya based on simple sequence repeats (SSRs) markers(Academic Journals, 2016) Muui, Catherine W.; Muasya, Reuben M.; Kirubi, Duncan T.; Runo, Steven; Karugu, ArthurThe aim of this study was to estimate the genetic variability of sorghum landraces grown in lower eastern Kenya based on simple sequence repeats (SSRs) markers. A total of 44 landraces obtained from the farmers and four improved varieties were analyzed using 20 SSR markers. All markers were polymorphic with polymorphism information content (PIC) value ranging from 0.04 to 0.81 with a mean of 0.49. Heterozygosity ranged from 0.00 to 0.04 suggesting that each detected a single genetic locus and that each of the sorghum accession used was stable. The alleles ranged between 2 and 10 and an average of 5.05 alleles per primer pair. The gene diversity ranged from 0.04 to 0.83 with a mean value of 0.53. All possible allele combinations ranged from 2 to 10, while major allele frequency ranged from 0.32 to 0.98. Genetic distances varied from 0.15 to 0.90 with two genotypes Karuge 1 and Karuge 2 obtained from Kiritiri in Mbeere having the minimum (0.15) and indication of very close relatedness. The diversity of the landraces was structured more on geographical locations and on seed colorations than agroecological conditions. Such intraregional genetic proximity in sorghum landraces would arise through seed exchanges among farmers. Analysis of molecular variation indicated higher variation within populations than among the groups. The genetic diversity can be exploited in hybridization programs to improve sorghum varieties grown by farmers in semi arid areas.Item Genomics of Sorghum Local Adaptation to A Parasitic Plant(PNAS, 2020-01) Bellisa, Emily S.; Kellya, Elizabeth A.; Lortsa, Claire M.; Gaoe, Huirong; DeLeoa, Victoria L.; Rouhanf, Germinal; Buddeng, Andrew; Bhaskarah, Govinal B.; Hui, Zhenbin; Muscarell, Robert; Timkok, Michael P.; Nebiel, Baloua; Chilcoat, N. Doane; Juengerh, Thomas E.; Morrisi, Geoffrey P.; dePamphilisa, Claude W.; Lasky, Jesse R.; Runo, StevenHost–parasite coevolution can maintain high levels of genetic diversity in traits involved in species interactions. In many systems, host traits exploited by parasites are constrained by use in other functions, leading to complex selective pressures across space and time. Here, we study genome-wide variation in the staple crop Sorghum bicolor (L.) Moench and its association with the parasitic weed Striga hermonthica (Delile) Benth., a major constraint to food security in Africa. We hypothesize that geographic selection mosaics across gradients of parasite occurrence maintain genetic diversity in sorghum landrace resistance. Suggesting a role in local adaptation to parasite pressure, multiple independent loss-of-function alleles at sorghum LOW GERMINATION STIMULANT 1 (LGS1) are broadly distributed among African landraces and geographically associated with S. hermonthica occurrence. However, low frequency of these alleles within S. hermonthica-prone regions and their absence elsewhere implicate potential trade-offs restricting their fixation. LGS1 is thought to cause resistance by changing stereochemistry of strigolactones, hormones that control plant architecture and below-ground signaling to mycorrhizae and are required to stimulate parasite germination. Consistent with tradeoffs, we find signatures of balancing selection surrounding LGS1 and other candidates from analysis of genome-wide associations with parasite distribution. Experiments with CRISPR–Cas9-edited sorghum further indicate that the benefit of LGS1-mediated resistance strongly depends on parasite genotype and abiotic environment and comes at the cost of reduced photosystem gene expression. Our study demonstrates long-term maintenance of diversity in host resistance genes across smallholder agroecosystems, providing a valuable comparison to both industrial farming systems and natural communities.Item Genotyping of Kenyan Passiflora Edulis Flavicarpa Hybrid Accessions and their Parents using SSR Markers(Society for Plant Research, 2016) Matheri, F.; Teya, F.; Kioko, F.; Mawia, A.M.; Mwangi, M.; Kirubi, D.T.; Ngugi, M.; Runo, StevenThe Passion fruit is a significant income earner for Kenya, ranking third after avocado and mango in terms of foreign exchange earnings. Increased research activities targeting variety improvement and development of new varieties have led to development of new passion fruit varieties by KALRO. These varieties are KPF-4, KPF-11 and KPF-12 which are hybrids of natural crosses of the coastal yellow varieties and purple variety. Despite these gains in breeding, there is little information on molecular variability of the hybrids as well as the parents. The present study aimed at evaluating the genetic variation of the hybrid and parent varieties using SSR markers. DNA was extracted using a modified CTAB protocol followed by PCR and analysis done using DARWIN 6 and GenAlex 6.502 software. The resulting dissimilarity matrix showed existing genetic variability among accessions within and among the varieties studied. Further, the results of principal component analysis and phylogenetic tree showed genetic homogeneity within the assigned populations. The findings of this study will supplement the existing body of knowledge in passion fruit breeding.Item Habits of a highly successful cereal killer, Striga(MDPI, 2018-01-11) Runo, Steven; Kuria, Eric K.Item How well do malaria tests correlate with disease severity? Comparison of parasite density in children with mild and severe malaria(2014) Waitumbi, John N.; Runo, Steven; Ngeranwa, J.J.N.; Nyakoe, Nancy; Kituyi, Sarah N.Background. Accurate diagnosis of malaria is key to proper management and control and an ideal diagnostic parameter that correlates to disease outcome is required. The former would be helpful in correctly identifying patients that need hospitalisation versus those that can be managed at home. This study determined how well the density estimates by microscopy, qPCR and PfHRP-2 correlate to malaria severity. Materials and Methods. Patients aged ≤ 5 yrs with severe (n = 60, Hb ≤ 6 g/dl) and mild (n = 60, Hb > 6 g/dl) malaria were enrolled to take part in a case control study at Kisumu District Hospital, Western Kenya. Parasite load was determined by microscopy, qPCR targeting the 18s rRNA gene and PfHRP-2 antigen ELISA. Results. The median parasite load and the 25th and the 75th percentile by microscopy in children with severe malaria (SM) was 49,958 parasites/μl (12,013-128,695) compared to 24,233 (6,122-103,886) in the group with mild malaria (MM), P = 0.10. By qPCR, the translated median parasite density was 31,550 parasites/μl (4,106-196,640) in the SM group compared to 24,365 parasites/μl (5,512-93,401) in the MM group (P = 0.73). According to PfHRP-2, the translated median parasite load in children with SM was 628,775 parasites/μl (332,222-1.165x106) compared to 150,453 (94,292-399,100) in children with MM (P < 0.0001). Conclusions. Unlike microscopy and qPCR, the parasite load detected by PfHRP-2 correlates with disease severity. Because of its unique attributes, PfHRP-2 is able to account for trophozoites and schizonts that are sequestered away from peripheral circulation. Because it persists in circulation, it also serves as an indicator of the magnitude of current and recent infections.Item Interspecific RNA interference of SHOOT MERISTEMLESS-like disrupts Cuscuta pentagona plant parasitism.(Plant Cell., 2012-07) Alakonya, Amos E.; Kumar, R; Koenig, D; Kimura, S; Townsley, B; Runo, Steven; Garces, HM; Kang, J; Yanez, A; David-Schwartz, R; Machuka, Jesse; Sinha, NInfection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA-mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.Item Lack of GFP Trafficking from Transgenic Nicotiana benthamiana to Parasitizing Cuscuta pentagona(IDOSI Publications, 2010) Alakonya, Amos E.; Runo, Steven; Dan, K; Monda, E. O.; Machuka, Jesse; Sinha, N.This work reports lack of GFP transport from transgenic N. bethamiana under the control of constitutive promoter CAMV 35S to Cuscuta pentagona. The data on N. bethamiana and C. pentagona host-parasite system suggests that macromolecular trafficking from host to parasite is host–parasite specific and such results should not be generalized outside the system studied. This data further suggests that molecular trafficking from host to parasite through either symplastic or apoplastic mechanisms is not fully understood. This is so for as especially the gating mechanisms involved. Macromolecular trafficking between host and parasite holds potential application in parasitic plant management and in basic research. It is therefore important to investigate why such contradictions exist in even very closely related host parasite systems like the genera Nicotiana and Cuscuta.Item Maize Bioengineering with C-Repeat Binding Factor 1 (CBF1) as a Technique for Desiccation Toleration(Society for Indonesian Biodiversity, 2019-06) Kuria, Eric Kimani; Machuka, Jesse; Runo, StevenKuria EK, Machuka J, Runo S. 2019. Maize bioengineering with c-repeat binding factor 1 (CBF1) as a technique for desiccation toleration. Trop Drylands 3: 1-10. Africa is a desiccation inclined continent leading to riskful cultivation particularly to small-scale cultivators who rely on rain-fed agriculture. Maize is the most widely cultivated main crop in Africa with more than 300 million people relying on it as their principal dietdiet fount. Desiccation causes crop fiasco, famine and poverty and this is being aggravated by climate change. There is therefore an obligation to flourish desiccation tolerablish maize. Traditional propagation techniques have been implementedcarried out in the establishment of desiccation tolerablish plants but are restricted by their requirement for labour, time and space, suggesting a limited genetic diversity within genotypes and transition of undesired traits along with the wanted ones. These restrictions are handled by utilizing these techniques along with bioengineering. Desiccation triggers a range of physiological and biochemical reactions in plants at cellular and molecular levels. These reactions include initiation of genes with several usefulness. Plant alteration for expanded desiccation toleration is generally based on the administration of either transcription and/or signaling factors or genes that directly secure plant cells contra water shortage. C-repeat binding factor (CBF) is a transcriptional factor that interacts with the desiccation responsive element (DRE), a cis-acting promoter element that governs gene expression in reaction to desiccation, brine and freezing stress. Over expression of these transcription factors, escalates stress toleration to freezing, desiccation and high brininess. In this study, three maize inbred lines and one hybrid were altered with CBF1 gene and appointed with mannose utilizing the Phosphomannose isomerase (PMI) gene. Genetic alteration was conducted through Agrobacterium tumefaciens and PCR was utilized to ascertain altered plants. Alteration frequency, alteration effectiveness and regeneration effectiveness were equated among the distinct genotypes altered. There were no remarkable dissimilarities in alteration frequency among the four maize genotypes. CML216 had the highest alteration effectiveness and regeneration effectiveness followed by A188. No alleged transgenic plants were regenerated from TL27 and A188×TL18 under the circumstances implemented on acount of their low regenerability. Further molecular analysis and desiccation stress tentatives on the expanded transgenic maize are significant prior to commercial release. Availability of desiccation tolerablish maize would bear a considerable positive collision contra famine particularly in Africa.Item Molecular Characterization and Genetic Variation of Root-knot Nematode (Meloidogyne spp.,) in Selected Legume Production Areas of Eastern Kenya(2016) Kavulukoa, Jacinta; Gichuki, Charity; Wanjohi, Waceke J.; Runo, StevenRoot-knot nematodes are sedentary endoparasites of plant roots and the primary nematode pathogens of most cultivated crops worldwide, including legumes. Root- Knot Nematodes of the genus Meloidogyne is the most economically important nematode pests affecting cowpea and pigeon pea in Eastern Kenya. This study sought to identify the Meloidoigyne species of root-knot nematodes on selected legumes in Mbeere district and characterize the genetic diversity of the species using small subunit (SSU) rDNA. PCR amplifications of the extracted purified DNA were carried out using primers specific for the intergenic spacer region between the 5S and 18S ribosomal DNA and the expected size of about 720bp was obtained. Purified PCR products were then sequenced and thirteen 5S-18S rDNA sequences obtained. The sequences were aligned using CLUSTALW2, Sequence statistics, pairwise differences, and estimates of divergence were determined with MEGA5. Nucleotide diversities were estimated in DnaSPv5. Phylogenetic tree was drawn using Phylowin and edited in MEGA5. From the findings of the study it has been established that root knot nematodes affecting the cowpea and pigeon pea in Mbeere district are M. javanica, M. incognita and M. arenaria. Judging from the extent of differences in base composition biases between sequences, it was concluded that the sequences under study have not evolved with the same pattern of substitution. Sequences from the species under study were closely related to sequences retrieved from sequences databases especially those sequences which were less divergent due to less substitutions, deletions and insertions. It can be concluded that SSUrDNA are useful in identification, inferring genetic diversity and phylogenetic relationships between the isolated root knot nematodes. There is need for a rapid and reliable method to identify populations of root-knot nematodes in order to design effective control programs. Keywords: Phylogenetic relationships between the Meloidogyne species; intergenic region between 18S and 5S genes; SSU rDNA analysis; Mbeere.Item Morphological Characterisation of Selected Ugandan Sweet potato (Ipomoea batatas L) Varieties for Food and Feed(OMICS International, 2016) Muinde, Mbithe J.; Runo, Steven; Agili, Sammy; Musembi, Kivuva B.; Wambua, Kioko F.; Kuria, EricSweet potato Ipomoea batatas L. (Lam.) is a symbol in the fight for a global nutrition plan that can save millions of children and help build a healthier and more productive future. However, characterisation of sweet potato varieties with optimal morphological features suitable for both food and feed has not been done. A population of 10,000 first filial generations (F1) sweet potato lines derived from seeds was generated through polycross crossing design in Uganda using 11 parents. Preliminary evaluation for the suitability of dual use of the F1’s led to selection of 11 varieties which were the basis of this study. This study therefore sought to morphologically characterise selected Ugandan sweet potato varieties to identify those with superior characteristics suitable for food and feed purposes. Sweet potato plants were raised from seeds after scarification. A selection of seedlings possessing single leaf lobes was done, after which they entered observation yield trials (OT). This was done in order to discard those that clearly did not meet the lowest acceptable gross morphological descriptors. The data were subjected to analysis of variance in order to find out if significant differences exist between the varieties based on morphological characterization. Cluster analysis was done using Minitab version 17 software. This study enables the selection of sweet potato varieties with optimal characteristics for both food and feed use. The data generated from this study could help recommend to farmers on how dual-purpose sweet potato could be produced, in order to provide enough healthier food to millions of children in Uganda and in the world, and better feed for live-stock farmersItem A new double right border binary vector for producing marker-free transgenic plants.(BMC Research Notes, 2013-11-08) Runo, Steven; Matheka, J. M.; Anami, Sylvester; Gethi, James; Omer, Rasha A; Alakonya, Amos E.; Machuka, JesseOnce a transgenic plant is developed, the selectable marker gene (SMG) becomes unnecessary in the plant. In fact, the continued presence of the SMG in the transgenic plant may cause unexpected pleiotropic effects as well as environmental or biosafety issues. Several methods for removal of SMGs that have been reported remain inaccessible due to protection by patents, while development of new ones is expensive and cost prohibitive. Here, we describe the development of a new vector for producing marker-free plants by simply adapting an ordinary binary vector to the double right border (DRB) vector design using conventional cloning procedures. We developed the DRB vector pMarkfree5.0 by placing the bar gene (representing genes of interest) between two copies of T-DNA right border sequences. The beta-glucuronidase (gus) and nptII genes (representing the selectable marker gene) were cloned next followed by one copy of the left border sequence. When tested in a model species (tobacco), this vector system enabled the generation of 55.6% kanamycin-resistant plants by Agrobacterium-mediated transformation. The frequency of cotransformation of the nptII and bar transgenes using the vector was 66.7%. Using the leaf bleach and Basta assays, we confirmed that the nptII and bar transgenes were coexpressed and segregated independently in the transgenic plants. This enable separation of the transgenes in plants cotransformed using pMarkfree5.0. The results suggest that the DRB system developed here is a practical and effective approach for separation of gene(s) of interest from a SMG and production of SMG-free plants. Therefore this system could be instrumental in production of "clean" plants containing genes of agronomic importance.Item Novel Sources of Witchweed (Striga) Resistance from Wild Sorghum Accessions(Frontiers Media, 2017) Mbuvi, Dorothy A.; Masiga, Clet W.; Kuria, Eric; Masanga, Joel; Wamalwa, Mark; Mohamed, Abdallah; Odeny, Damaris A.; Hamza, Nada; Timko, Michael P.; Runo, StevenSorghum is a major food staple in sub-Saharan Africa (SSA), but its production is constrained by the parasitic plant Striga that attaches to the roots of many cereals crops and causes severe stunting and loss of yield. Away from cultivated farmland, wild sorghum accessions grow as weedy plants and have shown remarkable immunity to Striga. We sought to determine the extent of the resistance to Striga in wild sorghum plants. Our screening strategy involved controlled laboratory assays of rhizotrons, where we artificially infected sorghum with Striga, as well as field experiments at three sites, where we grew sorghum with a natural Striga infestation. We tested the resistance response of seven accessions of wild sorghum of the aethiopicum, drummondii, and arundinaceum races against N13, which is a cultivated Striga resistant landrace. The susceptible control was farmer-preferred variety, Ochuti. From the laboratory experiments, we found three wild sorghum accessions (WSA-1, WSE-1, and WSA-2) that had significantly higher resistance than N13. These accessions had the lowest Striga biomass and the fewest and smallest Striga attached to them. Further microscopic and histological analysis of attached Striga haustorium showed that wild sorghum accessions hindered the ingression of Striga haustorium into the host endodermis. In one of the resistant accessions (WSE-1), host and parasite interaction led to the accumulation of large amounts of secondary metabolites that formed a dark coloration at the interphase. Field experiments confirmed the laboratory screening experiments in that these same accessions were found to have resistance against Striga. In the field, wild sorghum had low Area under the Striga Number Progressive curve (AUSNPC), which measures emergence of Striga from a host over time. We concluded that wild sorghum accessions are an important reservoir for Striga resistance that could be used to expand the genetic basis of cultivated sorghum for resistance to the parasite.Item Optimization of Genetic Transformation Protocol for Selected Banana and Plantain (Musa spp.) Cultivars Preferred in Africa(2013-12-14) Wanja, K. E.; Runo, Steven; Oduor, R.; Tripathi, L.Bananas and plantains (Musa spp.) are important staple food for rural and urban consumers and provide a source of income for resource poor farmers in the humid tropics of sub-Saharan Africa. However, banana production is severely limited by several pests and diseases, such as banana Xanthomonas wilt (BXW), Banana bunchy top virus, Banana streak virus, black leaf streak, Fusarium wilt, weevils and nematodes. The application of conventional breeding for both disease and pest resistance has resulted only in limited success due to the long generation times and the high sterility and triploidy of most cultivated bananas and plantains. Genetic transformation offers an alternative and viable means for introduction of agronomically important traits into these cultivars. However, to be successful, these applications require a rapid and efficient plant regeneration and transformation protocols for both banana and plantain. Currently, most transformation protocols for banana use cell suspensions. However, establishing cell suspensions is a lengthy process, highly cultivar-dependent and most farmer-preferred banana and plantain cultivars are recalcitrant to generation of embryogenic cell suspensions. Thus optimization of cultivar-independent transformation protocol using meristematic tissues becomes a prerequisite for agronomic improvement of bananas and plantains. The objective of this study will be to optimize a genetic transformation protocol of banana and plantain cultivars using mcristematic tissues and also develop transgenic plants resistant against BXW. Multiple bud clumps (MBCs) and intercalary meristematic tissues of 10 cultivars (Grande naine, Gross Michel, Gonja Manjaya, Nusu Ngombe, Ngombe, Mpologoma, Uganda green, Kayinja, Zebrina and Calcutta 4) will be co-cultivated with Agrobacterium strain EHA 105 harboring a binary vector pCAMBIA23 0 1 or modified pCAMBIA2300-GFP, followed by selection and regeneration of kanamycin-resistant plantlets. The effect of different parameters including acetosyringone concentration, length of infection time, sonication and vacuum infiltration on transformation efficiency will be determined. Transgenic plants will be subcultured for several cycles under selection to di Ill!e chimeras and progenies will be tested for presence of transgene. Histochemical GUS and GFP assays at different stages of transformation will be used to test the uniformity of transformed plants. The presence and integration of the nplIl and gusA genes in the progenies will be confirmed by PCR and Southern blot analysis, respectively. The optimized protocol will be used to transform' cultivars Mpologoma and Kayinja with hypersensitive response assisting protein (Hrap) gene. Hrap gene has been shown to intensify the harpin-., mediated hypersensitive response and consequently conferring resistance to a wide range of pathogens in plants. The trans genes have been reported to enhance the hypersensitive response induced by virulent pathogens that act through the release of proteinceous elicitor harpinj; in tobacco, Arabidopsis and banana .~'1BCs and meristematic tissues will be used for the transformation followed by selection, regeneration and evaluation of the resultant transgenic lines Lor resistance against nxw. This study will augment the ongoing genetic improvement of bananas and plantains and contribute to the food security of communities living in Africa.Item Parental Genome Contribution in Maize DH Lines Derived from Six Backcross Populations using Genotyping by Sequencing(Springer Verlag, 2015) Ogugo, V.; Semagn, K.; Beyene, Y.; Runo, Steven; Olsen, M.; Warburton, M. L.Molecular characterization of doubled haploid (DH) maize lines and estimation of parental genome contribution (PGC) may be useful for choosing pairs of DH lines for hybrid make up and new pedigree starts. Six BC1-derived DH populations created by crossing two donor with three recurrent parents were genotyped with 97,190 polymorphic markers with the objectives of: (i) understanding genetic purity, genetic distance and relationship among 417 maize DH lines; (ii) estimating PGC of the DH lines derived from different genetic backgrounds; and (iii) understanding the correlation between donor parent introgression and testcross performance for grain yield and anthesis-silking interval (ASI) under managed drought and optimum environments. The DH lines were 97 % genetically pure, with\2 % heterogeneity; only two DHlines showed heterogeneity[5 %,which is likely to be due to errors during seed multiplication or maintenance. Genetic distance between pairwise comparisons of the 417 DH lines ranged from 0.055 to 0.457; only 0.01 % showed a genetic distance\0.100, indicating large genetic differences among the DH lines. Both populations 1 and 6 showed significantly lower (p \ 0.001) donor introgression than the other four populations. Donor parent contribution was significantly (p\0.001) higher in the CML444 genetic background than CML395 and CML488. The average donor and recurrent PGC across all 417 DH lines was 31.7 and 64.3 %, respectively. Donor genome introgression was higher than expected in 82 %of the DH lines in the BC1 generation, possibly due to artificial selection during the DH process, during the development of F1 or BC1 seed, or during initial agronomic evaluation of the DH lines. Donor parent introgression up to 32 % showed significant positive correlation with grain yield under drought (r = 0.312, p\0.001) and optimum (r = 0.142, p\0.050) environments but negative correlation with ASI under drought (r = -0.276, p\0.001).Additional multi-environment phenotype data under managed drought are needed to confirm the correlations reported in this study and to map the specific genomic regions associated with such correlations.