CW-Department of Biochemistry and Biotechnology

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In Vivo Toxicological and Histopathological Effects of Aflatoxin B1 Exposure and Related Risk
(ICHWB (International Conference on Health & Well-Being), 2016) Ochieng, P.J.; Okun, D.; Mugenya, I.; Njagi, N.J.; Mugai, J.
Aflatoxin B1 are toxic metabolites of Aspergilus flavas and Aspergilus parasiticus which usually contaminant foods such peanuts, corn, and other grains as well as animal feeds resulting into intoxication. Studies have been conducted to elucidated the mechanism of AFB1 toxicity however, there is still a challenge explore the risk associated with AFB1. Therefore,the main objective of this research was to performed toxicological and histopathological analysis of aflatoxin B1 and related risk. Populations of mice were treated with ascending dosed of 3mg/Kg, 6mg/Kg, 9mg/Kg and 12mg/Kg of AFB1. The LD50 was then recorded, the liver biopsy from scarified and dead mice were screened for analysis of distribution of AFB1.Enzyme transaminases activity and total bilirubin content was then analysed by spectrometry,histology was then on performed on biopsy lastly; prothrobin time analysis conducted to assess the effect of AFB1 on blood clotting factors. From the results death occurred within 48hours for most mice treated with doses of 9mg/Kg and 12mg/Kg, biochemical test showed significant increase transaminases (ALT, AST and AP) activity with fluctuation of bilrubin content with gradual increases in prothrobin time (PTT). Liver biopsy showed bile duct proliferation, vacuolation of hepatocytes, enlargement of hepatic cells, fatty infiltration,necrosis, hemorrhage, and apoptosis. We concluded that prolonged consumption of AFB1contaminated feed or food at dose range of 3-6 mg/Kg may result to development of hepatocellular carcinoma while 9-12mg/Kg AFB1 may lead to server liver injury. Thus there are higher risk of AFB1 to induce hepatocellular carcinoma (HCC), mutagenic andImmunesuppression to both humans and animals.
Use of a non-mist propagation system to vegetatively propagate 12 E. grandis X E. Camadulensis hybrid clones
(Kenyatta University, 2009) Mwaniki, F.N.; Muluvi, G. M.; Gichuki, C.; Oeba, V.; Kanyi, B.
Evolution and diagnostic utility of major surface proteases from trypanosoma vivax, t. Brucei brucei and t. Congolense
(Kenyatta University, 2009) Machuka, Eunice M.; Masiga, Daniel K.; Ouma, Johnson O.; Gichuki, Charity W.
Subclinical nephrotoxicity caused by smoking and occupational silica exposure among male Kenyan industrial workers
(Moi University, 2007) Mwangi, Daniel M.; Ngeranwa, J.J.N.; McLigeyo, Seth O.; Orinda, George O.; Njagi, E.N.M.
Progress in transformation and regeneration of tropical inbred maize lines in Kenya
(Maize Genetics Cooperative Newsletter, 2008) Mgutu, Allan Jalemba; Anami, E. S.; Hanley-Bowdoin, L.; Rasha, A. O.; Nelissen, H.; Inzé, D.; Van, L. M.; Machuka, J.
Tropical inbred maize lines have a reputation of being difficult to transform, mainly as a result of their inherent limitations associated with resistance to Agrobacterium infection and their recalcitrance to in vitro regeneration. To enhance the capacity for public sector maize transformation, the Plant Transformation Facility at Kenyatta University, Kenya, embarked on a program to improve transformation of diverse tropical inbred maize lines using Agrobacterium tumefaciens. We evaluated both N6 (Frame et al., Plant Physiol. 129:13-22, 2002) versus LS (Negrotto et al., Plant Cell Rep.19:798-803, 2000) media with different hormone regimes and optimized transformation and regeneration protocol for tropical inbred maize lines. Using immature embryos as explants, four Kenyan tropical inbred lines TL21, TL22, TL23 and TL18; two Sudanese inbred lines IL1, IL2 and CIMMYT inbred lines CML 216 and CML 244 have been investigated for their tenability to transformation and regeneration. Transformation frequencies (callus resistant events over total explants) and efficiencies (plantlet regenerating events over total explants) for the recovered events were used to evaluate successful transformation
Optimisation of parameters for agrobacterium-mediated transformation of sweet potato
(2010) Machuka, Jesse; Njagi, W.; Gichuki, S. T.; Macharia, C.; Muluvi, G. M.
An Agrobacterium-mediated transformation and somatic regeneration protocol was adapted for a Kenyan sweet potato variety, KSP36. A model cultivar, CTP560 was used as a control. For selection of transformed explants paramomycin was found to be effective at 25mg/L while kanamycin was effective at 20mg/L. The lower concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations proved better for regeneration as opposed to the higher 2,4-D concentrations. Zeatin/ IAA (indole acetic acid) was more effective at embryo production as opposed to kinetin/ 2,4-D medium in both cultivars. Out of the 18 KSP36 plants tested by PCR, 11 tested positive for the coat protein gene while 9 out of the 19 CPT560 plants tested positive. This protocol can be recommended for other sweet potato varieties.
Effect of 2, 4-d levels on callus induction of leaf and stem explants of 5 local farmer-preferred sweet potato varieties in Kenya
(2010) Machuka, Jesse; Bett, B.; Gichuki, S. T.; Ateka, E.
Five local and popular sweet potato (Ipomoea batatas(Lam.) varieties were selected from the major sweet potato growing areas, based on farmer-preferences and desirable characteristics. Both leaf and stem explants were incubated for callus induction with various concentrations of an auxin 2,4 Dichlorophenoxyacetic acid (2,4-D) at 0, 0.5, 1.0, 2.0, and 5.0 mg/L. The highest percentage of callus induction was realized at 2.0 mg/L and 5.0 mg/L 2,4-D in all varieties for both leaf and stem explants. Callus was induced at 3 weeks after incubation of leaf and stem explants in all tested varieties. Stem explants demonstrated a better response to callus induction at all 2,4-D concentrations and in all varieties as compared to leaf explants. All varieties responded differently to callus induction.
Prospecting for bacillus thuringiensis for the control of prostephanus truncatus (horn) and sitophilus zeamais motschulsky
(2010) Machuka, Jesse; Likhayo, P.; Nang’ayo, F.; Maniania, N.
Three hundred twenty nine samples from different parts of Kenya which had not previously been treated with bio-pesticide were collected for Bacillus thuringiensis isolation. The δ-endotoxin crystals were isolated from nutrient broth culture by medium speed centrifugation. From the colony samples examined, 77.1% (236) were non crystal forming Bacillus, 16% (49) Bacillus thuringiensis and 6.9% (21) Bacillus sphaericus. The B. thuringiensis isolates were evaluated for insecticidal activity against Prostephanus truncates and Sitophilus zeamais using artificial seeds incorporating spore – crystal toxins at the rate of 4% w/w. Twenty five unsexed 2-week-old P. truncates and S. zeamais adults were separately fed for 14 days with ten seedsof each isolate contained in ventilated glass tubes. Seeds prepared with sterile distilled water only served as controls. None of the isolates showed satisfactory control of the two coleopteran species. The results demonstrate minimal prospects of finding more potent Bacillus thuringiensis from local sources.
Interaction of cassava mosaic disease and cassava brown streak disease in nicotiana benthamiana
(2010) Ngeranwa, J.J.N.; Irungu, J.; Miano; Mbogo, E.; Monjero, K.; Gichuki, S. T.
The interaction of Cassava mosaic geminivirus (CMG)and Cassava brown streak virus (CBSV) were tested in Nicotiana benthamiana. The inoculated virus and/or virus species were CMGs species (ACMV and EACMV-UgV), CBSV and dual CMGs and CBSV. The inoculum source was N. benthamianaplants pre-infected with viruses from diseased cassava cultivars collected from the western region of Kenya. Leaf samples from virus-infected N. benthamiana plants were ground in inoculation buffer [0.1M K-phosphate buffer, pH 7.0, containing 0.01% (w/v) βME and Na2SO3]. The leaf homogenates were rubbed on 2-3 carborundum-dusted leaves of Nicotiana benthamiana plants at 3-5 leaf stage. Four treatments, CMGs (UgV+ACMV); CBSV; combination of CMGs+CBSV and non inoculated control plants were applied with 10 plants per each treatment inoculated in two trials. The plants were grown in a screen house and data recorded on severity of symptoms on leaves, days to symptom appearance and the percentage of infected plants. Highly significant p<0.05 difference were observed on the severity of the disease but differences in days to first appearance of symptoms and the number of infected plants were not significant p<0.05. The most severe symptoms of the disease and number of infected plants were observed in plants infected with both CMGs and CBSV (4.7 and 8) respectively. The earliest symptoms appeared in plants infected with CBSV (5 dpi) and dual infection took the longest period before symptoms were observed (9dpi). None of the plants in the control exhibited leaf symptoms. The dually infected plants exhibited more severe symptoms compared to single infections indicating synergistic interaction when the two viruses occur in combination
Regeneration and transformation potential of elite Kenyan highland maize inbred lines
(2010) Machuka, Jesse; Taracha, C.; Nangayo, F.; Ombakho, G.
Agrobacterium-mediated transformation of tropical maize has been manipulated in only a limited number of genotypes, because a majority of maize germplasm isrecalcitrant toin vitroresponse. Establishment of a highly efficiency and widely used tissue culture system for maize will accelerate the application of transformation technology in breeding programs, and the study of the functions of maize specific genes. Out of the three media evaluated, it was established that two media could guarantee the production and proliferation of a large number of embyogenic calli with high regeneration capacityfrom immature zygotic embryos representing different maize germplasm. The results suggest that the evaluated tissue system will be widely applicable for the tissue culture of Elite Kenyan Highland inbred maize lines
Prospects of biofuel production from microalgae in Kenya
(2012) Khamis, F. K.; Abubakar, L. U.; Mutie, A. M.; Kenya, E.U.
With the depletion of oil resources as well as the negative environmental impact associated with the use of fossil fuels, there is a renewed interest in seeking alternative energy sources. The present study was conducted to determine the microalgae biodiversity in Kenyan aquatic environment and their potential in bio-fuel production. Microalgae species were collected from 3 lakes in Kenya (Lake Turkana, Baringo and Magadi)and identified morphologically. The abundant species were cultured in BBM and BG-11 media to obtain pure clones and lipids (oil) extracted by the Bligh and Dyer method. The results showed that the blue-greenalgae (Cyanophytes) were widespread and dominated the algal community in all the 3 lakes. However, Lake Turkana exhibited the highest species biodiversity, followed by Lake Magadi, while Lake Baringo had the least. Screening for lipid/oil content identified high oil yielding algae species abundantly distributed naturally in the Kenyan aquatic environment. The peak lipid content ranged from 1.5 – 10.5% of algal biomass. Chlorellaspecies showed the highest yields (10.5%), followed by Euglenaacus (5.78), Nitzschia (3.68%), Ankistrodesmus falcatus(1.58%) and Scenedesmus acuminatus(1.575%). The lipid profiles revealed high concentrations of oleic acid which is a main constituent of biodiesel production. DNA barcode for the species with highest lipid content was developed using cytochrome oxidase subunit I (COI). Phylogenetic analyses by MEGA 4 indicated that Nitzschiaspp and Chlorellaspp are different evolutionally, Scenedesmusspp and Chlorella spp both being of the same phyla show great similarity with a 71% score compared to the Nitzschiaspp which is a diatom. These species can therefore be potential candidates for biofuel production.
Efficacy of transgenic kernels harbouring the cry3a gene from bacillus thuringiensis in the control of the larger grain borer and maize weevil
(2012) Taracha, Catherine Ongecha; Ombakho, G.; Nan’gayo, F.; Machuka, Jesse
Insect bioassays were conducted to evaluate the efficacy of maize seed kernels expressing protein of aδ-endotoxin gene of Bacillus thuringiensis tenebrionis Cry3A against economically important coleopteran pests in stored maize. Results from infestation of transgenic seed kernel assays conducted in the laboratory indicate that the kernels had no influence on the survival of the adult larger grain borer Prostephanus truncatus (Horn.) and maize weevil Sitophilus zeamais respectively. There was no adult mortality recorded. There was high population reduction in the T0 transgenic kernels (61%-88%) for both the beetles when compared to the non-transgenic control , in the T1 generation the population reduction was 37%-76% for P. truncatus and 61%-80% for S. zeamais except for inbred A04 which had no effect on population reduction. The lowest kernel damage and weight loss was observed in maize inbredCML 395 for P. truncatus in both T0 and T1 maize generations and in inbred I04 for S.zeamais.
Genotype independent somatic embryogenesis and plant regeneration from immature zygotic embryos of tropical maize inbred lines
(2012) Mgutu, Allan Jalemba; Akoyi, J.; Sande, O.; Machuka, Jesse
Maize is one of the most important cereal crops in Sub-Saharan Africa and an important source of energy for humans. However, the difference in the dedifferentiation frequency of immature embryos among various genotypes indicates that callus induction and genetic transformation is dependent on the genotype. This phenomenon is an impediment in the fundamental process of improving tropical maize germplasm especially through genetic engineering. Here, five tropicalmaize (Zea maysL.) genotypes were tested for callus induction and subsequent regeneration on MS medium supplemented with a growth regulator dicamba. Genotype-independent embryogenic calli were inducedfrom immature zygotic embryos, 12 days after pollination, of maize inbred lines, CML216, CML144,A04, E04 and TL21 on MS medium supplemented with different levels (1 to 5 mg/L) of plant growth regulator dicamba. The optimal concentration of dicambafor induction of embryogenic callus in all the genotypes was 3 mg/L, which was also the concentration at which non embryogenic callus formation was lowest. The frequency of embryogenic callus induction ranged from34%–79%, with genotype CML 216 having the highest frequency at this optimal concentration of dicamba. Somatic embryos obtained germinated plantlets and produced normal R1 progeny of regenerants (R0). This regeneration method is expected to facilitate the development of a more efficient genotype independent Agrobacterium mediated transformation system for Kenyan adopted tropical inbred lines.
Macropropagation Technique for Production of Healthy Banana Seedlings.
(African Crop Science Society, 2011) Mwangi, M.; Njau, N.; Kahuthia-Gathu, R.; Muasya, R.; Mbaka, J.; Tenywa, J.S.; Taulya, G.; Kawube, G.; Kawuki, R.; Namugwanya, M.; Santos, L.
Banana (Musa spp) is one of the most important crops providing food security, nutrition and income for many smallholder farmers in sub-Saharan Africa. However, production is hindered by scarcity of high quality seedlings, insect pests and diseases. To improve availability of quality seedlings macropropagation technology is being introduced in Kenya. This study was carried out to generate knowledge to enhance understanding and adoption of the technology. A survey was done initially to identify the key pests and pathogens of banana in Central and eastern regions of Kenya. Macropropagation nurseries were established in six sites in farmers’ fields representing different agroecological zones. Corms of banana varieties Kampala, Sweet banana, Cavendish and Uganda Green obtained in accordance with established quality assurance protocols were propagated. The health of the seedlings produced was monitored. Fusarium oxysporum f. sp. cubense was isolated from less than 1% corms of sweet banana and Kampala varieties. Radopholus similis was isolated from all the varieties but its incidence was highest (46%) in the Cavendish variety. Endophytes and non pathogenic microorganisms were isolated from more than 90% of the corms. Over 98% of the propagated corms produced healthy seedlings and only less than 1% of the corms propagated rotted in the propagation media due to non pathogenic causes. In areas with high weevil infestation it was difficult to obtain corms with the standard required for macropropagation. The information obtained shows that macropropagation technique effectively produces healthy banana seedlings. Practitioners will need to observe quality control measures to ensure production of high quality seedlings.
Banana Distribution and their Seed Systems in Central and Eastern Kenya.
(African Crop Science Society, 2011) Mwangi, M.; Kasyoka, M.R.; Kori, N.; Mbaka, J.J.; Gitonga, N.; Tenywa, J.S.; Taulya, G.; Kawube, G.; Kawuki, R.; Namugwanya, M.; Santos, L.
Bananas (Musa spp.) serve as food, income resource and animal feed in addition to other environmental benefits. In Kenya, the crop is mainly grown and managed by smallholder farmers. Over the last two decades, banana production has been on the decline. Pests, diseases and limited access to adequate clean planting materials constitute priority problems. This study aimed to determine distribution of bananas varieties, and the availability and sources of planting materials in Central and Eastern provinces of Kenya. Use of naturally regenerated suckers as planting materials exceeded 90% and continuously perpetuated the spread of banana diseases and pests that substantially reduce yields. Prospects for increasing seedlings supply through micropropagation has not been successful due to high cost of tissue cultured seedlings leading to low adoption (<60%). There is a gap between farmers with varying resource capabilities in accessing and using good quality planting materials. This can be resolved by interventions that supply farmers with healthy and affordable banana seedlings. Macropropagation, which is a simple, cost effective method that has been used successfully in other countries has great potential to address issues.
Preferred Banana Varieties and their Seed Systems in Eastern and Central provinces of Kenya
(Ruforum, 2010) Mwangi, M.; Kasyoka, M.R.; Mbaka, J.; Kori, N.; Gitonga, N.; Muasya, R.; Adipala, E.; Tusiime, G.; Majaliwa, J.G.M.
Banana is an important crop for food security in Kenya. Its production has been hindered by scarcity of seedlings and pests, among other factors. A recent survey in eastern and central Kenya showed that desert varieties are prefered due to market demand. Natural regeneration is also preferred by most farmers (>85%) but it is inefficient and a source of pests and diseases to new plantations. On the other hand, adoption of tissue culture has been hindered by high cost of seedlings. Farmers need to be educated on shortcomings of natural regeneration and affordable seedling delivery system implemented. Macropropagation supported by an efficient delivery system is proposed to boost banana production in Kenya.
In vitro selection and characterization of salinity tolerant somaclones of tropical maize (Zea mays L.)
(2014-05-12) Machuka, Jesse; Matheka, J. M.; Magiri, E.; Omer, Rasha A
Tolerance to salinity was obtained in an open pollinated variety (KAT) widely grown in the East African region and a dryland hybrid (PH01) by applying in vitro selection and regeneration procedures. Immature zygotic embryos of KAT and PH01 plants were cultured on N6 medium supplemented with 2mg/l 2,4-dichlorophenoxyacetic acid to initiate embryogenic calli. Calli were then maintained for one month after which time they were subjected to increasing concentrations of NaCl (between 0 and 2.9%) to determine the appropriate concentrations of selection pressure. The survival and regeneration capacity of KAT and PH01 calli were significantly lower (p<0.05) than those of their controls after exposure on both levels of NaCl. The genotype did not influence the survival capacity of selected calli. However, KAT and PH01 were found to differ significantly (p<0.05) in regeneration capacity

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