Optimisation of in vitro techniques for cassava brown streak virus elimination from infected cassava clones

Cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV), is an economically important disease of cassava (Manihot esculenta Crantz) in East Africa. The objective of this study was tooptimise in vitro techniques for CBSV elimination from infected Ugandan cassava cultivars. Using semi-solid halfstrength Murashige and Skoog (MS) basal medium, hormone concentration and heat treatment regimes wereoptimised for micropropagation of farmer’s preferred cassava cultivars and CBSV elimination. Single nodes from young cassava stems were cultured for four weeks on ½MS medium supplemented with 6-benzyl amino purine(BAP) and 2,4-dichlorophenoxy acetic acid (2,4-D). The BAP and 2,4-D concentration (mg l-1) combinations used were 0.0 and 0.0, 0.5 and 0.1, 1.0 and 0.2, 1.5 and 0.3, and 2.0 and 0.4, respectively. The optimum medium was used for in vitro thermotherapy using four temperature regimes, namely 30-34, 34-38, 36-40 and 38-42 oC for 8 hoursdarkness and 16 hours light, respectively, for four weeks. The best plantlet growth in terms of height was observed on MS medium supplemented with 0.5 mg l-1 BAP and 0.1 mg l-1 2,4-D. Highest CBSV elimination efficiency of 40%, with 49% plantlet survival was observed at 36 oC for 8 hours darkness and 40 oC for 16 hours light. These results indicate that in vitro techniques can greatly enhance CBSV elimination and, thus, provide a means of CBSD management through dissemination and conservation of popular but CBSD susceptible cultivars

TDZ AND 4-CPPU in Gamborg B5 salts with MS vitamins doubles embryogenic 191 response from male flowers of EA-AAA banana.

Conventionally, auxins have been used in MS medium in combination or without purine-based cytokinins for induction of embryogenesis in EA-AAA banana (Musa spp.). Besides, low embryogenic response, it has been rare for more than two cultivars to respond similarly to a single treatment. This study investigated the efficacy of urea-type cytokinins, N-phenyl-N’-1,2,3-thidiazol-5-ylurea (TDZ) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4-CPPU); and salt formulations, Chu (N6), Eriksson, Gamborg B5, MS, Nitsch, NLN, SH and White for embryogenic callus induction in different EA-AAA banana cultivars. Immature male flowers of cultivars Mpologoma, Mbwazirume, Nakabululu, Nakinyika and Nfuuka were cultured on callus induction medium, supplemented with different TDZ and 4-CPPU combinations. Most of the cultivars had embryogenic response to the medium with 10μM TDZ+10μM CPPU. Cultivar Nakabululu recorded 22.2% embryogenic response, followed by Mwazirume (5.7%), Nakinyika (5.3%) and Mpologoma (4.6%). Cultivar Nfuuka had 9.1% embryogenic response on 15μM TDZ+15μM CPPU. When cultivars Mpologoma and Nakinyika were cultured on the same medium containing 10μM TDZ+10μM CPPU, but the MS salts substituted with the other salt formulations, their cultures recorded 11.4 and 8.3% embryogenic response, respectively to Gamborg B5 salts; which was almost twice their response to MS medium. The results suggested that TDZ and 4-CPPU, particularly in Gamborg B5 salt formulation, could increase percentage of embryogenic callus induced from male flowers of EA-AAA banana cultivars, and would improve plant regeneration and consequently help in the process of genetic improvement of EA-AAA banana.Key Words: Cytokinins, embryogenic response, Musa spp., Thidiazuro

Agrobacterium mediated transformation of banana (Musa sp.) cv. Sukali Ndiizi (ABB) with a modified Carica papaya cystatin (CpCYS) gene

Conventional banana breeding for pest and disease resistance is a very difficult and slow process due to the limited sources of resistance, sterility of cultivated banana varieties, high polyploidy levels, long cropping cycle and the lack of rapid screening methods. Molecular breeding using the transgenic approach with candidate genes such as cystatins offers an alternative method to banana improvement. Cystatin proteins inhibit the activity of cysteine proteases responsible for the breakdown of dietary proteins in the gut of many pests including nematodes resulting in protein deficiency. In this study, the papaya cystatin gene was introduced into the banana genome. Embryogenic cell suspension (ECS) cultures of the banana cultivar Sukali Ndiizi (ABB) were used as explants material for the successful transformation of banana. The Carica papaya cystatin gene (CpCYS-Mut89) previously modified to improve its inhibitory potential against banana pests was introduced into this cultivar using Agrobacterium tumefaciens, strain LBA4404 and the gus reporter gene was used to observe successful transformation process. We report the successful protocol for routine transformation of this cultivar, which was completed in six months with plant regeneration observed at a frequency of 23%. An additional four months was required to multiply the regenerant lines in order to have at least 20 plants per line for downstream challenging studies. Putatively transgenic plants were analyzed by PCR using hpt and CpCYS-Mut89 specific primers to confirm the presence of transgenes. Out of 28 selected lines, 27 were positive for both hpt and CpCYS-Mut89 transgenes giving 96.4% transformation efficiency. Five lines were then selected on the basis of putative PCR positives and a Southern blot analysis gave hybridization signals with 1 to 4 copy number integration patterns characteristic of Agrobacterium mediated transformation. These results confirm stable gene integration in East African banana cultivar cv. Sukali Ndiizi (genome group ABB) through an efficient Agrobacterium-mediated transformation protocol described for routine use in future improvement of this crop with genes of economic importance.Keywords: Cystatins, banana, Agrobacterium mediated transformation, southern blotAfrican Journal of Biotechnology Vol. 12(15), pp. 1811-181

Production of friable embryogenic callus and regeneration of Ugandan farmer-preferred cassava genotypes

Generation 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.Key words: Cassava (Manihot esculenta Crantz), somatic embryogenesis, amino acids, carbon sources

Prevalence of viruses infecting cowpea in Uganda and their molecular detection

The main areas for cowpea cultivation in Uganda were surveyed in June and October 2006 for viruses affecting the crop. Seed and leaf samples from symptomatic and asymptomatic plants were collected from farmers’ fields and analysed for infecting viruses using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The viruses detected in the leaf and seed samples were: cucumber mosaic cucumovirus (CMV), cowpea mild mottle calarvirus (CPMMV), cowpea mottle carmovirus (CPMoV), Cowpea chlorotic mottle bromovirus (CCMV), Cowpea yellow mosaic comovirus (CYMV), cowpea severe mosaic comovirus (CPSMV), cowpea aphid-borne mosaic potyvirus (CABMV) and Southern bean mosaic sobemovirus (SBMV). CPMV was detected only in leaf samples. CMV and CABMV were later confirmed using reverse transcription polymerase chain reaction (RT-PCR). Of the viruses detected in leaf samples, 53.26% occurred as single infections, 24.46% dual and 22.28% multiple infections. Similarly, analysis of seed samples revealed infection of 40.6, 34.6 and 24.8% for single, dual and multiple infections, respectively. Multiple virus infections were associated with more disease severity and higher yield losses. The seed transmission levels of 23.0, 20.3 and 16.4% were recorded for CMV, CPMMV and CABMV, respectively. This study identified six more viruses in addition to what was previously reported in the country, of which eight were seed-borne. This necessitates the need for the production and use of virus-free seeds, development of virus resistant genotypes and adoption of efficient seed certification systems.Keywords: Vigna unguiculata, disease incidence, seed-borne viruses, ELISA, (RT-PCR

Induction of somatic embryogenesis in recalcitrant sweetpotato (Ipomoea batatas L.) cultivars

Genetic transformation is considered as one of the most promising options for improvement of crop traits. Current transformation methods for sweetpotato depend on plant regeneration through organogenesis or somatic embryogenesis. Somatic embryogenesis and plant regeneration at a high frequency has been  restricted to a few sweetpotato varieties. Three auxins namely: 2,4-dichlorophenoxyacetic acid (2,4-D),  4-fluoroamphetamine (4-FA) and 4,5-trichlorophenoxyacetic acid (2,4,5-T) were investigated in this study for enhancing somatic embryogenesis from various plant organs of recalcitrant African sweetpotato cultivars.  2,4-D was found to be the best (p . 0.05) for induction of embryogenic callus. Cultivar Bwanjule had the highest  (20.2%) embryogenic callus frequency among the five African cultivars tested. The highest number of plants in this study was regenerated from the non-African cultivar variety Jonathan on media supplemented with 0.2 mg Zeatin. The emergence of roots from callus of recalcitrant Ugandan cultivars and the comparable high embryogenic responses in this work demonstrate the potential for regenerating plants from African  cultivars that have not been regenerated before. The regeneration of roots in this work could be useful for the initiation of root cultures. The most important application of this work is in genetic transformation of sweet potato, particularly for improvement of resistance to weevils.Key words: Embryogenesis, plant growth regulators, plant regeneration, Ipomoea batatas

Inflammatory arthritis in HIV positive patients: A practical guide

Background: Musculoskeletal manifestations of the human immunodeficiency virus (HIV) have been described since the outset of the global HIV epidemic. Articular syndromes that have been described in association with HIV include HIV-associated arthropathy, seronegative spondyloarthropathies (SPA) (reactive arthritis, psoriatic arthritis (PsA) and undifferentiated SPA), rheumatoid arthritis (RA) and painful articular syndrome. Methods: We carried out a computer-assisted search of PubMed for the medical literature from January 1981 to January 2015 using the keywords HIV, acquired immune-deficiency syndrome, rheumatic manifestations, arthritis, spondyloarthropathy, anti-TNF and disease modifying antirheumatic drugs. Only English language literature was included and only studies involving adult human subjects were assessed. Results: There are challenges in the management of inflammatory arthritis in patients who are HIV-positive, including difficulties in the assessment of disease activity and limited information on the safety of immunosuppressive drugs in these individuals. Conclusions: This review focuses on the clinical characteristics of the inflammatory articular syndromes that have been described in association with HIV infection and discusses the therapeutic options for these patients

Determinants of infant growth in Eastern Uganda: a community-based cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Child under-nutrition is a leading factor underlying child mortality and morbidity in Sub-Saharan Africa. Several studies from Uganda have reported impaired growth, but there have been few if any community-based infant anthropometric studies from Eastern Uganda. The aim of this study was to describe current infant growth patterns using WHO Child Growth Standards and to determine the extent to which these patterns are associated with infant feeding practices, equity dimensions, morbidity and use of primary health care for the infants.</p> <p>Methods</p> <p>A cross-sectional survey of infant feeding practices, socio-economic characteristics and anthropometric measurements was conducted in Mbale District, Eastern Uganda in 2003; 723 mother-infant (0–11 months) pairs were analysed. Infant anthropometric status was assessed using z-scores for weight-for-length (WLZ), length-for-age (LAZ) and weight-for-age (WAZ). Dependent dichotomous variables were constructed using WLZ < -2 (wasting) and LAZ < -2 (stunting) as cut-off values. A conceptual hierarchical framework was used as the basis for controlling for the explanatory factors in multivariate analysis. Household wealth was assessed using principal components analysis.</p> <p>Results</p> <p>The prevalences of wasting and stunting were 4.2% and 16.7%, respectively. Diarrhoea during the previous 14 days was associated with wasting in the crude analysis, but no factors were significantly associated with wasting in the adjusted analysis. The adjusted analysis for stunting showed associations with age and gender. Stunting was more prevalent among boys than girls, 58.7% versus 41.3%. Having brothers and/or sisters was a protective factor against stunting (OR 0.4, 95% CI 0.2–0.8), but replacement or mixed feeding was not (OR 2.7, 95% CI 1.0–7.1). Lowest household wealth was the most prominent factor associated with stunting with a more than three-fold increase in odds ratio (OR 3.5, 95% CI 1.6–7.8). This pattern was also seen when the mean LAZ was investigated across household wealth categories: the adjusted mean difference between the top and the bottom wealth categories was 0.58 z-scores, p < 0.001. Those who had received pre-lacteal feeds had lower adjusted mean WLZ than those who had not: difference 0.20 z-scores, p = 0.023.</p> <p>Conclusion</p> <p>Sub-optimal infant feeding practices after birth, poor household wealth, age, gender and family size were associated with growth among Ugandan infants.</p

Evidence for oxygenic photosynthesis half a billion years before the Great Oxidation Event

The early Earth was characterized by the absence of oxygen in the ocean–atmosphere system, in contrast to the well-oxygenated conditions that prevail today. Atmospheric concentrations first rose to appreciable levels during the Great Oxidation Event, roughly 2.5–2.3 Gyr ago. The evolution of oxygenic photosynthesis is generally accepted to have been the ultimate cause of this rise, but it has proved difficult to constrain the timing of this evolutionary innovation. The oxidation of manganese in the water column requires substantial free oxygen concentrations, and thus any indication that Mn oxides were present in ancient environments would imply that oxygenic photosynthesis was ongoing. Mn oxides are not commonly preserved in ancient rocks, but there is a large fractionation of molybdenum isotopes associated with the sorption of Mo onto the Mn oxides that would be retained. Here we report Mo isotopes from rocks of the Sinqeni Formation, Pongola Supergroup, South Africa. These rocks formed no less than 2.95 Gyr ago in a nearshore setting. The Mo isotopic signature is consistent with interaction with Mn oxides. We therefore infer that oxygen produced through oxygenic photosynthesis began to accumulate in shallow marine settings at least half a billion years before the accumulation of significant levels of atmospheric oxygen

Oligodendroglial neoplasms with ganglioglioma-like maturation: a diagnostic pitfall

Although oligodendroglial neoplasms are traditionally considered purely glial, increasing evidence suggests that they are capable of neuronal or neurocytic differentiation. Nevertheless, ganglioglioma-like foci (GGLF) have not been previously described. Herein, we report seven examples where the primary differential diagnosis was a ganglioglioma with an oligodendroglial component. These five male and two female patients ranged in age from 29 to 63 (median 44) years at initial presentation and neuroimaging features were those of diffuse gliomas in general. At presentation, the glial component was oligodendroglioma in six and oligoastrocytoma in one; one was low-grade and six were anaplastic. A sharp demarcation from adjacent GGLF was common, although some intermingling was always present. The GGLF included enlarged dysmorphic and occasionally binucleate ganglion cells, Nissl substance, expression of neuronal antigens, GFAP-positive astrocytic elements, and low Ki-67 labeling indices. In contrast to classic ganglioglioma, however, cases lacked eosinophilic granular bodies and CD34-positive tumor cells. Scattered bizarre astrocytes were also common and one case had focal neurocytic differentiation. By FISH analysis, five cases showed 1p/19q codeletion. In the four cases with deletions and ample dysmorphic ganglion cells for analysis, the deletions were found in both components. At last follow-up, two patients suffered recurrences, one developed radiation necrosis mimicking recurrence, and one died of disease 7.5 years after initial surgery. We conclude that GGLF represents yet another form of neuronal differentiation in oligodendroglial neoplasms. Recognition of this pattern will prevent a misdiagnosis of ganglioglioma with its potential for under-treatment