Regeneration of Sudanese maize inbred lines and open pollinated varieties

Eight maize inbred lines and three open pollinated varieties from Sudan were evaluated for their response to tissue culture. Immature embryos obtained 16 days after pollination were used as explants for callus induction. Calli were induced on LS medium supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid. Callus induction capacity was highest in inbred lines IL3, IL15 and IL1. The Varieties Hudiba-2 and Hudiba-1 were not statistically different (p >0.05) in callus induction. Thecapacity for embryogenic callus formation was highest in inbred line IL3 followed by IL1 and IL38 and in varieties Hudiba-2 and Hudiba-1. Inbred lines IL16, IL42, IL43 and IL28 had the lowest embryogeniccallus formation capacity. Plant regenerating genotypes were IL3, IL38, IL15, IL1, Hudiba-2 and Mojtamaa-45. Inbred line IL3 was the most regenerable genotype with a shoot formation frequency of 76% averaging 6 shoots per callus. The highest regenerating variety was Mojtamaa-45, which averaged 5 shoots per callus

Striga parasitizes transgenic hairy roots of Zea mays and provides a tool for studying plant-plant interactions

Background Striga species are noxious root hemi-parasitic weeds that debilitate cereal production in sub-Saharan Africa (SSA). Control options for Striga are limited and developing Striga resistant crop germplasm is regarded as the best and most sustainable control measure. Efforts to improve germplasm for Striga resistance by a non-Genetic Modification (GM) approach, for example by exploiting natural resistance, or by a GM approach are constrained by limited information on the biological processes underpinning host-parasite associations. Additionaly, a GM approach is stymied by lack of availability of candidate resistance genes for introduction into hosts and robust transformation methods to validate gene functions. Indeed, a majority of Striga hosts, the world’s most cultivated cereals, are recalcitrant to genetic transformation. In maize, the existing protocols for transformation and regeneration are tedious, lengthy, and highly genotype-specific with low efficiency of transformation. Results We used Agrobacterium rhizogenes strain K599 carrying a reporter gene construct, Green Fluorescent Protein (GFP), to generate transgenic composite maize plants that were challenged with the parasitic plant Striga hermonthica. Eighty five percent of maize plants produced transgenic hairy roots expressing GFP. Consistent with most hairy roots produced in other species, transformed maize roots exhibited a hairy root phenotype, the hallmark of A. rhizogenes mediated transformation. Transgenic hairy roots resulting from A. rhizogenes transformation were readily infected by S. hermonthica. There were no significant differences in the number and size of S. hermonthica individuals recovered from either transgenic or wild type roots. Conclusions This rapid, high throughput, transformation technique will advance our understanding of gene function in parasitic plant-host interactions

Dicamba growth regulator promotes genotype independent somatic embryogenesis from immature zygotic embryos of tropical maize inbred lines

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 tropical maize (Zea mays L.) genotypes, CML 216, CML 144, A 04, E 04 and TL 21, were evaluated for callus induction on MS medium supplemented with the growth regulator dicamba. Embryogenic and non embryogenic callus induction was independent of genotype when young immature embryos, 12 days after pollination (DAP) were used for tissue culture in combination with dicamba. The optimal concentration of dicamba for 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 from 35% to 79% among the five genotypes and somatic embryos regenerated R0 shoots that produced normal R1 progenies. This regeneration method is expected to facilitate the development of a more efficient genotype independent Agrobacterium- mediated transformation system for tropical inbred lines

Enhancing resistance against African weevils through development of transgenic sweetpotato cultivars (Ipomoea batatas (L.) Lam.) expressing cry7Aa1, cry3Ca1 and ET33-34 genes.

Sweetpotato (Ipomoea batatas) is one of the most important food crops in tropical and subtropical countries. In Sub-Saharan Africa, sweetpotato is mainly produced for consumption and as a source of income by resource-poor farmers. However, their production is limited by severe damage caused by pests and diseases. The African weevils Cylas puncticollis and C. brunneus are the main biological constraints that may cause losses between 50 and 100%. Biotechnological approaches to control weevils include the introduction of genes encoding Cry proteins found to be active against these pests. To that end, several protocols for sweetpotato regeneration and transformation by organogenesis or somatic embryogenesis have been developed but their efficiency remains largely genotype-dependent and time-consuming. In this study, 31 African sweetpotato cultivars from CIP genebank were screened for regeneration and transformation efficiencies by organogenesis and somatic embryogenesis. Additionally, “Jewel” and “Jonathan” cultivars were used as organogenic and embryogenic controls, respectively. Regeneration by organogenesis was conducted using a two-step protocol including 2,4-D then thidiazuron, zeatin or kinetin while regeneration by embryogenesis was performed using a three-step protocol, each one using a different hormone (2,4,5-T, ABA and AG3). Higher than 40% regeneration efficiencies were obtained for 8 cultivars (Jewel, Imby, Kawogo, Luapula, Mafutha, CIP440163, Zambezi and Ukerewe) with an organogenesis protocol and 8 cultivars (Jonathan, Imby, K51/3251, Bwanjule, CIP440163, SPK004, New Kawogo and KSP 11) with an embryogenesis protocol. Genetic transformation of sweetpotato with Jewel by organogenesis and Imby, CIP440163 and Jonathan by somatic embryogenesis has been achieved using chimerical genes coding for three of the most active proteins (Cry7Aa1, ET33-34, and Cry3Ca1) against African weevils. Transgenic events have been confirmed by kanamycin resistant calli test, PCR and Southern blot. Transcriptional activity and Cry protein accumulation are being tested in leaves and storage roots by Real time PCR and DAS-ELISA respectively

RECOVERY OF amiRNA3-PARP1 TRANSGENIC MAIZE PLANTS USING A BINARY VECTOR HAVING THE BIOSAFE PMI GENE

Positive plant selectable marker genes are commonly used in plant transformation because they not only enhance the frequency of generation transgenic tissues but are considered biosafe, unlike antibiotic or herbicide resistance genes. In this study, the binary vector pNOV2819-ubiamiRNA3PARP1, harbouring the phosphomannose isomerase (pmi) gene was developed and used in recovery of transgenic maize ( Zea mays L.) plants containing the drought tolerance gene, amiRNA3-PARP1. The pre-amiRNA3-PARP1 and Tnos transgenes were sequentially PCR-cloned upstream the ubiquitine promoter in the Ubi/NC1300 plasmid. The pre-amiRNA3-PARP1 expression cassette was transferred into the pmi gene-containing pNOV2819 plasmid to produce the pNOV2819-ubiamiRNA3PARP1 vector. Transgenic IL3 and A188 plants containing pre-amiRNA3-PARP1 were generated through transformation with LBA4404 harbouring the pNOV2819-ubiamiRNA3PARP1 vector. The plants were confirmed transgenic by PCR. It is clear that the developed vectors are effective in recovery of amiRNA3-PARP1 transgenic tissues and plants containing the pmi gene, which has been shown to have no negative environmental or health effects.Les marqueurs g\ue9n\ue9tiques de s\ue9lection positive de plantes sont commun\ue9ment utilis\ue9s dans la transformation des plantes parce que, non seulement ils augmentent la fr\ue9quence de la g\ue9n\ue9ration des tissus transg\ue9niques, mais aussi sont consid\ue9r\ue9s comme biosains, \ue0 l\u2019inverse des g\ue8nes de r\ue9sistance aux antibiotiques et herbicides. Dans cette \ue9tude, le vecteur binaire pNOV2819-ubiamiRNA3PARP1 portant le g\ue8ne isom\ue9rase phosphomannose (pmi) a \ue9t\ue9 d\ue9velopp\ue9 et utilis\ue9 dans le recouvrement transg\ue9nique des plantes du ma\uefs ( Zea mays L.) contenant le g\ue8ne to tol\ue9rance \ue0 la s\ue9cheresse amiRNA3-PARP1. Les transg\ue8nes pre-amiRNA3-PARP1 et Tnos \ue9taient s\ue9quentiellement clon\ue9s par PCR dans la partie sup\ue9rieure du promoteur ubiquitine dans le plasmide Ubi/NC1300. L\u2019expression de la cassette de la pr\ue9-amiRNA3-PARP1 \ue9tait transf\ue9r\ue9e dans le g\ue8ne pmi contenant le plasmide pNOV2819 pour produire le vecteur pNOV2819-ubiamiRNA3PARP1. Les plants transg\ue9niques IL3 et A188 contenant le pre-amiRNA3-PARP1 \ue9taient g\ue9n\ue9r\ue9s \ue0 travers la transformation avec LBA4404 portant le vecteur pNOV2819-ubiamiRNA3PARP1. Les plants \ue9taient confirm\ue9es transg\ue9niques par PCR. Il est clair que les vecteurs d\ue9velopp\ue9s sont efficaces dans le recouvrement des tissus transg\ue9niques amiRNA3-PARP1 et les plants contenant le g\ue8ne pmi qui ne pr\ue9sentent aucun effet n\ue9gatif sur l\u2019environnement et la sant\ue9

African swine fever virus (ASFV): biology, genomics and genotypes circulating in sub-Saharan Africa

African swine fever (ASF) is a highly infectious and fatal haemorrhagic disease of pigs that is caused by a complex DNA virus of the genus Asfivirus and Asfarviridae African suids family. The disease is among the most devastating pig diseases worldwide including Africa. Although the disease was first reported in the 19th century, it has continued to spread in Africa and other parts of the world. Globally, the rising demand for pork and concomitant increase in transboundary movements of pigs and pork products is likely to increase the risk of transmission and spread of ASF and pose a major challenge to the pig industry. Different genotypes of the ASF virus (ASFV) with varying virulence have been associated with different outbreaks in several countries in sub-Saharan Africa (SSA) and worldwide, and understanding genotype circulation will be important for ASF prevention and control strategies. ASFV genotypes unique to Africa have also been reported in SSA. This review briefly recounts the biology, genomics and genotyping of ASFV and provides an account of the different genotypes circulating in SSA. The review also highlights prevention, control and progress on vaccine development and identifies gaps in knowledge of ASFV genotype circulation in SSA that need to be addressed

Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies

Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional breeding programs in order to efficiently optimize productivity

The Diagnostic Utility of Induced Sputum Microscopy and Culture in Childhood Pneumonia.

BACKGROUND.: Sputum microscopy and culture are commonly used for diagnosing the cause of pneumonia in adults but are rarely performed in children due to difficulties in obtaining specimens. Induced sputum is occasionally used to investigate lower respiratory infections in children but has not been widely used in pneumonia etiology studies. METHODS.: We evaluated the diagnostic utility of induced sputum microscopy and culture in patients enrolled in the Pneumonia Etiology Research for Child Health (PERCH) study, a large study of community-acquired pneumonia in children aged 1-59 months. Comparisons were made between induced sputum samples from hospitalized children with radiographically confirmed pneumonia and children categorized as nonpneumonia (due to the absence of prespecified clinical and laboratory signs and absence of infiltrate on chest radiograph). RESULTS.: One induced sputum sample was available for analysis from 3772 (89.1%) of 4232 suspected pneumonia cases enrolled in PERCH. Of these, sputum from 2608 (69.1%) met the quality criterion of <10 squamous epithelial cells per low-power field, and 1162 (44.6%) had radiographic pneumonia. Induced sputum microscopy and culture results were not associated with radiographic pneumonia, regardless of prior antibiotic use, stratification by specific bacteria, or interpretative criteria used. CONCLUSIONS.: The findings of this study do not support the culture of induced sputum specimens as a diagnostic tool for pneumonia in young children as part of routine clinical practice