Modulation of energy homeostasis in maize and Arabidopsis to develop lines tolerant to drought, genotoxic and oxidative stresses

Abiotic stresses cause crop losses worldwide that reduce the average yield by more than 50%. Due to the high energy consumed to enhance the respiration rates, the excessive reactive oxygen species release provokes cell death and, ultimately, whole plant decay. A metabolic engineering approach in maize (Zea mays) altered the expression of two poly(ADP-ribosyl)ation metabolic pathway proteins, poly(ADP-ribose) polymerase (PARP) and ADP-ribose-specifIc Nudix hydrolase (NUDX) genes that play a role in the maintenance of the energy homeostasis during stresses. By means of RNAi hairpin silencing and CRISPR/Cas9 gene editing strategies, the PARP expression in maize was downregulated or knocked down. The Arabidopsis NUDX7 gene and its two maize homologs, ZmNUDX2 and ZmNUDX8, were overexpressed in maize and Arabidopsis. Novel phenotypes were observed, such as significant tolerance to oxidative stress and improved yield in Arabidopsis and a trend of tolerance to mild drought stress in maize and in Arabidopsis. Key words: poly(ADP-ribose) polymerase, Nudix hydrolase, CRISPR/Cas9, maize, oxidative stress, drought stress

Physical and Chemical Characteristics of Five Hot Springs in Eritrea

Eritrea has a number of hot springs whose physicochemical characteristics are not documented. This study examined the thermal and chemical features of five hot springs located in the eastern escarpment of Eritrea. Field data and water samples were collected from five hot springs namely; Akwar and Maiwooi near Gahtelai, Garbanabra and Gelti near Irafayle at the Gulf of Zula and Elegedi in Alid volcanic center. The water temperatures at source varied from 49.5°C to 100°C while pH levels ranged from 6.97 to 7.54. Elegedi had significantly higher temperature (p < 0.05) than the other four hot springs. Strong correlation was observed between electrical conductivity (EC), total dissolved solid (TDS), salinity, sodium, potassium, calcium and chloride (R2 > 0.9) as well as between temperature and sulphate levels (R2 = 0.96). Evident clustering was noted at p < 0.05, using Non-metric multidimensional scaling (NMDS), between the three locations of the hot springs. Akwar and Maiwooi, situated close to each other, clustered together, Garbanabra and Gelti, which were characterized by higher salinity levels, formed a separate cluster. Elegedi, characterized by high temperature (100°C), sulphate (979.7 mg/l) and NH4+ (196.33 mg/l) levels, clustered separately. Akwar and Maiwooi had high bicarbonate (345 mg/l and 393 mg/l) and fluoride (8.20 mg/l and 6.48 mg/l) levels which are above WHO limits. Electrical conductivity (23,133 mS/cm), total dissolved solid (15,552 mg/l), sodium (3,800 mg/l), potassium (198 mg/l), calcium (1,653 mg/l) and chloride (5,946 mg/l) levels in Garbanabra and Gelti hot springs exceeded WHO limits. Bromine (74.8 mg/l in Garbanabra and 45.2 mg/l in Gelti) and boron (2.21 mg/l in Garbanabra and 1.55 mg/l in Gelti) levels were also above standard limits set for potable water. Maiwooi (1.20) and Elegedi (1.10) were depositional while Akwar water (-0.71) was slightly corrosive. The corrosive nature of the water sample from Akwar, is a public health concern. The waters from the five Eritrean hot springs are thus not fit for human consumption. In addition, the use of thermal spring water for recreational purposes should be closely monitored. Keywords: key words, hot springs, physicochemical, Eritre

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

Genetic Improvement of African Maize towards Drought Tolerance: A Review

Africa supports a population of over 1 billion people with over half of them depending on maize for food and feed either directly or indirectly.  Maize in Africa is affected by many stresses, both biotic and abiotic which significantly reduce yields and eventually lead to poor production.  Due to the high demand for maize in the region, different improvement strategies have been employed in an effort to improve production.  These include conventional breeding, molecular breeding, high throughput phenotyping techniques and remote sensing-based techniques.  Conventional breeding techniques such as open pollination have been used to develop drought avoiding maize varieties like the Kito open pollinated variety (OPV) of Tanzania and Guto OPV of Ethiopia.  A combination of conventional breeding and molecular biology techniques has led to improved breeding strategies like the Marker Assisted Back Crossing (MABC) and Marker Assisted Recurrent Selection (MARS).  These techniques have been used to improve drought tolerance in existing inbred maize lines like the CML 247 and CML 176.  Through genetic engineering, different genes including C4-PEPC, NPK1, betA, ZmNF-YB2, cspB, ZmPLC1 and TsVP have been cloned in maize.  Transgenic maize crops expressing these genes have shown increased tolerance to drought stress.  Although there is substantial progress towards developing drought tolerant maize, many African farmers are yet to benefit from this technology.  This is due to lack of an enabling policy framework as well as a limited financial investment in biotechnology research. Keywords: Maize, Drought tolerance, Genetic engineering; Biotechnology; Transgenic crop

Genetic Transformation of Sweet Potato for Improved Tolerance to Stress: A Review

The sweet potato (Ipomoea batatas Lam) is a major staple food in many parts of the world. Sweet potato leaves and tubers are consumed as food and livestock feed. Biotic and abiotic stresses affect yield leading to a reduction in production. This review analyzes factors limiting sweet potato production and the progress made towards stress tolerance using genetic transformation. Genetic transformation could enhance yield, nutritional value and tolerance to stress. Transgenic sweet potatoes tolerant to biotic and abiotic stress, improved nutritional value and higher yields have been developed. Sweet potato expressing the endotoxin cry8Db, cry7A1 and cry3Ca genes showed lower sweet potato weevil infestation than non-transformed lines. Transgenic cultivar ‘Xushu18’ expressing the oryzacystatin-1 (OC1) gene showed enhanced resistance to sweet potato stem nematodes. Sweet potato line ‘Chikei 682-11’ expressing the coat protein (CP) exhibited resistance to the sweet potato feathery mottle virus (SPFMV). Transgenics expressing the rice cysteine inhibitor gene oryzacystatin-1 (OC1) also exhibited resistance to the SPFMV. Transgenic cultivar ‘Kokei’ expressing the spermidine synthetase gene FSPD1 had higher levels of spermine in the leaves and roots, and displayed enhanced tolerance to drought and salt stress. ‘Shangshu’ variety expressing the IbMas has shown enhanced tolerance to salt stress. Transgenic ‘Lixixiang’ expressing IbMIPSI showed an up-regulation of metabolites involved in stress response to drought, salinity and nematode infestation. Transgenic ‘Yulmi’ sweet potato transformed with copper/zinc superoxide dismutase (CuZnSOD) gene showed an enhanced tolerance to methyl viologen induced oxidative and chilling stress. Similarly, transformation of cultivar ‘Sushu-2’ with betaine aldehyde dehydrogenase (BADH) gene resulted in transgenics tolerant to salt, chilling and oxidative stress. Sweet potato varieties ‘Kokei14’ and ‘Yulmi’ transformed with the bar gene were shown to be tolerant to application of the herbicide Basta. The development of stress tolerant varieties will immensely increase the area under sweet potato production and eventually promote the adoption of sweet potato as a commercial crop. Sweet potato research and breeding for stress tolerance still faces technical and socio-political hurdles. Despite these challenges, genetic transformation remains a viable method with immense potential for the improvement of sweet potato. Key words: Sweet Potato (Ipomoea batatas Lam), Stress, Genetic Transformation, Transgeni

Silencing approach using poly(ADP-ribose) polymerase gene to improve drought stress tolerance in maize

Current and predicted climatic conditions, such as prolonged drought and heat episodes affect plant growth and yield, cause annual losses estimated at billions of dollars and pose a serious challenge for agricultural production worldwide, (Boyer 1982, Mittler 2006). Progress in generating transgenic crops with enhanced tolerance to abiotic stress has nevertheless been slow. The complex field environment with its heterogenic conditions, abiotic stress combinations, and global climatic changes are but a few of the challenges of modern agriculture. A combination of approaches has contributed significantly towards improving abiotic stress tolerance in both the greenhouse and the field (Hirayama and Shinozaki 2010). However, the increasing unpredictability and vagaries of rainfall and temperature variations consequent to global climate change, the dwindling availability of irrigation water and phosphate fertilizer, the escalating population in developing countries indicate that new ways of improving maize yield stability and stress tolerance under suboptimal conditions must be found and employed. A novel strategy to improve abiotic stress tolerance in Arabidopsis and Brassica has been reported by De Block et al., (2005). In their study, poly(ADP-ribosyl) ation activity was downregulated and transgenic plants became tolerant to a broad range of abiotic stresses such as drought, high light and heat. The researchers noted that stress tolerance was obtained by maintaining energy homeostasis due to reduced stress-induced energy consumption by prevention of NADP breakdown. Under abiotic stress conditions, plants overexpressing hairpin RNAi against Arabidopsis APP gene preserve their energy homeostasis without an overactivation of the mitochondrial respiration and avoiding the production of reactive oxygen species. Hence, plants with a lowered PARP activity appear tolerant to multiple stresses. Furthermore, a genome-wide transcript analysis of stressed anti PARP2 transgenic Arabidopsis (hpAt-PARP2) plants revealed that the induction of specific ABA signalling pathways steered increased levels of the cyclic nucleotide ADP-ribose (cADPR) thereby contributing towards tolerance to abiotic stresses (Vanderauwera et al., 2007). Indeed, modulation of poly(ADPribosyl) ation (PAR) reaction by an Arabidopsis thaliana ADP-ribose/NADH pyrophosphohydrolase, AtNUDX7 led to plants becoming tolerant to oxidative stress (Ogawa et al., 2009). Under oxidative stress, AtNUDX7 serves to maintain NAD+ levels by supplying ATP via nucleotide recycling from free ADP-ribose molecules and thus regulates the defence mechanisms against oxidative DNA damage via modulation of the PAR reaction. Furthermore, energy use efficiency is characterized by an epigenetic component that can be directed through selection to increase yield on top of heterosis (Hauben et al., 2009). Arabidopsis findings have previously been found to be directly applicable to commercial crop improvement (Nelson et al., 2005). Therefore, engineering crop plants for high NAD+ regeneration by an efficient 8 upregulation of the NAD+ salvage pathway or by a reduced NAD+ consumption under stress conditions, is a valuable approach to enhance overall stress tolerance in crops. The aim of this study was to assess a similar approach in the model species Zea mays through silencing maize PARP1 gene, a homolog of the Arabidopsis APP gene. This was achieved by establishing tropical maize regeneration system from immature embryo through somatic embryogenesis as a prerequisite for effective genetic transformation mediated by Agrobacterium. Our next goal was to engineer two hairpin constructs targeting maize PARP1 gene within the specific region at the 5’-end of the gene because silencing of the Arabidopsis APP gene using hairpin RNA constructs proved to be a valuable approach to obtain drought tolerance in Arabidopsis and canola (De Block et al., 2005). However, hairpin constructs are not so stable in extreme temperatures in the greenhouse which results in problems with stability in subsequent progenies (Szittya et al., 2003). Therefore, we aimed at evaluating the utility of artificial micro- RNAs (amiRNA) to silence the maize PARP1 gene. This technology is expected to be more effective and stable than the hairpin RNA approach because temperature does not affect the accumulation of microRNA (Szittya et al., 2003). Three amiRNAs were designed targeting maize PARP1 gene. As a complementary approach to the amiRNA and RNAi transgene technology we sought for mutants in the maize PARP1 gene in the Uniform Mu collections mutagenized by the transposon Mutator. Transgene technology and efficient transformation are important to fully exploit a species as a model for functional genomics studies. We developed an efficient and standardized maize transformation method based on the following criteria: (1) Agrobacterium tumefaciens co-cultivation was preferred over particle bombardment because usually intact T-DNA’s are transferred at low copy number, (2) a maize inbred line was favoured over a hybrid line because homogeneous progenies allow transgene testing already in T1 or T2, (3) Gateway vectors were optimized for use in monocots and provide a toolbox for rapid gene cloning. And finally, the transgenic maize plants overexpressing amiRNA constructs against maize PARP1 gene were evaluated for drought and Methyl Viologen-induced oxidative stress tolerance

Molecular improvement of tropical maize for drought stress tolerance in sub-Saharan Africa

The C4 grass Zea mays (maize or corn) is the third most important food crop globally after wheat and rice in terms of production and the second most widespread genetically modified (GM) crop, after soybean. Its demand is predicted to increase by 45% by the year 2020. In sub-Saharan Africa, tropical maize has traditionally been the main staple of the diet, 95% of the maize grown is consumed directly as human food and as an important source of income for the resource-poor rural population. However, its growth, development and production are greatly affected by environmental stresses such as drought and salinization. In this respect, food security in tropical sub-Saharan Africa is increasingly dependent on continuous improvement of tropical maize through conventional breeding involving improved germplasm, greater input of fertilizers, irrigation, and production of two or more crops per year on the same piece of land. Integration of advances in biotechnology, genomic research, and molecular marker applications with conventional plant breeding practices opens tremendous avenues for genetic modifications and fundamental research in tropical maize. The ability to transfer genes into this agronomically important crop might enable improvement of the species with respect to enhanced characteristics, such as enriched nutritional quality, high yield, resistance to herbicides, diseases, viruses, and insects, and tolerance to drought, salt, and flooding. These improvements in tropical maize will ultimately enhance global food production and human health. Molecular approaches to modulate drought stress tolerance are discussed for sub-Saharan Africa, but widely applicable to other tropical genotypes in Central and Latin America. This review highlights abiotic constraints that affect growth, development and production of tropical maize and subsequently focuses on the mechanisms that regulate drought stress tolerance in maize. Biotechnological approaches to manage abiotic stress tolerance in maize will be discussed. The current status of tropical maize transformation using Agrobacterium as a vehicle for DNA transfer is emphasized. This review also addresses the present status of genetically modified organisms (GMOs) regulation in sub-Saharan Africa

Higher plant transformation: principles and molecular tools

In higher plants, genetic transformation, which is part of the toolbox for the study of living organisms, had been reported only 30 years ago, boosting basic plant biology research, generating superior crops, and leading to the new discipline of plant biotechnology. Here, we review its principles and the corresponding molecular tools. In vitro regeneration, through somatic embryogenesis or organogenesis, is discussed because they are prerequisites for the subsequent Agrobacterium tumefaciens-mediated transferred (T)-DNA or direct DNA transfer methods to produce transgenic plants. Important molecular components of the T-DNA are examined, such as selectable marker genes that allow the selection of transformed cells in tissue cultures and are used to follow the gene of interest in the next generations, and reporter genes that have been developed to visualize promoter activities, protein localizations, and protein-protein interactions. Genes of interest are assembled with promoters and termination signals in Escherichia coli by means of GATEWAY-derived binary vectors that represent the current versatile cloning tools. Finally, future promising developments in transgene technology are considered

The Isolation, Screening, and Characterization of Polyhydroxyalkanoate-Producing Bacteria from Hypersaline Lakes in Kenya

Extremophilic microorganisms such as those that thrive in high-salt and high-alkaline environments are promising candidates for the recovery of useful biomaterials including polyhydroxyalkanoates (PHAs). PHAs are ideal alternatives to synthetic plastics because they are biodegradable, biocompatible, and environmentally friendly. This work was aimed at conducting a bioprospection of bacteria isolated from hypersaline-alkaliphilic lakes in Kenya for the potential production of PHAs. In the present study, 218 isolates were screened by Sudan Black B and Nile Red A staining. Of these isolates, 31 were positive for PHA production and were characterized using morphological, biochemical, and molecular methods. Through 16S rRNA sequencing, we found that the isolates belonged to the genera Arthrobacter spp., Bacillus spp., Exiguobacterium spp., Halomonas spp., Paracoccus spp., and Rhodobaca spp. Preliminary experiments revealed that Bacillus sp. JSM-1684023 isolated from Lake Magadi had the highest PHA accumulation ability, with an initial biomass-to-PHA conversion rate of 19.14% on a 2% glucose substrate. Under optimized fermentation conditions, MO22 had a maximum PHA concentration of 0.516 g/L from 1.99 g/L of cell dry weight and 25.9% PHA conversion, equivalent to a PHA yield of 0.02 g/g of biomass. The optimal PHA production media had an initial pH of 9.0, temperature of 35 °C, salinity of 3%, and an incubation period of 48 h with 2.5% sucrose and 0.1% peptone as carbon and nitrogen sources, respectively. This study suggests that bacteria isolated from hypersaline and alkaliphilic tropical lakes are promising candidates for the production of polyhydroxyalkanoates