The development and evaluation of single cell suspension from wheat and barley as a model system; a first step towards functional genomics application

<p>Abstract</p> <p>Background</p> <p>The overall research objective was to develop single cell plant cultures as a model system to facilitate functional genomics of monocots, in particular wheat and barley. The essential first step towards achieving the stated objective was the development of a robust, viable single cell suspension culture from both species.</p> <p>Results</p> <p>We established growth conditions to allow routine culturing of somatic cells in 24 well microtiter plate format. Evaluation of the wheat and barley cell suspension as model cell system is a multi step process. As an initial step in the evaluation procedure we chose to study the impact of selected abiotic stress elicitors at the physiological, biochemical and molecular level. We report the results of osmotic stress imposed by NaCl and PEG. As proline is an important osmoprotectant of the cereal cells, colorimetric assay for proline detection was developed for small volumes (200 μl). We performed RT-PCR experiments to study the change in the expression of the genes encoding Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and Δ¹-pyrroline-5-carboxylate reductase (PC5R) in response to abiotic stress.</p> <p>Conclusions</p> <p>We found differences between the wheat and barley suspension cultures, barley being more tolerant to the applied osmotic stresses. We suggested a model to explain the obtained differences in stress tolerance between the two species. The suspension cell cultures have proven useful for determining changes in proline concentration and expression level of genes (P5CS, P5CR) under various treatments and we suggest that the cells can be used as a model host system to study gene expression and regulation in monocots.</p

BIOTECHNOLOGY ISSUES FOR DEVELOPING COUNTRIES - Biotechnology in Developing Countries: Harnessing the Potential of High-TECH SMES in the Face of Global Competition

In the global market, the emergence of new technologies are representing opportunities and challenges to both industry and the public sector of the Latin American Countries (LAC). Some of these technologies have far reaching implications for a large number of industrial sectors, creating wealth and employment, resulting in an increased competitiveness of the developed economies around the world. Modern biotechnology is one of these key emerging technologies which LAC must harness successfully to sustain economic growth and competitiveness. The ability to commercially exploit research in this area is also of prime importance. Developed countries have made important progress in recent years, with a strong leadership of USA in this sector. Unless substantial steps are taken now by LAC the gap between developed and developing economies will only continue to widen in the short, medium and long term. In this, the role of Small and Medium size Enterprises (SMES) is of crucial importance, especially because "High-TECH SMES" firms, by their nature, are often start-ups where new products are developed. Therefore, it is up to public authorities and venture-capitalists, at both the national and regional level to help engender a more productive external business. This paper is aimed to provide an overview of the options to harness the potential of "High-TECH SMES" for generating and accessing to new technologies, both at the national and global levels, with particular emphasis on biotechnology sectors in order to improve the LAC competitiveness

Purity and structural composition of lignin isolated from Miscanthus x giganteus by sub-critical water extraction with associated modifiers

Lignin is an industrial by-product produced from the pulping and paper industry, where the process generates lignin in the form of lignosulphonates. While there are many applications for lignin, there are all low value and attempts to add value to lignin are hindered by its complex physicochemical nature and the presence of sulphur. Adopting the biorefining concept, the study evaluates the impact of direct (DE) and sequential extraction (SE) of Miscanthus x giganteus using sub-critical water with associated modifiers; ethanol and carbon dioxide on the physical and chemical properties of the extracted lignin. Isolated lignins were characterised by a Fourier Transform Infrared Spectroscopy (FTIR). Although higher delignification was achieved by DE and SE about 81.5% and 58.0%, respectively, the lignin recovered from the SE process showed remarkably higher purity with 91.5%. Lignin recovery did not differ considerably for either processing method. FTIR revealed a qualitative reduction in the intensity of bonds corresponding to hydroxyl groups for the lignin derived from DE rather than SE processing routes. These indicated that the lignin derived from SE had potential for subsequent preparation in lignin value-added bio-based materials

Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development

BACKGROUND: Cereal storage proteins represent one of the most important sources of protein for food and feed and they are coded by multigene families. The expression of the storage protein genes exhibits a temporal fluctuation but also a response to environmental stimuli. Analysis of temporal gene expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging. RESULTS: We designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and γ-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families, subfamilies or individual members. The specificity of the primer sets was validated before successfully applying them to a cDNA population derived from developing grains of field grown Hordeum vulgare cv. Barke. The results quantify the number of moles of transcript contributed to a particular gene family and its subgroups. More over the results indicate the genotypic specific gene expression. CONCLUSIONS: Quantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal expression of genes coding for barley storage proteins. The results imply that our rapid qRT-PCR system was sensitive enough to identify the presence of alleles and their expression profiles. It can be used to check the temporal fluctuations in hordein expressions or to find differences in their response to environmental stimuli. The method could be extended for cultivar recognition as some of the sequences from the database originated from cv. Golden Promise were not expressed in the studied barley cultivar Barke although showed primer specificity with their cloned DNA sequences

A pathway-specific microarray analysis highlights the complex and co-ordinated transcriptional networks of the developing grain of field-grown barley

The aim of the study was to describe the molecular and biochemical interactions associated with amino acid biosynthesis and storage protein accumulation in the developing grains of field-grown barley. Our strategy was to analyse the transcription of genes associated with the biosynthesis of storage products during the development of field-grown barley grains using a grain-specific microarray assembled in our laboratory. To identify co-regulated genes, a distance matrix was constructed which enabled the identification of three clusters corresponding to early, middle, and late grain development. The gene expression pattern associated with the clusters was investigated using pathway-specific analysis with specific reference to the temporal expression levels of a range of genes involved mainly in the photosynthesis process, amino acid and storage protein metabolism. It is concluded that the grain-specific microarray is a reliable and cost-effective tool for monitoring temporal changes in the transcriptome of the major metabolic pathways in the barley grain. Moreover, it was sensitive enough to monitor differences in the gene expression profiles of different homologues from the storage protein families. The study described here should provide a strong complement to existing knowledge assisting further understanding of grain development and thereby provide a foundation for plant breeding towards storage proteins with improved nutritional quality

Biotechnology in developing countries: harnessing the potential of high-TECH SMES in the face of global competition

In the global market, the emergence of new technologies are representing opportunities and challenges to both industry and the public sector of the Latin American Countries (LAC). Some of these technologies have far reaching implications for a large number of industrial sectors, creating wealth and employment, resulting in an increased competitiveness of the developed economies around the world. Modern biotechnology is one of these key emerging technologies which LAC must harness successfully to sustain economic growth and competitiveness. The ability to commercially exploit research in this area is also of prime importance. Developed countries have made important progress in recent years, with a strong leadership of USA in this sector. Unless substantial steps are taken now by LAC the gap between developed and developing economies will only continue to widen in the short, medium and long term. In this, the role of Small and Medium size Enterprises (SMES) is of crucial importance, especially because "High-TECH SMES" firms, by their nature, are often start-ups where new products are developed. Therefore, it is up to public authorities and venture-capitalists, at both the national and regional level to help engender a more productive external business. This paper is aimed to provide an overview of the options to harness the potential of "High-TECH SMES" for generating and accessing to new technologies, both at the national and global levels, with particular emphasis on biotechnology sectors in order to improve the LAC competitiveness

Organosolv lignin aggregation behaviour of soluble lignin extract from Miscanthus x giganteus at different ethanol concentrations and its influence on the lignin esterification

Background: Lignin is the second most abundant naturally occurring biopolymer from lignocellulosic biomass. While there are several lignin applications, attempts to add value to lignin are hampered by its inherent complex and heterogenous chemical structure. This work assesses the organosolv lignin aggregates behaviour of soluble lignin extract derived from Miscanthus × giganteus using diferent ethanol concentrations (50%, 40%, 30%, 20%, 10% and 1%). The efect of two diferent lignin concentrations using similar ethanol concentration on the efcacy of esterifcation was studied. Results: Overall, particle size of lignin analysis showed that the particle size of lignin aggregates decreased with lower ethanol concentrations. 50% ethanol concentration of soluble lignin extract showed the highest particle size of lignin (3001.8 nm), while 331.7 nm of lignin particle size was recorded at 1% ethanol concentration. Such fndings of particle size correlated well with the morphology of the lignin macromolecules. The lignin aggregates appeared to be disaggregated from population of large aggregates to sub-population of small aggregates when the ethanol concentration was reduced. Light microscopy images analysis by ImageJ shows that the average diameter and circularity of the corresponding lignin macromolecules difers according to diferent ethanol concentrations. The dispersion of lignin aggregates at low ethanol concentration resulted in high availability of hydroxyl group in the soluble lignin extract. The efcacy of the lignin modifcation via esterifcation was evidenced directly via FTIR using the similar ethanol concentration of soluble lignin extract at diferent lignin concentrations. Conclusion: This study provided the understanding of detail analysis on particle size determination, microscopic properties and structural insights of lignin aggregates at wider ethanol concentrations. The esterifed lignin derived at 5 mg/mL is suggested to expand greater lignin functionality in the preparation of lignin bio-based materials

Effects of ethanol concentration on organosolv lignin precipitation and aggregation from Miscanthus x giganteus

This work assesses the behavior of organosolv lignin aggregates derived from Miscanthus x giganteus using different ethanol concentrations (10%, 25%, 50%, and 75% by volume). The percentage of lignin recovery was found to decrease from 75.8% to 71.4% and 25.1%, as the ethanol concentration was increased from 10% to 25% and 50%, respectively. Increasing the ethanol concentration further to 75% led to zero recovery. The purity of the precipitated lignin was consistently found to be ≥90%. Lignin derived from the dried supernatant obtained at 50% ethanol concentration resulted in high lignin purity (51.6%) in comparison with the other ethanol concentrations used. Fourier transform infrared spectroscopy analysis showed that the precipitated lignin and dried supernatant at 50% ethanol concentration possessed the highest peak intensity apportioned to wavenumber of lignin as compared to that of at 25% and 10% ethanol concentrations, and the results linked with the percentage of lignin purity. The results of particle size analysis for precipitated lignin demonstrated particle sizes of 306, 392, and 2050 nm for 10%, 25%, and 50% ethanol concentrations, respectively, and the remaining supernatant with average particle sizes of 1598, 1197, and 875 nm, respectively. These results were verified with the morphology of lignin macromolecules in scanning electron microscopy images. Results of the particle size distribution of lignin revealed that the overall size of lignin aggregates decreased with decreasing ethanol concentration. In summary, these findings suggest that ethanol concentration affected the behavior of lignin aggregates in water–ethanol solution

Subcritical Water Processing of Proteins: An Alternative to Enzymatic Digestion?

Subcritical water is an emerging tool in the processing of bioorganic waste. Subcritical water is an environmentally benign solvent which has the potential to provide an alternative to traditional methods of protein hydrolysis without the inclusion of expensive acids or enzymes. To date, most studies on the subcritical water mediated hydrolysis of proteins have focused on the production of amino acids, rather than the intermediate peptides. Here, we investigate the specificity of subcritical water with respect to the production of peptides from three model proteins, hemoglobin, bovine serum albumin, and β-casein, and compare the results with enzymatic digestion of proteins by trypsin. In addition, the effect of subcritical water (SCW) treatment on two protein post-translational modifications, disulfide bonds and phosphorylation, was investigated. The results show that high protein sequence coverages (>80%) can be obtained following subcritical water hydrolysis. These are comparable to those obtained following treatment with tryspin. Under mild subcritical water conditions (160 °C), all proteins showed favored cleavage of the Asp-X bond. The results for β-casein revealed favored cleavage of the Glu-X bond at subcritical water temperatures of 160 and 207 °C. That was similarly observed for bovine serum albumin at a subcritical water temperature of 207 °C. Subcritical water treatment results in very limited cleavage of disulfide bonds. Reduction and alkylation of proteins either prior to or post subcritical water treatment improve reported protein sequence coverages. The results for phosphoprotein β-casein show that, under mild subcritical water conditions, phosphorylation may be retained on the peptide hydrolysis products