MicroTom—a high-throughput model transformation system for functional genomics

We have developed a high-throughput Agrobacterium-mediated transformation model system using both nptII and the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (cp4) based selections in MicroTom, a miniature rapid-cycling cherry tomato variety. With the NPTII selection system, transformation frequency calculated as independent transgenic events per inoculated explant ranged from 24 to 80% with an average of 56%, in industrial production scale transformation experiments. For CP4, with glyphosate selection, the average transformation frequency was 57%. Stable transformation frequency was positively correlated with transient expression (R=0.85), and variable with the genes of interest. DNA integration and germline transformation were confirmed by biological assay, Southern Blot analysis, and R₁ phenotype segregation. Transgene expression was observed in leaf, root, stem, flower, and fruit tissues of the transgenic plants. Ninety-five percent of transgenic events coexpressed two introduced genes based on β-glucuronidase (GUS) and neonmycin phosphotransferase II (NPTII) expression. Seventy-five percent of transgenic events contained one to two copies of the introduced uidA (GUS) gene based on Southern analysis. Transgenic plants from the cotyledon explants to the transgenic plants transferred to soil were produced within about 2–3 months depending on the genes of interest. The utility of this MicroTom model transformation system for functional genomic studies, such as identification of genes related to important agricultural traits and gene function, is discussed.     

TableButler – a Windows based tool for processing large data tables generated with high-throughput methods

Background  

High-throughput "omics" based data analysis play emerging roles in life sciences and molecular diagnostics. This emphasizes the urgent need for user-friendly windows-based software interfaces that could process the diversity of large tab-delimited raw data files generated by these methods. Depending on the study, dozens to hundreds of these data tables are generated. Before the actual statistical or cluster analysis, these data tables have to be combined and merged to expression matrices (e.g., in case of gene expression analysis). Gene annotations as well as information concerning the samples analyzed may be appended, renewed or extended. Often additional data values shall be computed or certain features must be filtered out.     

Development of a new hydrogen peroxide–based vaccine platform

Safe and effective vaccines are crucial for maintaining public health and reducing the global burden of infectious disease. Here we introduce a new vaccine platform that uses hydrogen peroxide (H(2)O(2)) to inactivate viruses for vaccine production. H(2)O(2) rapidly inactivates both RNA and DNA viruses with minimal damage to antigenic structure or immunogenicity and is a highly effective method when compared with conventional vaccine inactivation approaches such as formaldehyde or β-propiolactone. Mice immunized with H(2)O(2)-inactivated lymphocytic choriomeningitis virus (LCMV) generated cytolytic, multifunctional virus-specific CD8(+) T cells that conferred protection against chronic LCMV infection. Likewise, mice vaccinated with H(2)O(2)-inactivated vaccinia virus or H(2)O(2)-inactivated West Nile virus showed high virus-specific neutralizing antibody titers and were fully protected against lethal challenge. Together, these studies demonstrate that H(2)O(2)-based vaccines are highly immunogenic, provide protection against a range of viral pathogens in mice and represent a promising new approach to future vaccine development.     

The pistachio nut—a new crop for the Western United States

Importers claim that each year they could process and sell approximately 15,000 tons ot pistachio nuts it they were available. Presently, only a small quantity are produced commercially in Caliiornia. Experimental work with selected imported varieties, breeding, cultivating, pruning and other horticultural practises at the United States Department of Agriculture's Plant Introduction Station at Chico, California has demonstrated the economic feasibility of this crop for American growers.     

Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method

To isolate enantioselective α-hydroxyacid dehydrogenases (α-HADHs), a high-throughput screening method was established. 2,4-Dinitrophenylhydrazine solution forms a red-brown complex with ketoacid produced during the α-HADH-mediated oxidation of α-hydroxyacid. The complex can be easily quantified by spectrophotometric measurement at 458?nm. The enantioselectivity of α-HADH in each strain can be measured with this colorimetric method using (R)- and (S)-α-hydroxyacid concurrently as substrates to evaluate the apparent enantioselectivity (E _app). The E _app closely matches the value of true enantioselectivity (E _true) determined by HPLC analysis. With this method, a total of 34 stains harboring enantioselective α-HADHs were selected from 526 potential α-HADH-producing microorganisms. Pseudomonas aeruginosa displayed the highest (S)-enantioselective α-HADH activity. This strain appears promising for potential application in industry to produce (R)-α-hydroxyacids. The method described herein represents a useful tool for the high-throughput isolation of enantioselective α-HADHs.     

The OASIS® HLB μElution plate as a one-step platform for manual high-throughput in-gel digestion of proteins and peptide desalting

Separation or prefractionation of proteins by gel electrophoresis, followed by in-gel proteolytic digest and analysis by MS is a typical standard application in proteomics. For many laboratories, it is not cost-effective to use a robot with all its drawbacks. On the other hand, manual digest is time consuming and not free of error if many samples are processed at the same time. The OASIS? HLB μElution plate allows the handling of 96 samples for protein in-gel digest and peptide desalting in a semiautomatic way. Using multichannel pipettes, solutions are added quickly, and with the use of the positive pressure-96 stand all solutions are removed simultaneously. In this article, a complete and detailed protocol for the use of the OASIS? HLB μElution plate is introduced and its effectiveness is shown with 2D- and 1D-gel samples.     

Aquaporins in the honeybee crop—a new function for an old organ

Nectar used by bees as a food source is collected and stored in the crop, where it is transported and converted into honey. The production of honey involves water uptake from nectar. However, the crop is a portion of the insect foregut that has been characterized solely as a food storage organ. Aquaporins are integral membrane proteins that function as specific canal for water transport and are abundant in tissues with high water permeability. In this study, we detected five predicted genes for aquaporins in the gut of the honeybee Apis mellifera. We evaluated the aquaporins’ localization in the crop by using an anti-aquaporin antibody produced against the peptide sequence from one of the expressed genes, which was detected in the crop epithelium, particularly in the apical portions of the cells. Furthermore, we also showed an increase in sugar concentration in a sucrose solution collected from the crop lumen a few minutes after feeding, indicating that water uptake occurs during storage of nectar in the crop, suggesting a previously unidentified function for the honeybee crop.     

Crop 3D—基于激光雷达技术的作物高通量三维表型测量平台

作物表型测量技术发展的滞后已成为当前育种领域的发展瓶颈,高通量的精确表型测量有助于加速育种进程.激光雷达是一种新兴的主动遥感技术,能够精确获取作物的空间形态数据,在高通量作物表型监测中有广阔应用前景.然而,目前我国基于激光雷达技术的作物表型监测仍存在较大空白.因此,本课题组自主研发了一套以激光雷达为主,集成高分辨率相机、热成像仪、高光谱成像仪等传感器的高通量作物测量平台—Crop3D.与传统作物表型测量技术相比,Crop 3D优势在于能够通量化同步地对作物各生长时期进行多源表型数据的获取并提取株高、株幅、叶长、叶宽、叶倾角和叶面积等参数,可为植物生物学和基因组学分析提供数据支持.本文重点对Crop 3D平台的整体规划设计、传感器集成、运行模式及平台获取的表型参数做了详细描述,并对其潜在应用领域做了简要探讨.本课题组认为,激光雷达与传统表型测量技术相结合的集成型平台有望成为未来作物表型参数获取的趋势所在.     

Plant genome sequencing — applications for crop improvement

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QuantPrime – a flexible tool for reliable high-throughput primer design for quantitative PCR

Background  

Medium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays.     

Lablab purpureus—A Crop Lost for Africa?

In recent years, so-called ‘lost crops’ have been appraised in a number of reviews, among them Lablab purpureus in the context of African vegetable species. This crop cannot truly be considered ‘lost’ because worldwide more than 150 common names are applied to it. Based on a comprehensive literature review, this paper aims to put forward four theses, (i) Lablab is one of the most diverse domesticated legume species and has multiple uses. Although its largest agro-morphological diversity occurs in South Asia, its origin appears to be Africa. (ii) Crop improvement in South Asia is based on limited genetic diversity. (iii) The restricted research and development performed in Africa focuses either on improving forage or soil properties mostly through one popular cultivar, Rongai, while the available diversity of lablab in Africa might be under threat of genetic erosion. (iv) Lablab is better adapted to drought than common beans (Phaseolus vulgaris) or cowpea (Vigna unguiculata), both of which have been preferred to lablab in African agricultural production systems. Lablab might offer comparable opportunities for African agriculture in the view of global change. Its wide potential for adaptation throughout eastern and southern Africa is shown with a GIS (geographic information systems) approach.     

Penman-Monteith approaches for estimating crop evapotranspiration in screenhouses—a case study with table-grape

In arid and semi-arid regions many crops are grown under screens or in screenhouses to protect them from excessive radiation, strong winds, hailstorms and insects, and to reduce crop water requirements. Screens modify the crop microclimate, which means that it is necessary to accurately estimate crop water use under screens in order to improve the irrigation management and thereby increase water-use efficiency. The goal of the present study was to develop a set of calibrated relationships between inside and outside climatic variables, which would enable growers to predict crop water use under screens, based on standard external meteorological measurements and evapotranspiration (ET) models. Experiments were carried out in the Jordan Valley region of eastern Israel in a table-grape vineyard that was covered with a transparent screen providing 10 % shading. An eddy covariance system was deployed in the middle of the vineyard and meteorological variables were measured inside and outside the screenhouse. Two ET models were evaluated: a classical Penman-Monteith model (PM) and a Penman-Monteith model modified for screenhouse conditions by the inclusion of an additional boundary-layer resistance (PMsc). Energy-balance closure analysis, presented as a linear relation between half-hourly values of available and consumed energy (1,344 data points), yielded the regression Y?=?1.05X–9.93 (W m^?2), in which Y = sum of latent and sensible heat fluxes, and X = net radiation minus soil heat flux, with R ²?=?0.81. To compensate for overestimation of the eddy fluxes, ET was corrected by forcing the energy balance closure. Average daily ET under the screen was 5.4?±?0.54 mm day^?1, in general agreement with the model estimates and the applied irrigation. The results showed that measured ET under the screen was, on average, 34 % lower than that estimated outside, indicating significant potential water saving through screening irrigated vineyards. The PM model was somewhat more accurate than the PMsc for estimating ET under the screen. A model sensitivity analysis illustrates how changes in certain climatic conditions or screen properties would affect evapotranspiration.     

Computer controlled cryo-electron microscopy--TOM² a software package for high-throughput applications

Automated data acquisition expedites structural studies by electron microscopy and it allows to collect data sets of unprecedented size and consistent quality. In electron tomography it greatly facilitates the systematic exploration of large cellular landscapes and in single particle analysis it allows to generate data sets for an exhaustive classification of coexisting molecular states. Here we describe a novel software philosophy and architecture that can be used for a great variety of automated data acquisition scenarios. Based on our original software package TOM, the new TOM(2) package has been designed in an object-oriented way. The whole program can be seen as a collection of self-sufficient modules with defined relationships acting in a concerted manner. It subdivides data acquisition into a set of hierarchical tasks, bonding data structure and the operations to be performed tightly together. To demonstrate its capacity for high-throughput data acquisition it has been used in conjunction with instrumentation combining the latest technological achievements in electron optics, cryogenics and robotics. Its performance is demonstrated with a single particle analysis case study and with a batch tomography application.     

Quinoa – a Model Crop for Understanding Salt-tolerance Mechanisms in Halophytes

Quinoa (Chenopodium quinoa Willd.) is an ancient Andean crop that produces edible seeds and leaves. Quinoa's tolerance to salinity and other types of abiotic stresses provides it with high potential in a world where scarcity of water and increased soil salinization are important causes of crop failures. Due to its traditionally broad cultivation area (from Colombia to southern Chile), there is a wide range of quinoa cultivars adapted to specific conditions displaying a broad genetic variability in stress tolerance. In addition, being practically unique as a halophytic seed-producing crop with amazing nutritional properties, it is ideal as a model species for investigating morphological, cellular, physiological, and bio-molecular mechanisms of salinity tolerance. This review summarizes current knowledge of genotype-dependent variability in salinity responses and adaptive salt-tolerance mechanisms in quinoa. These include anatomical features and physiological aspects, such as osmotic adjustment through accumulation of ions, osmoprotectants, and sodium loading, transport, and storage, including the activity and gene expression of plasma and vacuolar membrane transporters. Finally, current knowledge regarding the effect of salinity on the nutritional properties of quinoa is discussed.