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Transgenesis technologies, such as overexpression or RNA interference-mediated suppression, have often been used to alter the activity of target genes. More recently developed targeted genome modification methods using customizable endonucleases allow for the regulation or knockout mutation of target genes without the necessity of integrating recombinant DNA. Such approaches make it possible to create novel alleles of target genes, thereby significantly contributing to crop improvement. Among these technologies, the Cas9 endonuclease-based method is widely applied to several crops, including barley (Hordeum vulgare). In this chapter, we describe an Agrobacterium-based approach to the targeted modification of grain dormancy genes in barley using RNA-guided Cas9 nuclease., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Temperature-induced elongation of hypocotyls, petioles, and roots, together with hyponastic leaf responses, constitute key model phenotypes that can be used to assess a plant's capacity for thermomorphogenesis. Phenotypic responses are often quantified at a single time point during seedling development at different temperatures. However, to capture growth dynamics, several time points need to be assessed, and ideally continuous measurements are taken. Here we describe a general experimental setup and technical solutions for recording and measuring seedling phenotypes at single and multiple time points. Furthermore, we present an R-package called "rootdetectR," which allows easy processing of hypocotyl, root or petiole length, and growth rate data and provides different options of data presentation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The FAIR data principle as a commitment to support long-term research data management is widely accepted in the scientific community. However, although many established infrastructures provide comprehensive and long-term stable services and platforms, a large quantity of research data is still hidden. Currently, high-throughput plant genomics and phenomics technologies are producing research data in abundance, the storage of which is not covered by established core databases. This concerns the data volume, for example, time series of images or high-resolution hyperspectral data; the quality of data formatting and annotation, e.g., with regard to structure and annotation specifications of core databases; uncovered data domains; or organizational constraints prohibiting primary data storage outside institutional boundaries. To share these potentially dark data in a FAIR way and master these challenges the ELIXIR Germany/de.NBI service Plant Genomic and Phenomics Research Data Repository (PGP) implements an on-premise approach, which allows research data to be kept in place and wrapped in FAIR-aware software infrastructure. In this chapter, the e!DAL infrastructure software and the PGP repository are presented as best practice on how to easily setup FAIR-compliant and intuitive research data services., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fluorescence in situ hybridization (FISH) has been widely used to visualize target DNA sequences in fixed chromosome samples by denaturing the dsDNA to allow complementary probe hybridization, thus damaging the chromatin structure by harsh treatments. To overcome this limitation, a CRISPR/Cas9-based in situ labeling method was developed, termed CRISPR-FISH. This method is also known as RNA-guided endonuclease-in situ labeling (RGEN-ISL). Here we present different protocols for the application of CRISPR-FISH on acetic acid: ethanol or formaldehyde-fixed nuclei and chromosomes as well as tissue sections for labeling repetitive sequences in a range of plant species. In addition, methods on how immunostaining can be combined with CRISPR-FISH are provided., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The crop species barley is a genetic model for the small grain temperate cereals. Thanks to the availability of whole genome sequence and the development of customizable endonucleases, site-directed genome modification has recently revolutionized genetic engineering. Several platforms have been established in plants, with the most flexible one offered by the clustered regularly interspaced short palindromic repeats (CRISPR) technology. In this protocol, commercially available synthetic guide RNAs (gRNAs), Cas enzymes, or custom-generated reagents are used for targeted mutagenesis in barley. The protocol has been successfully used with immature embryo explants to generate site-specific mutations in regenerants. As the double-strand break-inducing reagents are customizable and can be efficiently delivered, pre-assembled ribonucleoprotein (RNP) complexes allow efficient generation of genome-modified plants., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Varietal differences within a species with agronomic importance are often based on minor changes in the genomic sequence. For example, fungus-resistant and fungus-susceptible wheat varieties may vary in only one amino acid. The situation is similar with the reporter genes Gfp and Yfp where two base pairs cause a shift in the emission spectrum from green to yellow. Methods of targeted double-strand break induction now allow this exchange precisely with the simultaneous transfer of the desired repair template. However, these changes rarely lead to a selective advantage that can be used in generating such mutant plants. The protocol presented here allows a corresponding allele replacement at the cellular level using ribonucleoprotein complexes in combination with an appropriate repair template. The efficiencies achieved are comparable to other methods with direct DNA transfer or integration of the corresponding building blocks in the host genome. They are in the range of 35 percent, considering one allele in a diploid organism as barley and using Cas9 RNP complexes., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The transient expression of veterinary vaccines in plants is a promising tool because of its low cost connected with a practically unlimited scale-up. To achieve these goals, two major challenges, high immunogenicity of vaccines and minimal of down-stream processing cost, have to be overcome. Here we present and discuss protocols enabling to generate highly immunogenic H5 influenza candidate vaccines as H5 oligomers, by transient expression in Nicotiana benthamiana plants and to perform analytical experiments as Western blot, ELISA, and hemagglutination and hemagglutination inhibition assays., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The cell cycle is a complex sequence of events by which cells grow and divide mitotically or meiotically. Mitosis results in the generation of two identical daughter cells, while meiosis generates gametes as a prerequisite for sexual reproduction. To study the localization and dynamics of proteins involved in the regulation and proceeding of the cell cycle, life cell imaging of proteins fused to fluorescent tags can be performed. However, in some cases this approach cannot be applied, e.g., due to low fluorescence intensity, fast bleaching, or degradation of recombinant proteins by the proteasome pathway. Instead, immunolabeling with protein-specific antibodies offers a useful approach for the analysis of intact cells. Alternatively, immunolabeling can also be applied to isolated and/or flow-sorted nuclei of particular cell cycle stages (G1, S, and G2) or of different endopolyploidy levels. The following chapter will detail indirect immunolabeling protocols to analyze the subcellular localization and distribution of cell cycle-specific proteins in Arabidopsis thaliana., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bimolecular fluorescence complementation (BiFC) assay is one of the sensitive techniques that allows to investigate direct protein-protein interactions (PPI) in vivo and visualize the subcellular localization of interacting proteins. It is based on splitting of a fluorescent protein into two nonfluorescent parts accordingly fused to two putative interacting partners. If interaction between studied proteins is possible, nonfluorescent parts come to close proximity resulting in reconstitution of the functional fluorescent protein and giving fluorescence under certain wavelength. BiFC analysis implies transient or stable expression of the proteins of interest and can be used as a method to test or validate the direct PPI in various biological pathways, including the regulation of gametogenesis, which is the main focus of this book. In our protocol we give detailed information for beginners about three main steps of BiFC analysis of centromeric protein interactions. These steps include (1) generation of appropriate expression clones with the help of Gateway cloning technology, (2) infiltration of Nicotiana benthamiana plants by Agrobacteria containing generated constructs, and (3) microscopic analysis of plants under fluorescence microscope. Also, we discuss appropriate negative controls that can be used for evaluation as well as recommendable vector systems, possible artifacts and measures to avoid artifactual interactions for BiFC assay., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

To unlock the genetic potential in crops, multi-genome comparisons are an essential tool. Decreasing costs and improved sequencing technologies have democratized plant genome sequencing and led to a vast increase in the amount of available reference sequences on the one hand and enabled the assembly of even the largest and most complex and repetitive crops genomes such as wheat and barley. These developments have led to the era of pan-genomics in recent years. Pan-genome projects enable the definition of the core and dispensable genome for various crop species as well as the analysis of structural and functional variation and hence offer unprecedented opportunities for exploring and utilizing the genetic basis of natural variation in crops. Comparing, analyzing, and visualizing these multiple reference genomes and their diversity requires powerful and specialized computational strategies and tools., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cryopreservation of shoot tips facilitates long-term storage of plant genetic resources which can otherwise only be propagated vegetatively. The vitrification approach using the cryoprotectant plant vitrification solution 3 (PVS3, 50% sucrose and 50% glycerol) is easy to handle, has shown to produce high regrowth percentages in a number of potato, mint, garlic, and shallot accessions, and is, thus, highly suitable for routine cryopreservation of plant genetic resources. In the current chapter, the vitrification procedure is described for potato, mint, garlic, and shallot and includes details about modifications for the different plant species. Special emphasis is given on the preparation of the different culture media, solutions, the culture conditions prior and post-cryopreservation, and the preparation of the shoot tips from different sources. Furthermore, protocols to introduce plants into in vitro culture and methods to estimate cryopreservation success are provided.

The generation of doubled haploid barley plants by means of the so-called "Bulbosum" method has been practiced for meanwhile five decades. It rests upon the pollination of barley by its wild relative Hordeum bulbosum. This can result in the formation of hybrid embryos whose further development is typically associated with the loss of the pollinator's chromosomes. In recent years, this principle has, however, only rarely been used owing to the availability of efficient methods of anther and microspore culture. On the other hand, immature pollen-derived embryogenesis is to some extent prone to segregation bias in the resultant populations of haploids, which is due to its genotype dependency. Therefore, the principle of uniparental genome elimination has more recently regained increasing interest within the plant research and breeding community. The development of the present protocol relied on the use of the spring-type barley cultivar Golden Promise. The protocol is the result of a series of comparative experiments, which have addressed various methodological facets. The most influential ones included the method of emasculation, the temperature at flowering and early embryo development, the method, point in time and concentration of auxin administration for the stimulation of caryopsis development, the developmental stage at embryo dissection, as well as the nutrient medium used for embryo rescue. The present protocol allows the production of haploid barley plants at an efficiency of ca. 25% of the pollinated florets., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

In this chapter we describe protocols for immunolabeling and FISH of pollen grains undergoing postmeiotic mitosis using Aegilops speltoides, Secale cereale, and Hordeum vulgare as models. Tissue sectioning of pollen overcomes the problem of the pollen grain wall impermeability that interferes with immunolocalization and in situ hybridization. The crucial element of the protocol is the generation and immobilization of tissue sections. Our method facilitates the investigation of the microspore cell divisions and pollen grain maturation.

Changing environmental conditions greatly affect the accumulation of many proteins; therefore, the analysis of alterations in the proteome is essential to understand the plant response to abiotic stress. Proteomics provides a platform for the identification and quantification of plant proteins responsive to cold stress and taking part in cold acclimation. Here, we describe the preparation of proteins for LC-MS measurement to monitor the changes of protein patterns during cold treatment in Arabidopsis thaliana. In our protocol, proteins are precipitated using TCA/acetone, quantified with 2D Quant Kit and digested with trypsin using a filter-based method and analyzed using an LC-MS approach. The acquired results can be further applied for label-free protein quantification.

Here, we describe a protocol for in planta delivery of chemical compounds into meiocytes of different barley genotypes not impacting plant fertility allowing to harvest seeds from treated plants. Compound uptake into meiocytes is assessed by determining 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Similar to EdU, other compounds being soluble in an aqueous solution can be delivered in planta before/during meiosis to decipher their impact on meiosis and meiotic recombination.We give practical advice on how to deliver EdU as compound example (delivery via injection or needle and thread, addition of detergents or surfactants to increase compound uptake), how in planta compound delivery can be established for your plant material under specific growing conditions, how to generate and characterize barley hybrid plants, and how to conduct a meiotic cytological study of (treated) barley plants.

Meiosis is a specialized cell division during which homologous chromosomes can exchange genetic material through recombination. This mechanism generates novel allelic combinations, which can be exploited by plant breeders to achieve crop improvement. Pollen grains are the haploid products of meiosis required in fertilization. Here, we describe two approaches to measure meiotic recombination in single haploid pollen nuclei. Pollen nuclei are first separated by fluorescence-activated cell-sorting. Afterwards, the DNA of single pollen nuclei can be amplified by multiple-displacement-amplification using Phi29 DNA polymerase and meiotic recombination events can be measured using KASP markers. Alternatively, the PicoPLEX DNA-seq kit can be used to amplify the DNA of single pollen nuclei followed by library preparation for whole-genome sequencing and subsequent bioinformatic analysis.

Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells. Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique uses the inactive variants of Cas9 combined with different fluorescent proteins (GFP and mRuby) and telomere-specific guide RNA to target telomeric repeats in Nicotiana benthamiana. Our immuno-FISH data revealed that signals arising from the CRISPR/dCas9 method are specifically belonging to telomeric regions.

Yeasts, like Arxula adeninivorans, Hansenula polymorpha, Pichia pastoris, Debaryomyces hansenii, Debaryomyces polymorphus, Schwanniomyces occidentalis, Yarrowia lipolytica, and Saccharomyces cerevisiae are frequently used producers of recombinant enzymes, particularly when posttranslational modifications are mandatory to obtain full functionality. The wide-range transformation/expression platform presented in this chapter can be used to select the optimal yeast host for high-level synthesis of the desired enzyme with favorable biochemical properties. This platform is composed of a selection marker and up to four expression modules in a linearized cassette. Here we describe the protocols for the assembly as well as the transformation of yeast strains with the respective cassettes, screening of transformants, the isolation and biochemical characterization of the enzymes, and finally a simple fermentation strategy to achieve maximal yields of the chosen recombinant enzyme.

Genome engineering involves methods of genetic modification of cells at predefined genomic sites. Here, we used transcription activator-like effector nucleases (TALENs) for the site-directed mutagenesis in barley. Target gene-specific TALEN-encoding expression units were designed and delivered to totipotent cells of either cultivated embryogenic pollen or immature embryos. The analysis of resulting transgenic plants revealed that the described approach allows for the generation of site-specific, heritable mutations at reasonable efficiency.

Recently, long noncoding RNAs (lncRNAs) are shown to be implicating nuclear domain organization and gene regulation by mediating long-range chromatin interactions. Chromosome conformation capture (3C) is a method used to study such long-range interaction between two different loci in the 3D nuclear space. Through successive improvement in resolution and throughput, 3C, chromosome conformation capture on chip (4C), and chromosome conformation capture carbon copy (5C) to Hi-C methods were developed to study interactions between loci from one versus one scale to an unprecedented genome-wide resolution. In situ Hi-C is a variant of Hi-C in which proximity ligation is performed at the intact nuclei to improve the signal-to-noise ratio and throughput of the experiment to provide useful genome-wide contact frequency matrix/maps. The contact frequency maps obtained could be used for physical ordering of scaffolds in complex genome assembly projects, in deducing the nuclear domain organization in high resolution and in identifying specific long-range interactions between genomic regions of interest. In this chapter, we describe in detail a protocol for in situ Hi-C used on crops like barley, wheat, rye, oat, and evening primrose.

TILLING (Targeting Induced Local Lesions IN Genomes), a popular reverse genetics approach in barley research, combines plant mutagenesis with efficient mutation detection for studying biological function of a specific gene. The high mutation frequency within a TILLING population principally enables the identification of induced variations in (almost) all genes of a given species (more precisely a given genotype of a species) of interest, which can be tested for their functional impact on morphological and/or physiological characteristics of the plant. Several TILLING populations induced by chemical mutagenesis were established for barley (Talame et al., Plant Biotechnol J 6:477-485, 2008; Gottwald et al., BMC Res Notes 2:258, 2009; Caldwell et al. Plant J 40:143-150, 2004) and showed the possibility for adapting protocols to develop further populations. This chapter describes a chemical mutagenesis protocol for barley seeds and two independent procedures for efficient single nucleotide polymorphism (SNP) detection in a large number of mutagenized plants either by slab-gel- or capillary gel-based electrophoreses on the LI-COR 4300 DNA Analyzer and the AdvanCE FS96 instruments, respectively.

Microscopic investigations of biological objects are an integral part in plant research and most fields of life sciences. They allow the description of morphological, histological, and structural aspects of individual cells or tissues. Based on various cell biological tools and methods it is possible to characterize different plant genotypes or study their adaptation to changing environmental conditions. In combination with antibodies raised against specific antigens and epitopes light and electron microscopy enable investigation of the function of single genes/proteins in plant growth and development or their role related to abiotic or biotic stresses.Here, we describe sample preparation of barley roots for cell biological investigations using light and electron microscopy, to characterize morphological, structural, and functional aspects on root sections and the root surface.

In situ nucleus and chromatin analyses rely on microscopy imaging that benefits from versatile, efficient fluorescent probes and proteins for static or live imaging. Yet the broad choice in imaging instruments offered to the user poses orientation problems. Which imaging instrument should be used for which purpose? What are the main caveats and what are the considerations to best exploit each instrument's ability to obtain informative and high-quality images? How to infer quantitative information on chromatin or nuclear organization from microscopy images? In this review, we present an overview of common, fluorescence-based microscopy systems and discuss recently developed super-resolution microscopy systems, which are able to bridge the resolution gap between common fluorescence microscopy and electron microscopy. We briefly present their basic principles and discuss their possible applications in the field, while providing experience-based recommendations to guide the user toward best-possible imaging. In addition to raw data acquisition methods, we discuss commercial and noncommercial processing tools required for optimal image presentation and signal evaluation in two and three dimensions.

The investigation of biological systems from different perspectives leads, due to novel -omics technologies, to large-scale, heterogeneous, and complex datasets. To elucidate molecular programs that control biological systems growth and development the integration and analysis of these -omics data remains challenging. Network-integrated visualizations based on graphical standards support intuitive exploration and interpretation of -omics data within the functional context. This integrated vision of the biological system to be studied tries to extract all hidden information for deepening our understanding and reveals new biological insights.The method described here gives detailed instructions on the generation of such an integrative visualization of -omics data in the context of networks presented in the Systems Biology Graphical Notation (SBGN) using VANTED; a software tool for systems biology applications. An example illustrates the application of the method for metabolomics and proteomics data integration and analysis using a primary metabolic pathway, for the model crop potato.

Plasma membrane (PM) proteins are of special interest due to their function in exchanging material and information with the external environment as well as their role in cellular regulation. In quantitative proteomic studies PM proteins are underrepresented mostly because they constitute only small percent of all membrane proteins. Strong demand is placed on plasma membrane enrichment methods. For decades two-phase partitioning Dextran T500/PEG 3350 isolation protocols were applied for many different animal and plant species and also a variety of tissue types. The typical quantity of material used in the enrichment protocols is 10-30 g of fresh weight. The main difficulty of working with in vitro cultivated plants is the low amount of material, especially when roots are examined. In addition, roots are frequently characterized by low protein concentrations. Our protocol established for roots of in vitro cultivated potato plants is adjusted to amounts of fresh weight not exceeding 7.5 g and allows studying the plasma membrane proteome by LC-MS.

Spatiotemporal patterning throughout the plant body depends to a large degree on cell- and tissue-specific expression of genes. Subsequently, for a better understanding of cell and tissue differentiation processes during plant development it is important to conduct transcript analyses in individual cells or tissue types rather than in bulk tissues. Laser capture microdissection (LCM) provides a useful method for isolating specific cell types from complex tissue structures for downstream applications. Contrasting to mammalian cells, the texture of plant cells is more critical due to hard, cellulose-rich cell walls, large vacuoles, and air spaces which complicates tissue preparation and extraction of macromolecules, like DNA and RNA. In particular, developing barley seeds (i.e. grains) depict cell types with differences in osmomolarity (meristematic, differentiating and degenerating tissues) and contain high amounts of the main storage product starch. In this study, we report about methods allowing tissue-specific transcriptome profiling by RNA-seq of developing barley grain tissues from low-input RNA amounts. Details on tissue preparation, laser capture microdissection, RNA isolation, and linear mRNA amplification to produce high-quality samples for Illumina sequencing are provided. Particular emphasis was placed on the influence of the mRNA amplification step on the transcriptome data and the fidelity of deduced expression levels obtained by the developed methods. Analysis of RNA-seq data confirmed sample processing as a highly reliable and reproducible procedure that was also used for transcriptome analyses of different tissue types from barley plants.

Wide hybridization is a one of the important techniques in plant breeding. Oat (Avena sativa L.) and pearl millet (Pennisetum glaucum L.) belong to different subfamilies of Poaceae. In generally, such distant relative species show uniparental chromosome elimination after successful fertilization. However, all seven pearl millet chromosomes are retained beside the genome of oat during embryogenesis. Hybrid seedlings develop, but show necrosis after light irradiation. Here, a detailed protocol for wide hybridization between oat and pearl millet is described.

Methods of the Agrobacterium-mediated transformation of bread wheat (Triticum aestivum L.) have been improved in recent years so that genetic engineering can be routinely used for functional genomics as well as for wheat breeding. In the protocol described here, immature embryos of the spring-type model genotype Bobwhite SH 98 26 have been used. Preculture and temperature pretreatment of these explants have led to the reproducible generation of transgenic plants at efficiencies between 5 and 15%. Whereas primary transgenic plants regenerated in vitro commonly show reduced fitness and fertility, no apparent variations with regard to morphology and grain set in their transgenic progeny as compared to wild-type counterparts were observed.

Conventional oxygen (micro-) sensors assess oxygen concentration within a particular region or across a transect of tissue, but provide no information regarding its bidimensional distribution. Here, a novel imaging technology is presented, in which an optical sensor foil (i.e., the planar optode) is attached to the surface of the sample. The sensor converts a fluorescent signal into an oxygen value. Since each single image captures an entire area of the sample surface, the system is able to deduce the distribution of oxygen at a resolution level of few micrometers. It can be deployed to dynamically monitor oxygen consumption, thereby providing a detailed respiration map at close to cellular resolution. Here, we demonstrate the application of the imaging tool to developing plant seeds; the protocol is explained step by step and some potential pitfalls are discussed.

The high-throughput analysis of respiratory activity has become an important component of many biological investigations. Here, a technological platform, denoted the "MultiSense tool," is described. The tool enables the parallel monitoring of respiration in 100 samples over an extended time period, by dynamically tracking the concentrations of oxygen (O 2 ) and/or carbon dioxide (CO 2 ) and/or pH within an airtight vial. Its flexible design supports the quantification of respiration based on either oxygen consumption or carbon dioxide release, thereby allowing for the determination of the physiologically significant respiratory quotient (the ratio between the quantities of CO 2 released and the O 2 consumed). It requires an LED light source to be mounted above the sample, together with a CCD camera system, adjusted to enable the capture of analyte-specific wavelengths, and fluorescent sensor spots inserted into the sample vial. Here, a demonstration is given of the use of the MultiSense tool to quantify respiration in imbibing plant seeds, for which an appropriate step-by-step protocol is provided. The technology can be easily adapted for a wide range of applications, including the monitoring of gas exchange in any kind of liquid culture system (algae, embryo and tissue culture, cell suspensions, microbial cultures).

Wheat zygotes can be mechanically isolated and cultivated to continue their development in vitro. Since each zygote needs to be individually isolated, only relatively few of these cells are available per experiment. To facilitate embryonic growth despite of this limitation, the zygotes are kept within a culture insert placed in a larger dish which itself contains embryogenic pollen cocultivated for continuous medium conditioning. This setup ensures that the two cultures, while being physically separated from one another, can exchange essential intercellular signal molecules passing through the bottom of the insert which is made of a permeable membrane. Thanks to the natural fate of zygotes, which is to form an embryo followed by the generation of a plant, embryogenesis and plant regeneration are achieved at much higher efficiency as compared to other single-cell systems. While the method is largely independent of the genotype, it allows for the nondestructive observation, manipulation, and individual analysis of zygotes and very young embryos.

The transient and stable expression of potentially therapeutic proteins in plants is a promising tool for the efficient production of vaccines and antibodies at low cost connected with a practically unlimited scale-up. To achieve these goals, two major challenges, inadequate production levels and non-scalable purification technologies, have to be overcome. Here we present and discuss protocols enabling to perform influenza vaccine production by transient expression in tobacco plants, to perform analytical experiments as Western blot, ELISA, and hemagglutination assays and to purify the antigens by classical affinity chromatography and scalable membrane-based Inverse Transition Cycling.

The cell cycle is a complex sequence of events by which cells grow and divide mitotically or meiotically. Mitosis results in the generation of two identical daughter cells, while meiosis generates gametes as a prerequisite for sexual reproduction. To study the localization and dynamics of proteins involved in the regulation and proceeding of the cell cycle, life cell imaging of proteins fused to fluorescent tags can be performed. However, in some cases this approach cannot be applied, e.g., due to low fluorescence intensity, fast bleaching or degradation of recombinant proteins by the proteasome pathway. Instead, immunolabeling with protein-specific antibodies represents a useful approach for the analysis of intact cells. Alternatively, immunolabeling can also be applied to isolated and/or flow-sorted nuclei of particular cell cycle stages (G1, S, and G2) or of different endopolyploidy levels. This chapter details indirect immunolabeling protocols to analyze the subcellular localization and distribution of cell cycle-specific proteins in Arabidopsis thaliana.

The temperate cereal barley is grown as a source of food, feed, and malt. The development of a broad range of genetic resources and associated technologies in this species has helped to establish barley as the prime model for the other Triticeae cereals. The specific advantage of the transformation method presented here is that transgene homozygosity is attained in the same generation as the transgenic event occurred through the coupling of haploid technology with Agrobacterium-mediated transformation. Pollen is haploid and, following transformation, can be induced to regenerate into haploid plantlets, which can subsequently subjected to colchicine treatment to obtain diploid, genetically fixed plants. The routine application of the method based on the winter-type barley cultivar 'Igri' over a period of over 10 years has achieved an average yield of about two transgenic plants per donor spike. The whole procedure from pollen isolation to non-segregating transgenic, mature grain takes less than 12 months.

Barley is a major crop species, and also has become a genetic model for the small grain temperate cereals. A draft barley genome sequence has recently been completed, opening many opportunities for candidate gene isolation and functionality testing. Thanks to the development of customizable endonucleases, also site-directed genome modification recently became feasible for higher plants, which marks the beginning of a new era of genetic engineering. The development of improved binary vectors and hypervirulent Agrobacterium tumefaciens strains has raised the efficiency of genetic transformation in barley to a level where the technique has become relatively routine. The transformation method described here involves immature barley embryos cocultivated with Agrobacterium after removal of their embryo axis. Critical adjustments to the protocol have included the supplementation of the cocultivation medium with the polyphenolic signaling compound acetosyringone at comparatively high concentration and the use of cysteine to reduce the extent of cellular oxidation upon agroinfection. In addition, the use of liquid, rather than solid, cocultivation medium promotes the throughput of the method. The protocol has delivered well over 10,000 transgenic barley plants over the past 10 years. Routine transformation efficiency, calculated on the basis of the recovery of independent transgenics per 100 explants, has reached about 25 % in cultivar (cv.) "Golden Promise". The protocol has proven effective for more than 20 barley cultivars, although some adjustments to the culture conditions have had to be made in some cases. The transformation efficiency of cv. "Golden Promise" remains higher than that of any other cultivar tested.

Isotope labelling experiments with stable or radioactive isotopes have long been an integral part of biological and medical research. Labelling experiments led to the discovery of new metabolic pathways and made it possible to calculate the fluxes responsible for a metabolic phenotype, i.e., the qualitative and quantitative composition of metabolites in a biological system. Prerequisite for efficient isotope labelling experiments is a reliable and precise method to analyze the redistribution of isotope label in a metabolic network. Here we describe the use of the CORRECTOR program, which utilizes matrix calculations to correct mass spectral data from stable isotope labelling experiments for the distorting effect of naturally occurring stable isotopes (NOIs). CORRECTOR facilitates and speeds up the routine quantification of experimentally introduced isotope label from multiple mass spectral readouts, which are generated by routine metabolite profiling when combined with stable isotope labelling experiments.

DIGE (differential in-gel electrophoresis) is a modified version of the widely used 2-D gel electrophoresis (2-DE) for separation of complex protein samples. Two extracts to be compared are differentially labeled using fluorescent cyanine dyes and then separated together by 2-DE. An internal standard labeled using a third dye is included. This approach avoids the pitfalls of gel distortions frequently observed in the standard procedure, which hamper the subsequent gel image analysis. Inclusion of an internal standard improves the quantitative evaluation of the protein patterns. Using the advantages of the DIGE approach, impact of minor temperature differences during cold stress treatment could be quantitatively monitored. We will describe the application of DIGE to monitor the impact of cold stress on the proteome pattern of Arabidopsis. In addition to the separation of proteins, we will also outline how plant growth is performed. Finally, we will also give protocols how proteins of interest can be identified by MALDI-TOF- as well as ESI-MS/MS.

In this chapter we illustrate the methodology for high-throughput metabolic flux analysis. Central to this is developing an end to end data pipeline, crucial for integrating the wet lab experiments and analytics, combining hardware and software automation, and standardizing data representation providing importers and exporters to support third party tools. The use of existing software at the start, data extraction from the chromatogram, and the end, MFA analysis, allows for the most flexibility in this workflow. Developing iMS2Flux provided a standard, extensible, platform independent tool to act as the "glue" between these end points. Most importantly this tool can be easily adapted to support different data formats, data verification and data correction steps allowing it to be central to managing the data necessary for high-throughput MFA. An additional tool was needed to automate the MFA software and in particular to take advantage of the course grained parallel nature of high-throughput analysis and available high performance computing facilities.In combination these methods show the development of high-throughput pipelines that allow metabolic flux analysis to join as a full member of the omics family.

Garlic (Allium sativum L.) is a very important medicinal and spice plant. It is conventionally propagated by daughter bulbs ("cloves") and bulbils from the flower head. Micropropagation is used for speeding up the vegetative propagation mainly using the advantage to produce higher numbers of healthy plants free of viruses, which have higher yield than infected material. Using primary explants from bulbs and/or bulbils (shoot tips) or unripe inflorescence bases, in vitro cultures are initiated on MS-based media containing auxins, e.g., naphthalene acetic acid, and cytokinins, e.g., 6-γ-γ-(dimethylallylaminopurine) (2iP). Rooting is accompanying leaf formation. It does not need special culture phases. The main micropropagation methods rely on growth of already formed meristems. Long-term storage of micropropagated material, cryopreservation, is well-developed to maintain germplasm. The main method is vitrification using the cryoprotectant mixture PVS3.

Synthetic biology methods are routinely applied in the plant field as in other eukaryotic model systems. Several synthetic components have been developed in plants and an increasing number of studies report on the assembly into functional synthetic genetic circuits. This chapter gives an overview of the existing plant genetic networks and describes in detail the application of two systems for inducible gene expression. The ethanol-inducible system relies on the ethanol-responsive interaction of the AlcA transcriptional activator and the AlcR receptor resulting in the transcription of the gene of interest (GOI). In comparison, the translational fusion of GOI and the glucocorticoid receptor (GR) domain leads to the dexamethasone-dependent nuclear translocation of the GOI::GR protein. This chapter contains detailed protocols for the application of both systems in the model plants potato and Arabidopsis, respectively.

B chromosomes are dispensable elements of the genome that do not recombine with the A chromosomes of the regular complement and that follow their own evolutionary pathway. Here, we survey current knowledge on the DNA/chromatin composition, origin, and drive mechanisms of B chromosomes and discuss the potential research applications of supernumerary chromosomes.

Current microarray technologies allow high-density in situ synthesis of oligonucleotides or ex situ spotting of target molecules (cDNA) for conducting genome-wide comparative gene expression profiling studies. The avalanche of available microarray gene expression data from model plant species covering cell-related, tissue-specific, and developmental events, as well as perturbations to a variety of environmental stimuli has triggered many activities regarding the development of adequate bioinformatics tools for the analysis of these complex data sets. In this chapter we summarize the technical issues of different microarray technologies, discuss the availability of bioinformatics tools, and present approaches to extract biologically meaningful knowledge. For case studies of abiotic stress transcriptome analysis we highlight the unprecedented opportunities provided by these high-throughput technologies to understand networks of regulatory and metabolic pathway responses of plant cells to the application of abiotic stress stimuli.

Visualization is a powerful method to present and analyze a large amount of data. It is increasingly important in bioinformatics and is used for exploring different types of molecular biological data, such as structural information, high throughput data, and biochemical networks. This chapter gives a brief introduction to visualization methods for bioinformatics and presents two commonly used techniques in detail: heatmaps and force-directed network layout.

Embryonic stem (ES) cells, the pluripotent cells of early embryos have been successfully cultured as undifferentiated cells. The cells are characterized by two unique properties, unlimited self-renewal capacity and the ability to differentiate into all cells of the body. Because of the high in vitro differentiation potential, ES cells have been used as model system in cell and developmental biology. Here we present methods that use mouse embryonic stem cells for the in vitro differentiation and characterization of neuronal, cardiac, pancreatic and hepatic cells, derivatives of the ectoderm, mesoderm and endoderm, respectively. In the future, differentiated cells may be also generated from human ES cells by cultivation of early embryos or from reprogrammed cells derived by nuclear transfer. Such cells could represent potential sources for tissue repair of serious human diseases.