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The plant life cycle is characterized by the alternation of generations between genetically active diploid sporophytes and haploid gametophytes. The gametophytes of flowering plants are sexually dimorphic. While the male gametophyte consists of only three cells (two sperm and a vegetative cell) and is released by the parent sporophyte, the female gametophyte (or embryo sac) is more complex and remains imbedded within diploid sporophyte tissues. In maize, the female gametophyte is embedded in a large ovule surrounded with multiple nucellar cell layers impeding live-cell imaging approaches to study embryo sac functions. Here, we describe a simple protocol to visualize embryo sacs with hormonal fluorescent reporters by increasing accessibility of the female gametophyte. The method described is applicable for visualization of any fluorescent embryo sac reporter. The embryo sacs visualization method developed for maize could be extended to facilitate visualization of embryos sac in other important cereals like wheat, rice, and oats.

Much of meiosis research is focussed on Arabidopsis thaliana, largely due to the significant advantages it brings, having a small sequenced genome with comparatively little repetitive DNA, the ease of forward and reverse genetics, and a short life cycle. On the other hand, due the small genome size using Arabidopsis may be problematic for generating sufficient meiotic material for other types of analysis e.g., proteomics using prophase meiocytes and cytological analysis of the synaptonemal complex at the subcellular level. One solution is to use closely related species with larger genomes, in this case the Brassicas. This chapter contains methods for spreading of Brassica oleracea meiocytes for the analysis of the synaptonemal complex by silver staining and immunolocalization with gold-coupled antibodies using transmission electron microscopy.

Advances in the molecular biology and genetics of Arabidopsis thaliana have led to it becoming an important model for the analysis of meiosis in plants. Cytogenetic investigations are pivotal to meiotic studies and a number of technological improvements for Arabidopsis cytology have provided a range of tools to investigate chromosome behavior during meiosis (Jones et al. Chromosome Res 11:205-215, 2003). This chapter contains a detailed description of an immunological technique currently used in our lab for the preparation of meiotic chromosomes for immunolocalization.

Immunolocalization studies to visualize the distribution of proteins on meiotic chromosomes have become an integral part of studies on meiosis in the model organism Arabidopsis thaliana. This chapter describes fixation, sample preparation, and immunolocalization techniques used in our laboratory. These techniques have been used to visualize a wide range of meiotic proteins involved in different aspects of meiosis, including sister chromatid cohesion, recombination, synapsis, and chromosome segregation.

The success of meiosis depends on intricate coordination of a series of unique cellular processes to ensure proper chromosome segregation. Many proteins involved in these cellular events are directly or indirectly associated with chromosomes, especially those required for homologous recombination. These meiotic processes have been explored extensively by conventional light microscopy. However, many features of interest, such as chromatin organization, recombination nodules, or the synaptonemal complex are beyond the resolution of conventional wide-field microscopy. Moreover, in most sample preparation techniques for light microscopy, meiotic cells are squashed, which destroys the spatial organization of the nucleus. Here, I describe a protocol to analyze maize meiotic chromosomes by three-dimensional structured illumination microscopy (3D-SIM), a recently developed high-resolution microscopy technique. This protocol can be used to examine protein localizations at a high resolution level by immunofluorescence.

Transmission electron microscopy (TEM) of thin sections of meiocytes embedded in plastic resin allows analysis of structures in meiotic nuclei, such as the synaptonemal complex, at very high resolution while preserving the spatial organization of chromosomes.

Plant meiosis involves complex and dynamic processes that occur within the space inside the nucleus. Direct inspection of meiotic chromosomes by fluorescence microscopy has been used to investigate many of these processes. In particular, optical sectioning microscopy of fluorescence in situ hybridization (FISH)-stained nuclei provides three-dimensional spatial information about the organization and distribution of specific sequences and chromosomal loci within the nucleus. Here we provide a fully detailed three-dimensional (3D) acrylamide FISH method for the analysis of plant meiotic nuclei. Several examples illustrate the versatility of this technique for the investigation of meiotic telomere dynamics in maize, Arabidopsis, and oat. Additional examples of 3D FISH include chromosome painting in a maize chromosome-addition line of oat and telomere FISH with maize nuclei from plants expressing a fluorescently tagged fusion protein, histone H2B-mCherry.

Meiosis is a key step in sexual plant reproduction. Cytological analysis of meiosis enables hypotheses of gene function during meiosis to be confirmed or discarded. Observation of meiosis in the female reproductive organs in many higher plants is technically challenging because the cells are enclosed deep within the nucellus and ovule tissues. In this chapter, we describe a simple staining procedure using Schiff's reagent optimized for use with confocal microscopy to observe meiosis within intact ovules of Zea mays (maize). Using this procedure we present images taken by confocal laser scanning microscopy of wild-type meiotic cells in whole-mount maize ovules.

Development of techniques to analyze pachytene chromosomes has greatly overcome most of the difficulties in cytological studies of rice chromosomes caused by their small size. Visualization of meiotic chromosomes has now become routine in cytogenetic studies in this species. This chapter provides protocols on basic meiotic chromosome preparation, FISH analysis, and immunocytology in rice.

Advances in molecular biology and in the genetics of Arabidopsis thaliana have led to it becoming an important model for the analysis of meiosis in plants. Cytogenetic investigations are pivotal to meiotic studies and a number of technological improvements for Arabidopsis cytology have provided a range of tools to investigate chromosome behavior during meiosis. This chapter contains a detailed description of cytological techniques currently used in our laboratory for the basic preparation of meiotic chromosomes for investigation of the female and male meiotic pathway and fluorescence in situ hybridization (FISH) analysis for the frequency and distribution of crossovers (chiasmata) at metaphase I.

Progression of meiosis has been traditionally reconstructed from microscopic images collected from fixed cells. However, studies conducted in a number of species, including plants, indicate that this approach has clear shortcomings in accurately portraying the dynamic nature of meiotic processes. Here, we describe two methods to study chromosome dynamics in live meiocytes in maize, a protocol to observe chromosomes during meiotic prophase I and a technique to monitor chromosome segregation in anaphase I and anaphase II. The first method relies on culturing intact maize anthers and observing meiocytes embedded in the anthers with multiphoton excitation (MPE) microscopy. This approach circumvents difficulties in culturing isolated prophase I meiocytes in plants. The second technique uses culturing isolated meiocytes, which is possible with anaphase cells. Both methods can be fairly easily adapted for use in other plant species. We also detail the kinds of time-lapse movies that can be captured and analyzed using this technique, and describe software that can be utilized for analysis of movies chromosome dynamic in live meiocytes.

This chapter contains a detailed description of the immunological and cytological techniques developed to determine factors involved in crossover control during meiosis in barley. The immunological technique involves digesting fresh anthers followed by chromosome spreading to remove cytoplasm that causes unwanted background, whilst protecting the fragile chromosome structure from being compromised. Specific antibodies raised against meiotic proteins are then incubated with nuclei, detected with secondary antibodies conjugated to fluorescent dyes, and visualized with either wide-field or confocal microscopes. In the cytological technique, barley inflorescences are fixed, followed by dissecting out the anthers, digesting the cell walls and then spreading the meiotic chromosomes. Both techniques can be used in conjunction with specific DNA probes for fluorescent in situ hybridization (FISH) to label telomeres, centromeres, and ribosomal DNA; to identify DNA modifications such as 5-methylcytosine; and to detect the incorporation of DNA base analogues such as 5-bromo-2'-deoxyuridine (BrdU) or 5-ethynyl-2'-deoxyuridine (EdU) to be used for a meiotic time-course or assaying newly synthesized DNA. Although these techniques have been specifically developed for barley, they should be directly transferable to other cereal crop species such as wheat and rice.

Plant meiosis studies have enjoyed a fantastic boom in recent years with the use of Arabidopsis thaliana as an important model species for developmental studies because of its small genome, short life cycle, and large mutant collections. Unlike other eukaryotic models, plant meiosis does not display strict checkpoints and rarely commits to apoptotic processes, which makes it possible to investigate the whole meiotic process (spanning from premeiotic interphase to spore formation) in knockout mutants. In this chapter we describe a protocol for immunolabelling Arabidopsis and Brassica meiotic proteins on robustly spread chromosomes. This protocol allows the detection of a large range of proteins on well-preserved chromosomes and throughout the entire meiotic process.