A set of sampling, preparation, and staining techniques for studying meiosis in cucumber.

The development of genetic and genomic resources for biological studies in cucumber has experienced an unprecedented boom in recent years. To investigate the function of putative meiotic genes and germplasm in breeding programs, an accurate cytogenetic characterization is required. Cytological methods and reference to investigate meiosis in cucumber are limited at present. Here we provide a set of cytological techniques that have been adapted for the study of meiosis in cucumber. The meiotic stages can be identified with high precision using hierarchical criteria from developing buds, undisturbed meiocytes, and freshly stained chromosomes. A meiotic cytological atlas of all stages is presented as a reference for identifying particular stages and for comparison of meiosis between normal and mutant plants. We performed a comparative analysis of the distribution of cytoplasmic organelles between cucumber and Arabidopsis, and we described a highly nonsynchronous condensation of chromosome parts during diplotene. A simplified fluorescence in situ hybridization (FISH) protocol, using robustly spread chromosomes, were developed. In addition, we designed a single oligonucleotide probe for 5S rDNA to use in karyotyping and monitoring of homologous chromosome pairing, which will make FISH analysis of 5S rDNA easier and more economical.

Comparison of Sample Preparation Techniques for Inspection of Leaf Epidermises Using Light Microscopy and Scanning Electronic Microscopy.

The micro-morphology of leaf epidermises is valuable for the study of leaf development and function, as well as the classification of plant species. There have been few studies comparing different preparation and imaging methods for visualizing the leaf epidermis. Here, four specimen preparation methods were used to investigate the leaf epidermis morphology of Arabidopsis, radish, cucumber, wheat, rice, and maize, under an inverted basic light microscope (LM), a laser scanning confocal microscope (LSCM), or a scanning electron microscope (SEM). Optical microscope specimens were obtained using either the direct isolation method or the chloral hydrate-based clearing method. SEM images were obtained using a standard stage for conventional dehydrated samples or a Coolstage for fresh tissue. Different parts of epidermis peels were well focused under the LM. Investigation of samples cleared by chloral hydrate is convenient and autofluorescence of cell walls can be detected in rice. The resolution of images of conventional SEM leaf samples was generally higher than the Coolstage images at the same magnification, whereas local collapse and shrinkage were observed in leaves with high water content when using the conventional method. However, stomatal apparatuses of Arabidopsis, cucumber, radish, and maize deformed and showed poor appearance when using the Coolstage. Moreover, we usually used glutaraldehyde as an SEM fixative when using t-butanol for freeze-drying, though methanol is considered a better fixative in recent studies. In addition, fresh samples were not stable on the Coolstage. Thus, we compared four different t-butanol freeze-drying methods and two Coolstage methods. The dimension and morphology of tissues were compared using the six different methods. The results indicate that methanol fixative obviously reduced shrinkage of SEM samples compared with glutaraldehyde and formaldehyde alcohol acetic acid (FAA) fixatives. The use of methanol and a graded series of steps improved the preservation of samples. Preparing samples with optimal cutting temperature compound and observing at -30°C helped to increase the stability of Coolstage samples. In summary, our results provide an overview of the shortcomings and merits of four different methods, and might provide some information about choosing an optimal method for visualizing epidermal morphology.