Premise: Fluorescence microscopy is an effective tool for viewing plant internal anatomy. However, using fluorescent antibodies or labels hinders throughput. We present a minimal protocol that takes advantage of inherent autofluorescence and aldehyde‐induced fluorescence in plant cellular and subcellular structures to markedly increase throughput in cellular and ultrastructural visualization.Methods and Results: Twelve species distributed across the plant phylogeny were each subjected to five fixative treatments: 1% paraformaldehyde and 2% glutaraldehyde, 2% paraformaldehyde, 2% glutaraldehyde, formalin‐acid‐alcohol (FAA), and 70% ethanol. Samples were prepared by embedding and mechanically sectioning or via whole mount. A confocal laser scanning system was used to collect micrographs. We evaluated and compared fixative influence on sample structural preservation and tissue autofluorescence.Conclusions: Formaldehyde fixation of Viridiplantae taxa samples generates useful structural data while requiring no additional histological staining or clearing. In addition, a fluorescence‐capable microscope is the only specialized equipment required for image acquisition. The minimal protocol developed in this experiment enables high‐throughput sample processing by eliminating the need for multi‐day preparations.

Understanding plant adaptation mechanisms to prolonged water immersion provides options for genetic modification of existing crops to create cultivars more tolerant of periodic flooding. An important advancement in understanding flooding adaptation would be to elucidate mechanisms, such as aerenchyma air-space formation induced by hypoxic conditions, consistent with prolonged immersion. Lysigenous aerenchyma formation occurs through programmed cell death (PCD), which may entail the chemical modification of polysaccharides in root tissue cell walls. We investigated if a relationship exists between modification of pectic polysaccharides through de-methyl esterification (DME) and the formation of root aerenchyma in select Fabaceae species. To test this hypothesis, we first characterized the progression of aerenchyma formation within the vascular stele of three different legumes—Pisum sativum, Cicer arietinum, and Phaseolus coccineus—through traditional light microscopy histological staining and scanning electron microscopy. We assessed alterations in stele morphology, cavity dimensions, and cell wall chemistry. Then we conducted an immunolabeling protocol to detect specific degrees of DME among species during a 48-hour flooding time series. Additionally, we performed an enzymatic pretreatment to remove select cell wall polymers prior to immunolabeling for DME pectins. We were able to determine that all species possessed similar aerenchyma formation mechanisms that begin with degradation of root vascular stele metaxylem cells. Immunolabeling results demonstrated DME occurs prior to aerenchyma formation and prepares vascular tissues for the beginning of cavity formation in flooded roots. Furthermore, enzymatic pretreatment demonstrated that removal of cellulose and select hemicellulosic carbohydrates unmasks additional antigen binding sites for DME pectin antibodies. These results suggest that additional carbohydrate modification may be required to permit DME and subsequent enzyme activity to form aerenchyma. By providing a greater understanding of cell wall pectin remodeling among legume species, we encourage further investigation into the mechanism of carbohydrate modifications during aerenchyma formation and possible avenues for flood-tolerance improvement of legume crops.

Classical histology describes the histological organization in Zea mays as having a “closed organization” that differs from Arabidopsis with the development of xylem conforming to predictable rules. We speculated that root apical meristem organization in a wild subspecies of Z. mays (a teosinte) would differ from a domestic sweetcorn cultivar (‘Honey Bantam’). Careful comparison could contribute to understanding how evolutionary processes and the domestication of maize have affected root development. Root tips of seedlings were prepared and sectioned for light microscopy. Most sections were treated with RNase before staining to increase contrast between the walls and cytoplasm. Longitudinal and serial transverse sections were analyzed using computer imaging to determine the position and timing of key xylem developmental events. Metaxylem development in mexicana teosinte differed from sweetcorn only in that the numbers of late-maturing metaxylem vessels in the latter are typically two-fold greater and the number of cells in the transverse section of procambium were greater in the latter, but parenchymatous cell sizes were not statistically different. Promeristems of both were nearly identical in size and organization, but did not operate quite as previously described. Mitotic activity was rare in the quiescent centers, but occasionally a synchronized pulse of mitoses was observed there. Our reinterpretation of histogen theory and procambium development should be useful for future detailed studies of regulation of development, and perhaps its evolution, in this species.

Premise: Young plant roots share a common architecture: a central vascular cylinder surrounded by enveloping cylinders of ground and dermal tissue produced by an apical promeristem. Roots with closed apical organization can be studied to explore how ontogeny is managed. The analysis of transverse and longitudinal sections has been the most useful approach for this, but suffers from limitations. We developed a new method that utilizes digital photography of transverse sections and three‐dimensional (3D) computer virtual reconstructions to overcome the limitations of other techniques.Methods: Serial transverse sections of teosinte root tips (Zea mays subsp. mexicana) were used to construct longitudinal images, 3D images, and an animated 3D model. The high‐resolution, high‐contrast, and low‐distortion sectioning method developed previously by the authors enabled high‐quality virtual image construction with the aid of a standard laptop computer.Results: The resulting 3D images allowed greater insight into root tissue ontogeny and organization, especially specific cellular structures such as histogen layers, xylem vessels, pericycle, and meristematic initials.Discussion: This new method has advantages over confocal laser scanning microscopy and magnetic resonance imaging for visualizing anatomy, and includes a procedure to correct for sectioning distortion. An additional advantage of this method, developed to produce better knowledge about the developmental anatomy of procambium in roots, is its applicability to a wide range of anatomical subjects.