Re-Evaluation of Vascular Histogenesis in the Root Tips of Selected Species in the Poaceae Using New Methods: Analysis of the Plerome, Vascular Initials, Pericycle and Late-Maturing Metaxylem Vessels.

Serial sectioning and 3D image reconstruction methods were applied to elucidate the structures of the apices of root vascular cylinders (VCs) in taxa of the Poaceae: Zea mays "Honey Bantam", Z. mays ssp. mexicana, Hordeum vulgare and Oryza sativa. The primary and nodal roots were investigated. Observations were performed using high-quality sectioning and 3D image-processing techniques improved and developed by the authors. We found that a quiescent uniseriate plerome was located at the most distal part of each VC. Vascular initials were located immediately basipetally to the plerome as a specific uniseriate layer that could be classified into central and peripheral initials that produced all the cells in the VC. No supplying of cells from the plerome to the vascular initials was observed. Numerical analysis revealed a "boundary point" along the root axis where the rate of increase of the vascular cell number markedly declined, and the VC diameter, number of vascular cells, and number of late-maturing metaxylem vessels (LMXs) at that point showed a similar relationship among the taxa and the types of roots examined (primary vs. nodal). The plerome and vascular initials layer can be considered independent after seed germination in these taxa. A boundary point at which procambial cell proliferation sharply declined was identified. The diameters of the VCs, number of LMXs, and number of vascular cells at the boundary point were found to be strongly related to each other.

Improved image processing for 3D virtual object construction from serial sections reveals tissue patterns in root tips of Zea mays.

Premise: Previously we described methods for generating three-dimensional (3D) virtual reconstructions of plant tissues from transverse thin sections. Here, we report the applicability of longitudinal sections and improved image-processing steps that are simpler to perform and utilize free applications. Methods: In order to obtain improved digital images and a virtual 3D object (cuboid), GIMP 2.10 and ImageJ 2.3.0 running on a laptop computer were used. Sectional views of the cuboid and 3D visualization were realized with use of the plug-ins "Volume Viewer" and "3D Viewer" in ImageJ. Results: A 3D object was constructed and sectional views along several cutting planes were generated. The 3D object consisted of selected tissues inside the cuboid that were extracted and visualized from the original section data, and an animated video of the 3D construct was also produced. Discussion: Virtual cuboids can be constructed by stacking longitudinal images along the transverse depth direction or stacking transverse images vertically along the organ axis, with both generating similar 3D objects. Which to use depends on the purpose of the investigation: if the vertical cell structures need close examination, the former method may be better, but for more general spatial evaluations or for evaluation of organs over longer tissue distances than can be accommodated with longitudinal sectioning, the latter method should be chosen.

Three-dimensional digital image construction of metaxylem vessels in root tips of Zea mays subsp. mexicana from thin transverse sections.

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.