In: Dashek, William V., ed. Methods in plant biochemistry and molecular biology. Boca Raton, FL: CRC Press: pp. 3-11. Chapter 1. 1997. Chapter Methods for and Analysis of Plant Cell Tissue Ultrastructure John E. Mayfield and William V. Dashek Contents 1.1 Introduction The balsam fir (Abies balsamea), a transcontinental gymnosperm, is economically important because of its value as a source for lumber and wood pulp. Besides its economic value, balsam fir, like many other conifers, contains the embryonic tissue that will produce the mature structures during the next growing season within relatively dormant vegetative buds. These buds have several advantages that make them very attractive for the preparation and study of plant ultrastructure. Thus, transmission electron spectroscopy (TEM) of buds is presented as an example of plant electron microscopy. Methods for the preparation of a wide variety of plant tissues can be found in the supplementary references to this chapter (Table 1.1). For a discussion of plant cell fractionation and the assay of organelle fractions for purity, the reader is referred to the review by Quail. 1 One advantage is the ease of handling because of the ideal size of the buds. They are large enough to treat as macroscopic structures, but small enough to allow good TEM fixation without a labor-intense effort. Terminal buds have a mean height of 2.60 mm. Because they have relatively well-described developmental cycles, gymnosperm buds can be collected and prepared at intervals that coincide with temporal-related cytological information. Since the preburst bud has a localized region (the apical dome) which contains an abundance of undifferentiated cells. many difficulties associated with the fixation and sectioning of differentiated plant tissues are avoided. Also. the 0-8493-9480-5/97/$0.00+$.50 1997 by CRC Press LLC 3
4 Methods in Plant Biochemistry and Molecular Biology apical region, before bud burst, will produce an abundance of celis within various stages of vegetative cell division. The apical portion of the vegetative balsam fir bud exhibits a cytological zonation similar to that described for other gymnosperms. 2 There is a change in cell morphology, cytochemistry, and mitotic activity throughout the apical zones and changes correlate with the annual growth cycle. An examination of the cells within these zones will yield a variety of cytological forms at the electron microscopic level. 1.2 Protocols 1.2.1 Collection and Fixation of Buds In this laboratory, vegetative balsam fir buds were collected in Vermont during April. Other related species such as Fraser fir from other locations can be used with an equal degree of success if the collection times are changed to account for species- or locality-related differences in temporal sequence. As in most biological materials, it is important to keep the interval between its natural environment and the TEM-fixed state as short as possible. Bud-containing terminal branch sections were removed from trees and stored in plastic bags for transportation to the laboratory. In the laboratory, the buds were dissected from the enclosing scales and placed into 3% glutaraldehyde in 0.05 M sodium cacodylate buffer, ph 7.4. While covered with buffered glutaraldehyde, the apical portion of the buds was-cut into l-mm 3 portions and fixation continued in fresh buffered glutaraldehyde for 2 to 24 h. After 2 h, we were unable to notice any differences in ultrastructural appearance. 1.2.2 Postfixation in Osmium Tetroxide and Dehydration Following glutaraldehyde fixation, the bud sections are washed in three changes of buffer during a 1-h period. While within fish buffer, the tissue-containing vials should be placed into an ice bucket in a fume hood. All preparations of osmium tetroxide solutions and subsequent fixation must be conducted in a chemical fume hood. The buds are now postfixed in cold 2% osmium tetroxide (OsO 4 ) in the same buffer for 2 h. The postfixation in OsO 4 is followed by ten changes of cold distilled water during a 1-h period. The final water wash is replaced by 0.5% uranyl acetate. To enhance the electron-dense staining contrast, the tissue should remain in uranyl acetate overnight. After urartyl acetate, tissue is placed into water for several minutes before the next step (dehydration). Dehydration is necessary for the tissue to become permeated by the embedding medium. The tissue is dehydrated in a graded series of ethyl alcohol that includes 50, 70, and 95%. Tissue is
Methods for Analysis of Plant Cell and Tissue UItrastructure 5 immersed in each solution for 10 min. Tissue is subsequently placed into two changes of 100% acetone with 10 min in each change. From acetone. the tissue is introduced to the embedding medium as a 1:1 mixture of acetone and Spurr s 3 low-viscosity embedding medium. Since acetone is highly volatile, it is necessary to make certain that the tissue is always completely covered with the liquid. 1.2.3 Infiltration and Embedding The next steps, infiltration and embedding, are started when the tissue is placed in a 1:1 mixture of acetone and Spurr s and immersed from 6 h to overnight. Infiltration is followed by embedding. We obtain best results when most of the infiltrating mixture is removed from tissue before placing into 100% Spurr s. A plastic Petri dish is lined with a piece of filter paper and about two to three drops of embedding medium are placed on the paper. Using a toothpick, individual sections are removed from the infiltration mixture and placed onto Spurr s-laden filter paper and rolled around on the filter paper to be certain that the tissue section remains covered with Spurr s. Several drops of embedding medium may be added before the tissue is removed from the filter paper and placed into a BEEM capsule. For observations in which orientation is not a concern, embedding may be earned out using BEEM capsules. The capsules should be placed into a suitable holder. Place a label (5 15 mm) that contains enough information to identify the specimen. Please use a pencil to record information on the label. Place about three drops (enough to fill the conical tip of the capsule) of Spurr s embedding medium into the BEEM capsule. Using a wooden toothpick, place the small piece of tissue into the capsule and gently tease it to the bottom. Completely fill the remainder of the capsule with Spurr s. If one is interested in embedding the tissue sections with a specific orientation, it will be necessary to use a flat-embedding procedure. A simple way of accomplishing this is by using the lid of the BEEM capsule as the support surface. For this, the capsule is prepared by removing the conical portion with a razor blade. The identification label is then inserted from the lid end of the capsule and pushed toward the cut end so that it will be removed from the vicinity of the tissue. Holding the capsule so that the lid is in a horizontal position, a drop of Spurr s is placed on the center of the surface of the lid. The desired tissue section is removed from the Spurr s-soaked filter paper, as previously described, and placed within the drop of Spurr s with the desired surface against the surface of the lid. The capsule is now closed in the inverted orientation by inserting the bottom portion into the lid. The closed capsule (with the cut end facing up) is placed into the capsule holder. The capsule is now filled with Spurr s from the cut end. The BEEM capsule lids fit snugly into the openings of the holders. If it is necessary to remove the capsule before curing of the Spurr s is completed, this can be accomplished by pushing the capsule in the direction away from the hinge of the lid. This will allow the removal of the capsule without leaking the embedding medium. For curing or polymerization of the Spurr s, all capsules are placed into a 60 to 70 C oven. After 30 min in the oven check to see if the tissue sections are remaining within the capsule tips or against the bottom of the lid. If they have moved away from their original position, a gentle push or relocation with a toothpick should be adequate. After overnight polymerization, the blocks are ready for trimming and sectioning. 1.2.4 Block Trimming and Sectioning Remove the cured block from the BEEM capsule by removing the snap lid and make a longitudinal cut with a single-edge razor blade and remove the block. Securely place the block into the chuck
Methods in Plant Biochemistry and Molecular Biology of a specimen holder and place the assembly on the stage of a dissecting microscope. With a singleedge razor blade, trim the end of the block to produce a trapezoid surface. Trim so that the surface of the block includes, as far as possible, only tissue (Figure 1.1). The width of the block face to be sectioned should be no greater than 0.5 mm. Edges (a) and (b) should be parallel. It is better to use a fresh double-edge razor blade for the final trim. It is extremely important that surface (b) has a very lustrous appearance. The trimmed blocks may be sectioned on an available ultramicrotome with either a glass or diamond knife. Although reasonably good sections may be obtained with glass knives, sectioning with a diamond will consistently yield thin sections. The use of glass or diamond knives will depend on both the resources and skill of the electron microscopist. We will not attempt to provide instructions for ultramicrotomy, since specific instructions must be provided for each type of ultramicrotome. Pale gold or silver sections are adequate for examination with the transmission electron microscope. These sections are floated onto 200-mesh copper grids and stained with lead citrate stain. 4 The samples in this article were viewed with a Philips 300 electron microscope with an accelerating voltage of 80 kv. 1.3 Expected Results Examples of cell ultrastructure are provided from apical dome cells of buds collected in April. 5 Some cells contained electron-dense inclusions and nuclei with highly condensed chromatin (Figure 1.2). Also, one may observe dictyosomes with associated highly distended vesicles (Figure 1.3). Often the vesicles were confluent with the plasma membrane. A dense population of distended vesicles is observed in association with a high density of endoplasmic reticulum (ER) (Figure 1.4). Besides dictyosome-vesicle complexes and ER, the cytoplasm also contains a relatively high density of free ribosomes and mitochondria with a matrix of reduced electron density (Figure 1.5). Many cells are characterized by an abundance of thin primary cell walls and nuclei with varying degrees of condensed chromatin (Figure 1.6). Also, microtubules are shown extending through developing ceil plates (phragmoplasts) (Figure 1.7). This stage of cell plate formation coincides with both nuclear membrane reconstitution in daughter nuclei and the occurrence of ribonucleoprotein granules within the nuclei.
Methods for Analysis of Plant Cell and Tissue Ultrastructure 7 Figure 1.2 Cell with nucleus (N) and starch-containing plastids (P). Bar= 2.0 µm Figure 1.3 Golgi body (G) with associated vesicles. Bar = 1.0 µm.
8 Methods in Plant Biochemistry and Molecular Biology Figure 1.4 Cell with high density of distended vesicles (V) and endoplasmic reticulum (ER). Bar = 2.0 µm. Figure 1.5 Free ribosomes and mitochondria (M). Bar = 0.5 µm.
Methods for Analysis of Plant Cell and Tissue Ultrastructure 9 Figure 1.6 Primary cell wall (W) close to nucleus with highly condensed chromatin. Bar = 0.5 µm.
10 Methods in Plant Biochemistry and Molecular Biology Figure 1.7 Cytoskeletal elements extending through developing cell plates. Bar = 1.0 µm. References
Methods for Analysis of Plant Cell and Tissue Ultrastructure 11