ANALYSIS of CAMBIUM and DIFFERENTIATING VESSEL ELEMENTS in KALOPANAX PICTUS USING RESIN CAST REPLICAS By

IAWA Journal, Vol. 22 (1), 2001: 15–28 ANALYSIS OF CAMBIUM AND DIFFERENTIATING VESSEL ELEMENTS IN KALOPANAX PICTUS USING RESIN CAST REPLICAS by Peter Kitin1, 2, Yuzou Sano1 & Ryo Funada1* SUMMARY A resin-casting method with subsequent scanning electron microscopy (SEM) was used to examine the three-dimensional (3-D) shapes of cells and the cell walls of cambium and differentiating xylem. Glutaralde- hyde-fixed and dehydrated specimens were embedded in polystyrene and then organic material was removed by digestion with acidic solutions or enzymes. The acidic solutions used for treatment were sulphuric acid and a mixture of acetic acid and hydrogen peroxide and the enzymes used for treatment were pectinase and cellulase, with a final treatment with sodium hypochlorite. Both methods could be used for studies of the differentiation of cambial cells; however, digestion with enzymes allowed better preservation of the 3-D organisation of the tissue. Negative replicas of inner surfaces of cell walls of differentiating vessel elements revealed the sequential stages of the development of bordered pits and perforation plates. Future bordered pits at the early stages of the differentiation of cell walls were demarcated by the accu- mulation of organic material between adjacent pit membranes. Subse- quent deposition of cell wall material resulted in formation of pit cavities and the rims of perforation plates. Key words: Cambium, differentiating vessel elements, Kalopanax pictus, pit formation, resin casting. INTRODUCTION Differentiating vessel elements are turgid cells with soft walls and large vacuoles. If a specimen is not embedded in resin, the cells often break or shrink during sectioning. In addition, it is difficult to observe cambial cells and differentiating xylem elements from one end to the other in a single histological section by conventional light microscopy because both ends might not appear on the same section or in the same focal plane (Kitin et al. 1999). 1) Department of Forest Science, Faculty of Agriculture, Hokkaido University, Sapporo 060- 8589, Japan. 2) Department of Dendrology, University of Forestry, Kliment Ohridski str. 10, Sofia 1756, Bulgaria. *) Corresponding author. E-mail: [email protected] Downloaded from Brill.com10/05/2021 07:44:30AM via free access 16 IAWA Journal, Vol. 22 (1), 2001 Kitin, Sano & Funada — Resin casting of differentiating vessel elements 17 Data on the three-dimensional (3-D) shapes of cambial and differentiating xylem cells in dicotyledonous species have been obtained from analyses of serial thin sections of resin-embedded specimens (Fujita et al. 1984; Fujita 1993). Preparation of such sections is, however, time-consuming and impractical for studies of the dimen- sions and shapes of large numbers of cells. In addition, due to the uneven thickness of sections and to errors during the manual ordering and stacking of serial images, mis- takes in the reconstruction of the 3-D images are easily made. The 3-D shapes and the lengths of fusiform cambial cells in Kalopanax pictus have been studied by confocal laser scanning microscopy (CLSM) with reconstruction from serial images of optical sections (Kitin et al. 1999, 2000). However, the resolution of CLSM is limited and is far below the level achieved by electron microscopy. Therefore, it is not possible to examine the details of micromorphological changes in differentiating cells, such as the changes in the minute structures associated with sculpturing of the cell wall. The resin-casting method allows the 3-D shapes of matured xylem cells and their spatial arrangement to be clearly reproduced as negative replicas (Fujii 1993; Mauseth & Fujii 1994; André 1995, 1998; Fujii & Hatano 1996, 2000; André et al. 1999; Kitin et al. 1999). Low-viscosity resins can penetrate the minute apoplastic spaces in plant tissues. Casts have high fidelity and, thus, negative replicas of the cell wall structures, such as pits, perforation plates and warts, can be studied in detail with the scanning electron microscope (Stieber 1981; Fujii 1993; Mauseth & Fujii 1994; André et al. 1999; Xu & Liese 1999). The potential of this method for plant anatomical studies seems to be considerable: it has been reported that not only xylem elements but all cell types can be infiltrated with styrene (Mauseth & Fujii 1994). However, preparation of resin cast replicas of differentiating tissues, which might provide important information for studies of developmental anatomy, has not yet been reported, to our knowledge. Kalopanax pictus (Araliaceae) is a ring-porous hardwood that grows in the tem- perate forests of East Asia. In this species, uniseriate, large-diameter earlywood vessels contrast strongly in terms of size with the small-diameter vessels and vascular tracheids of the latewood (Ohtani 2000). The process of differentiation of the second- ary xylem in Kalopanax pictus has been studied by light microscopy (Imagawa & Ishida 1972; Kitin et al. 1999). We examined the cambial and differentiating xylem cells using a resin-casting method with scanning electron microscope in an attempt to analyse their shapes, as well as cell wall structures, such as pits and perforation parti- tions, during sequential stages of differentiation. MATERIAL AND METHODS Plant material A single specimen of Kalopanax pictus (diameter at breast height, 76 cm), grow- ing on the campus of Hokkaido University, was used for all experiments. Samples, including cambium and adjacent phloem and xylem, were taken by chisel from the stem at breast height during the periods of cambial reactivation (April 3) and cambial activity (June 5). Radial slivers (of approximately 20 mm, 10 mm and 3 mm in the axial, radial and tangential directions, respectively) were cut from the sample blocks Downloaded from Brill.com10/05/2021 07:44:30AM via free access 16 IAWA Journal, Vol. 22 (1), 2001 Kitin, Sano & Funada — Resin casting of differentiating vessel elements 17 and immediately fixed in a 4% solution of glutaraldehyde. The solution was degassed briefly under a vacuum and then samples were left overnight in the fixative. Embedding in resin Glutaraldehyde-fixed slivers were trimmed into small blocks (of approximately 5 mm, 5 mm and 3 mm in the axial, radial and tangential directions, respectively) and dehydrated through a graded ethanol series. The resin was prepared by mixing styrene with benzoyl peroxide (99 : 1, v/v). Then the tissue blocks were infiltrated by immer- sion in increasing concentrations of the resin in ethanol (25%, 50%, 75%) for one hour per solution. Finally, they were incubated in 100% resin three times for one hour each. The specimens were then inserted into gelatine capsules filled with resin and baked in an oven at 60°C for three days for polymerisation of the resin. Removal of cell walls from embedded tissue Embedded tissues were exposed on the surface of individual resin blocks either by polishing or by splitting the block. Then the organic material was digested by alter- nate treatments with 95% sulphuric acid and a mixture of equal volumes of hydrogen peroxide and glacial acetic acid, as described by Fujii (1993) and Mauseth and Fujii (1994). Alternatively, digestion was performed with pectinase (pectinase from mould, 0.01 U mg -1; Fluka, Buchs, Switzerland), and then with cellulase (ʻOnouzukaʼ RS; Yakult Co. Ltd., Tokyo, Japan) and sodium hypochlorite. Samples were immersed in 2% pectinase in phosphate buffer (0.1 M, pH 6.5), then in 2% cellulase in phosphate buffer (0.1 M, pH 5.8) and, finally, in sodium hypochlorite solution (available chlo- rine, min. 5%). Treatment was continued for 72 h at 35 °C in each reagent. Solutions were renewed at 24 h-intervals. The surfaces of specimens were cleaned after digestion either by agitation of specimens in water or with sticky tape. Preparation of samples and observations The casts were rinsed in distilled water. Then they were dehydrated by passage through a graded ethanol series and drying in an oven at 35 °C for 1–2 h. The blocks were mounted on specimen stubs and coated with carbon and gold by vacuum evapo- ration. They were observed with a field-emission scanning electron microscope (JSM- 6301F; Jeol Co. Ltd., Tokyo, Japan) at an accelerating voltage of 2 to 3 kV (Sano et al. 1999). RESULTS AND DISCUSSION Infiltration of tissues with styrene The extent of infiltration and the stability of the resin are both factors of crucial importance for successful resin casting. We selected polystyrene for our experiments because Taneda et al. (1979) and Fujii (1993) showed that polystyrene casts were stable against the chemical reagents used for digestion of organic materials. Styrene was able to penetrate pit membranes of cells and was successfully used to produce polystyrene casts of various types of xylem and cortical cells by Mauseth and Fujii (1994). Downloaded from Brill.com10/05/2021 07:44:30AM via free access 18 IAWA Journal, Vol. 22 (1), 2001 Kitin, Sano & Funada — Resin casting of differentiating vessel elements 19 Fig. 1 — For legends, see page 20 Downloaded from Brill.com10/05/2021 07:44:30AM via free access 18 IAWA Journal, Vol. 22 (1), 2001 Kitin, Sano & Funada — Resin casting of differentiating vessel elements 19 Fig. 2 — For legends, see page 20 Downloaded from Brill.com10/05/2021 07:44:30AM via free access 20 IAWA Journal, Vol. 22 (1), 2001 Kitin, Sano & Funada — Resin casting of differentiating vessel elements 21 Our observations showed that styrene had fully infiltrated the lumens of cambial cells and differentiating cambial derivatives (Fig. 1A & 1B). By contrast, styrene did not infiltrate the cell walls. Thus, after digestion of cell walls, there were empty spaces between the cell casts. As observed in other studies (Mauseth & Fujii 1994; André et al. 1999; Kitin et al. 1999), styrene fully infiltrated the lumens of differentiated xylem elements (Fig. 1C). Minute cavities in cell walls, such as pits, were filled with resin and the casts replicated cell wall structures with high fidelity.

Load more