IAWA Journal, Vol. 19 (3),1998: 265-278 A STUDY OF THE VASCULAR ORGANIZATION OF BAMBOOS (POACEAE-BAMBUSEAE) USING A MICROCASTING METHOD by Jean-Pierre Andre INRA, P. O. Box 2078, 06600 Antibes, France SUMMARY A reliable and simple microcasting method is applied to the study of the vascular structure in bamboo nodes; it provides new insights into their complexity, revealing the exact arrangement of branched vessels and clustered tracheary elements. Axial differentiation gradients in the metaxylem cell files, probable relics of the intercalary meristem, can also be found using this method. This anatomical finding can be linked to arecent hypothesis on the continuum in the tracheary element differ­ entiation. Key words: Metaxylem, vascular bundle, vessel, bamboo, node-inter­ calary meristem. INTRODUCTION Numerous anatomical details on the bamboo vascular organization have been de­ scribed in the last 20 years (Zee 1974; Yulong & Liese 1997) but it is obvious that the classical histological methods - serial sections, macerated sampies, cleared prepara­ tions - hardly give an adequate three-dimensional understanding of such extensive and complex anatomical structures. A new casting method reaches this goal much more easily; it allows one to visual­ ize the vessels of a culm segment and to accurately examine cell file casts in their original arrangement (Andre 1993). This has been recently applied to 25 bamboo species in order to complete earlier observations (Andre 1996). The method reveals the exact features of the bundle ramifications in the form of tracheary element clus­ ters and their mode of mutual connections, allowing hypotheses on the bundle organi­ zation in the wh oie mature plant. Moreover, by these means it was found that the internodes of the aerial axes show a probable trace of their intercalary meristem in the form of a short differentiation gradient of the secondary wall thickening in the metaxylem vessels. Assumptions conceming these particular 'transition' structures, which have not as yet been describ­ ed for Poaceae, can be made on the basis of the concept of procambium - metacambium continuum according to Larson (1982) and of the differentiation continuum hypoth­ esis by Savidge (1996). Other casting methods have recently been published (Fujii 1993; Fujii & Hatano 1996) but, to our knowledge, have not been applied to this area. Downloaded from Brill.com09/25/2021 08:39:40PM via free access 266 IAWA Journal, Vol. 19 (3),1998 Table 1, Classification of the studied species. Subtribe Species Arundinariinae Arundinaria distichus (Mitf.) Muroi & Okamura* linearis (Hack.) Nakai* DrepanostachyumJalcatum (Nees) Keng f. Fargesia nitida (Mitf.) Nakai** Pseudosasa japonica Sieb. & Zucc. ex Steud. Sasa palmata var. nebulosa (Mak.) s. Susuki Shibateinae Chimonobambusa marmorea (Mitf.) Mak. quadrangularis (Munro Nakai) Fenzi Mak. Hibanobambusa tranquillans var. shirochima (Koidz.)*** Phyllostachys aurea Carr./ A. & C. Riv. aureosulcata MacClure bambusoi'des var. castilloni Madiar & Carr. Mak. var. violascens A. & C. Riv. flexuosa Carr./ A. & C. Riv. nigra (Lodd. ex Lindl.) Munro viridiglaucescens Carr./A. & C. Riv. viridis Robert Young Semiarundinaria kagamiana Mak. fastuosa (Marliac ex. Mitf.) Mak. ex Nakai Bambusinae Bambusa multiplex (Lour.) Reuschel ex. Schult. f. multiplex cv. eIe gans multiplex cv. golden goddess tuldoi'des (Munro) ventricosa MacClure Guaduinae Otatea aztecorum (Mac Cl ure & Smith) Calderon *: synonym Pleioblastus; **: synonym Sinarundinaria; ***: probably a hybrid of Sasa x Phyllostachys (Demoly 1996). MATERIALS AND METHODS Plant material The 25 bamboo speeies whieh were studied are from 4 of the 9 subtribes and from 11 of the 69 genera reeently reeognized for woody bamboos (Clark 1995) (Table 1). The speeies were eolleeted from three plaees in the South ofFranee ('Jardin Thuret' in Antibes, 'Bambouseraie du Mandarin' in Montauroux, and 'Bambouseraie de Prafranee' in Anduze). Two mature eulms ofless than three years old, plus a growing eulm of Bambusa tuldoides, were cut at soillevel and sampled for eaeh speeies. For one speeies, Phyllostachys viridis, the eulm was sampled with its rhizome. The spe­ eies originated from regions of similar climate, but differed in numerous morphologi- Downloaded from Brill.com09/25/2021 08:39:40PM via free access Andre - Microcasting of vascular bundles in bamboos 267 cal characters, namely the culm size and axis architecture, which are not analyzed here. The nodes were numbered from the soil level, positively for the aerial part, negatively for the underground part. The diameter and length of the internodes were measured. The nodes to be cast were sawn 2-3 cm above and below the leaf sheath scar. The sawn rims were carefully recut with a razor blade. Slices of adjacent internodes were kept for bundle counting. Methods For the casting, the air-dried segments were immersed in a liquid mixture of two silicon elastomer components (RTV 141, Rhöne Poulenc, France) and evacuated in a vacuum chamber at room temperature. As soon as the vacuum was broken, the elastomer penetrated each empty space, namely the intracellular lumina of the cut metaxylem vessels and the intercellular cavities which formed in the bundles of age­ ing or dried bamboos, between the partially collapsed protoxylem and protophloem cell files. The injection speed depended on the pore size. In order to limit the catalyzed setting of the mixture, the culm segments were kept at a low temperature (-18°) for 10 hours. The intracellular injection was stopped by intact pits, but easily passed through the perforations. Thus, it was possible to cast files containing several hun­ dred cells. After this step, the elastomer was cured at 50 oe for 4 hours and became a transpar­ ent, elastic and chemically inert material. The cell walls were then destroyed by suc­ cessive bathing in solutions of sulfuric acid (H 2S04, 2H20), sodium bicarbonate (l mole/l) and sodium hypochlorite (2 moles/l). The casts ofthe metaxylem vessels and the cavities where the protoxylem and part of the phloem cell files were embed­ ded, were shaped like tiny filaments soldered at each end to a small elastomer block. They were covered by a water film for optimal observation under a light microscope and photographed in a scanning electron microscope (SEM) after standard coating with gold (Andre 1993). RESULTS Macroscopical description The bamboos form vegetative axes from the buds, which are in distichous alter­ nate positions at the rhizome, culm and branch nodes. Each axis consists of similar metamers where diameters are relatively constant in the rhizome. They increase from one metamer to the next in the underground part of the culm, then decrease in the aerial part at the rate of approximately 3-5 mm/m of culm, and also in the branches, from the first to the apical metamer. Each internode is cylindrical and hollow with an externall internal diameter ratio depending on the bamboo species. Numerous axial, collateral, parallel bundles run in the intern ode wall, scattered in a ground tissue of parenchyma. From the outside to the inside of the wall, the bundle diameters increase whereas their density decreases, e. g., from approximately 50 to 5 bundles/mm2 in the basal internodes ofthe Phyllostachys species studied. In the ma­ ture culm, the main water conducting vessels are probably the metaxylem vessel pairs, Downloaded from Brill.com09/25/2021 08:39:40PM via free access 268 IAWA Journal, Val. 19 (3),1998 p)( 1 ph m'l( b - - -- -- ..... vessel . 2 C I 1 nb I, I [] [J 1 I I I ph .a. 1000 I I I I ~-------*- - * "- "- ~ " 1 "- o 1500 o 0 0 , * - - - 0 - 15 -- -- --, "- o '", C I ' *"­ , I o ' , , I , 0 , l:> *, 1000 , "- U.p. A.P. ~ , ''<t "- , 0 " *, I o ;:;., ' I , ' , I ".., , ~ O". I 0 ' , I V. O'\~J J.. ph . ~llill I I .2 10 12 14 16 118 20 node 10 ~ i i-~ " 3 ~4 7 8 • 11 J j 6 "1.., 10 Downloaded from Brill.com09/25/2021 08:39:40PM via free access Andre - Microcasting of vascular bundles in bamboos 269 whose diameters vary from 80-130 /illl in the internal bundles to 5 -15 /illl in the peripheral ones (Fig. 1). The nodal zones where each adjacent organ is connected are anatomically more complex than the intern odal ones. Moreover a parenchymatous nodal diaphragm is present with numerous transverse connecting bundles. Dur observations focus essen­ tially on the nodal vascularization and the connections between mature axes. The following description is based mainly on the metaxylem casts. Bundle and metaxylem vessel counting - Casting efficiency As observed in the transverse sections, the number of bundles remains constant along an internode, but varies from one internode to the next. It increases or decreases with the diameter of the culm, from its junction with the rhizome to its apex. Accurate counting of the bundles along successive metamers was limited to the aerial internodes of a culm of Phyllostachys aurea and to some internodes of the lower and under­ ground part of a culm of Phyllostachys viridis. These data were multiplied by 2 in order to give the total number ofthe metaxylem vessels (t). In addition, the number of the effectively cast metaxylem vessels (c) was counted in each segment of the Phyllostachys aurea culm. A relative constancy of t was observed in the first aerial internodes, where nodal buds and branches were absent. In the branched part ofthe Phyllostachys aurea culm, t decreased at the rate of about 100 vessels/node and in the underground part of the Phyllostachys viridis cu Im, it increased at the rate of about 250 vessels/node (Fig. 2). The casting efficiency was 75-80% for the Phyllostachys aurea culm. The deficit of 20-25% between c and t resulted from tyloses which partially or totally blocked several vessels and from the minute diameters of the most peripheral vessels. These c values indicate that a nodal cast thus gives reliable, albeit not entirely complete mate­ rials for observations.