Arch. Histol. Cytol., Vol. 55, Suppl. (1992) p. 217-224 Ultrastructural Organization of Two Tapetal Types in Angiosperms Susan H. BARNES and Stephen BLACKMORE The Natural History Museum, London, United Kingdom Received February 18, 1992 Summary. The development of preparation techniques As has been the case for other tissues within the that include freeze fracturing provide an ideal method developing anther, previous studies have documented for studying the differentiation of plant tissues in the the organization of the tapetum at the level of optical scanning electron microscope. This is illustrated with microscopy (see for example, SCHNARF,1923; UBISCH, reference to tapetal development in Catananche caer- 1927) and through the application of transmission ulea, which has a plasmodial tapetum, and in Lolium electron microscopy (see for example, DICKINSONand perenne, which has a secretory tapetum. LEWIS, 1973, PACINI and KEIJZER, 1989; EL-GHAZALY and NILSSON, 1991). Scanning electron microscope The tapetum is a specialised tissue concerned with studies had to await the development of techniques the nutrition of the developing spores, and is found in which permitted the examination of the internal sur- the sporangia of lower plants and anthers of higher faces of organs, tissues, cells and organelles. We have plants (for reviews, see PACINI et al.,1985; CHAPMAN, reviewed the historical development of such tech- 1987). Tapetal cells exhibit a variety of develop- niques in relation to plant ultrastructure (BARNES and mental pathways, especially in terms of nuclear BLACKMORE,1986a) and emphasised the importance divisions, the behaviour of the cell walls and the of the pioneering work of Professor Keiichi TANAKA. synthesis of pollen wall precursors. Two major He devised a series of techniques ranging from crack- ultrastructural classes of tapetum are found in the ing resin embedded blocks (TANAKA, 1974) through anthers of angiosperms. The secretory tapetum is to methods involving chemical fixation followed characterised by persistent cell walls and is also by freeze fracturing (TANAKA, 1981; TANAKA and referred to as glandular, parietal, or cellular. In NAGURO, 1981). Advances in specimen handling and contrast, the cells walls of the plasmodial tapetum processing have enabled the basic technique to be break down during microspore development. This applied to free cells suspended in culture medium second kind of tapetum is sometimes referred to as (FUKUDOME and TANAKA, 1986). invasive or amoeboid. The two major classes can Although initially applied to animal tissues, the basic themselves be subdivided on the basis of various technique proved readily adaptable to plant tissues criteria, particularly the sequence of events during by adopting an extended period of extraction in microsporogenesis (PACINI et al., 1985; PACINI, 1990; osmium tetroxide (BARNES and BLACKMORE, 1984a, PACINI and FRANCHI (1991). b). One area of botanical research where the tech- In the course of a programme of comparative nique, which we refer to as freeze-fracture and cyto- studies of pollen ontogeny aimed at determining the plasmic maceration, has proved particularly suitable morphogenetic pathways associated with taxon- has been the study of pollen and spore ontogeny specific features, we have examined representatives (BLACKMORE and BARNES, 1985, 1987, 1990; BARNES of the two major tapetal classes. Here, we describe and BLACKMORE, 1986b; DICKINSON and SHELDON, and illustrate the later, mainly post tetrad, develop- 1986; WILMS et al., 1986). mental stages of the secretory tapetum of Lolium As observations of the tapetum demonstrate, the perenne L. (Gramineae) and the plasmodial tapetum freeze fracture and cytoplasmic maceration tech- of Catananche caerulea L. (Compositae: Lactuceae), nique is particularly suited to the study of the spatial as studied by means of scanning electron microscopy. relations of membrane-bound organelles. 217 218 S. H. BARNES and S. BLACKMORE: plasmodial tapeta (ALBERTINIet al., 1987), the tapetal MATERIALS AND METHODS cells subsequently intrude into the spaces between developing microspores. In contrast, the tapetum Anthers at various stages of development were taken remains an organized cylindrical tissue in the anthers from plants of Lolium perenne L. (Gramineae) col- of flowering plants with secretory tapeta, recognized lected from wild populations in Sussex and from from 175 families (ALBERTINI et al., 1987). specimens of Catananche caerulea L. (Compositae: In Catananche, as in Cichorium (PACINI and KEIJZER, Lactuceae) cultivated at Chelsea Physic Gardens, 1989), the tapetum does not become invasive until London. after the tetrad stage (Fig. 2). During the tetrad stage The anthers were prepared by the freeze fracture the tapetal cell walls remain intact and distinct. The and cytoplasmic maceration technique as described nuclei of most, if not all, tapetal cells undergo mitosis by BARNES and BLACKMORE (1984a). Catananche during this stage. As in other tapetal cells (see for anthers at later stages of development were dissected example, CHAPMAN, 1987), the nuclear division is not from the bud and individually trimmed at each end followed by cytokinesis. The cells are therefore binu- to assist the penetration of solutions. Early stage cleate, with the two nuclei remaining in close contact. anthers were processed in buds or groups of anthers After the callose special cell wall around the tetrads to facilitate easy handling. Lolium anthers were disperses, the free microspores are released into the dissected and processed singly. Prepared anthers locule (Fig. 3). The developing microspores have a were fixed in 1% osmium tetroxide in M/15 phos- spiny exine by this stage, the outer layer of the exine phate buffer for 2-16h with continuous rotation. (the ectexine) is not fully differentiated (Fig. 4). The They were then washed in buffer and transferred tapetum starts to intrude between the microspores through 15%, 30% and 50% dimethyl sulphoxide for early in the free microspore stage. Each tapetal cell 30 min in each solution. The anthers were freeze- has a continuous plasma membrane and has an fractured on a liquid nitrogen-cooled metal block organelle-rich cytoplasm, with particularly abundant using a razor blade and hammer. The fragments were endoplasmic reticulum (Fig. 5). At this stage of devel- collected and thawed in fresh 50% dimethyl sulphox- opment the tapetum is generally considered to be a ide. After copious washing in buffer the specimens highly active secretory tissue producing sporopol- were transferred to 0.1% osmium tetroxide in M/15 lenin precursors which form the exine. Consistent phosphate buffer and left to macerate at 4C for 14 with this interpretation, numerous dictyosomes are days. During this period the specimens were regularly present in the tapetal cytoplasm (Fig. 6). checked and the solution replenished if it had dis- As the microspores develop (Figs. 7, 8) the exine coloured. To enhance electrical conductivity the spec- differentiates and becomes caveate and the micro- imens were fixed in 1% osmium tetroxide, washed, spore nuclei enlarge. At this stage, the tapetum treated with 2% tannic acid, washed again and intrudes between the microspores but retains a con- refixed in 1% osmium tetroxide. They were then tinuous plasma membrane around its convoluted dehydrated by transfer through an acetone series and inner surface. As the tapetal cells extend towards the critical point dried. The pieces were mounted on to centre of the anther locule their large nuclei maintain specimen stubs using Araldite adhesive and sputter a peripheral position. The tapetal cytoplasm (Fig. 8) coated with approximately 15nm of gold/palladium. continues to be actively synthetic and retains a highly Images were taken using a Hitachi 5800 field emis- organized system of endoplasmic reticulum. sion scanning electron microscope at an accelerating Once the exine reaches its mature morphology, the voltage of 8kV. microspores enter a characteristic vacuolate stage (Fig. 9) in which the mlcrospore cytoplasm is dis- placed to the periphery by a large vacuole. The tapetum, having completed its contribution to exine RESULTS AND DISCUSSION synthesis, begins to degenerate. The most conspicu- ous change, at this stage, is the absence of extensive Plasmodial tapetum sheets of endoplasmic reticulum (Fig. 10). Numerous When the anther tissues first differentiate, the tapetal vesicles are now present in the tapetal cytoplasm and cells form a cylinder surrounded by both the parietal the plasma membranes are no longer continuous. The cells and the epidermal cells and enclosing the micro- freeze fracture and cytoplasmic maceration tech- sporocytes. This configuration (Figs. 1, 2) initially nique reveals fine details of the pollen wall, such as occurs in both major classes of tapetum. In the Com- the minute cavities known as internal f oraminae positae, and in 31 other flowering plant families with (SKVARLA and TURNER, 1966) present within elements Two Tapetal Types in Angiosperms 219 I 4 2 5 3 6 Figs. 1-6. Catananche caerulea. Fig. 1. Premeiotic anther locule. x 960. Fig. 2. Locule at tetrad stage, tapetum binucleate. x 880. Fig. 3. Locule with free microspores. x 880. Fig. 4. Detail of Fig. 3. x 2,400. Fig. 5. Detail of microspore and tapetum from Fig. 3. x 8,800. Fig. 6. Detail of tapetal dictyosomes in Fig. 3. x 48,000 Abbreviations: A aperture, E exine, ER endoplasmic reticulum, D dictyosome, M microspore, MS microsporo- cyte, N nucleus, O orbicule, L lipid, T tapetum, V vacuole. 220 S. H. BARNES and S. BLACKMORF: of the ectexine. Catananche, in common with other members of the At a later stage (Figs. 11, 12), the nucleus of each Lactuceae, has tricellular pollen grains at maturity. microspore undergoes mitosis, giving rise to a gener- This situation results from a further mitotic division ative and a vegetative cell. Technically this division of the generative cell (Fig. 11), giving rise to the male marks the transition from microspore to pollen grain. germ unit (BARNES and BLACKMORE, 1987). By this 7 10 8 11 9 12 Fig. 7-12. Catananche cc rulea. Fig. 7. Locule with plasmodial tapetum and free microspores. x 800.