POLLEN ULTRASTRUCTURE IN ANTHER CULTURES OF DATURA INNOXIA

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1 J. Cell Set. 23, (1976) Printed in Great Britain POLLEN ULTRASTRUCTURE IN ANTHER CULTURES OF DATURA INNOXIA I. DIVISION OF THE PRESUMPTIVE VEGETATIVE CELL J.M. DUNWELL AND N. SUNDERLAND John Innes Institute, Colney Lane, Norwich, U.K. SUMMARY Ultrastructural features of embryogenic pollen in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generativecell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cell in Nicotiana tabacum. INTRODUCTION While the ultrastructural features of pollen embryogenesis have been studied in some detail in Nicotiana tabacum (Vazart, 19716,19730,6; Dunwell & Sunderland, 19740,6, 1975) little is known about the structural changes that occur in Datura innoxia as the pollen is diverted from its normal programme. Embryogenesis is readily induced in both these species and under very similar conditions (for review see Sunderland, 1974), yet the behaviour of their pollens during the initial stages is remarkably different. In both, the presumptive vegetative cell functions as an embryo mother cell, and gives rise to haploid embryos, but in D. innoxia the cell also contributes, in conjunction with the presumptive generative cell, to the formation of embryos of higher ploidy levels (Sunderland, Collins & Dunwell, 1974). D. innoxia also differs in being

2 47 J- M- Dunwell and N. Sunderland more prone to atypical microspore divisions in culture and these lead to the formation of anomalous* pollen grains which are susceptible to embryogenesis. We have therefore studied the ultrastructure of D. innoxia pollen during the early stages of culture in an attempt to elucidate the basis of its varied behaviour. The data will be presented in 3 parts each of which will deal with one specific aspect of the Datura situation. This first paper concerns the behaviour of the presumptive vegetative cell when it functions directly as an embryo mother cell. MATERIALS AND METHODS Plants of Datura innoxia Mill, were raised, and anther cultures initiated from them, in the manner previously described (Sunderland et al. 1974). Anthers were inoculated at a stage just prior to the microspore division (stage 3 as defined by Sunderland, 1974) and were harvested at 24, 48, and 72 h; they were each cut along the connective tissue into 2 longitudinal halves, and fixed, one for examination of the pollen in the light microscope, and the other for examination in the electron microscope. For comparative purposes, and for study of pollen development in vivo, fixations were also made of anthers covering stages 3-6 (pollen grains having a detached generative cell but lacking starch). Anther halves destined for light microscopy were fixed in a 113 v/v mixture of glacial acetic acid and ethanol, and the contents stained and mounted as described previously (Sunderland et al. 1974). The fixative used for electron microscopy consisted of a 5 % v/v solution of glutaraldehyde made up in cacodylate buffer (ph 6-8) containing 2 % w/v sucrose. After immersion in the glutaraldehyde for 2 h at room temperature, the anther halves were rinsed in several changes of buffer solution and postfixed in Caulfield's osmium fixative for a further hour at room temperature. Tissues were rinsed in distilled water, dehydrated in an acetone series, infiltrated and finally embedded in Spurr's resin (Spurr, 1969). During dehydration, anther halves were left for at least 30 min in each solution and at the 100 % step the acetone was saturated with uranyl acetate. Sections were cut on an LKB microtome, mounted on copper grids coated with a film of Parlodion, and counterstained in lead citrate according to the procedure of Reynolds (1963). As in the previous studies on Nicotiana tabacum (Dunwell & Sunderland, 1974a, b, 1975), most of the pollen in the cultured anthers degenerated rapidly. From observations made in the light microscope it was estimated that after only 24 h not more than 10-20% viable pollen The term 'anomalous' pollen grains is used here to avoid confusion with the 'irregular' pollen grains produced in this species in vivo and which arise by aberrant divisions at meiosis (see Collins, Dunwell & Sunderland, 1974). The observations referred to in this and the succeeding 2 papers all refer to the regular haploid pollen but since the non-haploid pollens also follow the same embryogenic pathways the remarks contained herein are equally applicable. Fig. 1. Section through the nuclear region of a microspore of D. innoxia just before division (stage 3). The mitotic spindle will form in a plane perpendicular to, or oblique to, the spore wall. The nucleus contains a prominent nucleolus (nu) and several heterochromatic spots. This particular section does not show starch in the plastids (p). x Fig. 2 Formation of the cell plate in a dividing microspore of D. innoxia in which the mitotic spindle has formed in a plane perpendicular to the spore wall. This section is from a cultured anther after 24 h and shows the same features as a dividing spore in vivo (stage 4). The plate has made contact with the intine but remnants of the phragmoplast (ph) can be seen where presumably plate deposition is still going on. Telophase chromosomes are present in the presumptive generative nucleus ign). Both nuclei have prominent nucleoli (nu). Note the osmiophilic material deposited on the tonoplast (arrows), x

3 Pollen ultrastructure in anther cultures. I 47i

4 472 J. M. Dunwell and N. Sunderland remained. Accordingly, only the most highly embryogenic anthers (as screened in the light microscope) were used. RESULTS In vivo observations In common with the microspores of Nicotiana tabacum (Vazart, 1971 a; Dunwell & Sunderland, 1974 a) and other dicotyledons which have been investigated at the ultrastructural level [Beta vulgaris, Hoefert, 1969; Linutn usitatissimum, Vazart, 1969), Datura microspores prior to mitosis show one large central vacuole separated from a thin layer of parietal cytoplasm by the tonoplast. The nucleus, with prominent nucleolus, lies close to the microspore wall (Fig. 1). Mitochondria and plastids are both structurally simple, the former having relatively few cristae and the latter occasional internal lamellae and starch grains. Immediately after completion of the microspore division 2 forms of pollen grain are observed. These differ with respect to the position of the presumptive vegetative nucleus. Depending on the orientation of the spindle during mitosis the vegetative nucleus comes to occupy either a central (Fig. 2), or a peripheral position. In both cases the nuclei are separated by a typical curved cell plate which partitions the cytoplasm of the microspore unequally. The 2 nuclei show similar interphase profiles and each contains a large, well defined nucleolus; indeed one nucleus can only be distinguished from the other with certainty by reference to the intervening wall. This is in contrast to N. tabacum in which the generative nucleus can be readily identified by the presence of highly condensed chromatin (see Dunwell & Sunderland, 1974 a). As the pollen grains develop the distinction between the two forms disappears. The vacuole is resorbed and the vegetative cell is packed with ribosomes produced by an enlarged nucleolus (Fig. 3). Plastids and mitochondria also increase both in number and electron density. The generative cell becomes detached from the intine and has a smaller nucleolus and a lower cytoplasmic ribosome concentration. Fig. 3. Section of a young pollen grain in D. innoxia during resorption of the vacuole and after detachment of the generative cell from the intine (stage 6). The outline of the generative cell (marked with asterisks) is obscured by the denseness of the vegetative-cell cy toplasm.the nucleolus (nu) in the vegetative cell is prominent compared to that in the generative cell and the cytoplasmic ribosomal density greater. Plastids and mitochondria are much more electron-dense in both cells (cf. Fig. 2). x Embryogenesis does not take place if anthers are cultured at this stage. Fig. 4. Section of an embryogenic pollen grain of D. innoxia showing the generative cell still attached to the intine and the vegetative nucleus at metaphase. The spindle has developed in the same plane as that of the microspore mitosis (cf. Fig. 2). Starchcontaining plastids and spherical lipid droplets (Z) are evident in the vegetative-cell cytoplasm. Stage 3 anther cultured for 48 h at 28 C in darkness. H medium without hormones, x 0000.

5 Pollen ultrastructure in anther cultures. I 473

6 474 J- M. Dunwell and N. Sunderland In vitro observations In anthers cultured just prior to the microspore division the microspores divide between o and 24 h. Some atypical pollen grains are produced (see Part III) but most of them are identical in structure to those produced in vivo. Both forms having either central or peripheral vegetative nuclei are present and they all show a thin layer of parietal cytoplasm and a large vacuole. It may be stressed that grains in more advanced stages of gametophytic differentiation (Fig. 3) were not observed in either the 24- or 48-h samples; nor were any of the various non-embryogenic forms of development as found in anther cultures of N. tabacum. (Dunwell & Sunderland, 1974a). The first division of the vegetative cell commences between 24 and 48 h, that is, the nucleus enters mitosis soon after completion of the microspore division. It does so while the generative cell is still attached to the intine (Fig. 4). The dividing vegetative cell still shows the same thin layer of parietal cytoplasm and large vacuole carried over from the microspore. This absence of typical postmitotic cytoplasmic synthesis indicates that the pollen embarks on its new programme as soon as the 2 gametophytic cells are laid down. If embryogenesis does not ensue the pollen degenerates. The spindle of the dividing vegetative nucleus is oriented in the same plane as that of the microspore mitosis and thus at cytokinesis cell-plate formation commences in a plane parallel to the wall of the attached generative cell (Fig. 5). As in the preceding division, aggregation of plate-forming vesicles begins within the spindle and subsequently radiates out into the cytoplasm until contact is made with the intine. However, the wall laid down between the 2 sporophytic cells does not follow the same regular course as occurs in the microspore division. The sporophytic wall sometimes makes contact with the generative-cell wall at the junction with the intine (Fig. 6), but in general the wall makes independent contact with the intine (Fig. 7). The plane of the division is such that the cell may be partitioned without the wall traversing the vacuole, so that a small cytoplasmic-rich cell is cut off from a large vacuolate cell. In many instances, however, the daughter cell adjacent to the generative cell contains a small vacuole (Fig. 7), suggesting that the developing cell plate traverses part of the vacuole presumably in association with a short phragmosome. The nature of the dividing wall is unclear. In profile the wall resembles that which separates the 2 gametophytic cells in consisting of 2 plasmalemmae on either side of an Fig. 5. Section, through the phragmoplast of a dividing embryogenic pollen grain, in D. innoxia at a slightly later stage than that illustrated in Fig. 4. Vesicles can be seen in the process of aggregation to form the cell-plate, deposition of which has commenced parallel to the attached generative-cell wall. The part of the section containing the generative cell is shown in Fig. 8. Only one of the two vegetative daughter-nuclei (vn), which is in late telophase, is present in the section. Stage 3 anther cultured for 72 h at 28 C. x Fig. 6. Section of a pollen proembryo in D. innoxia consisting of 2 vegetative daughtercells and an attached generative cell. The wall runs parallel to the generative-cell wall and joins the intine at the same point. Osmiophilic material coats the tonoplast (arrow). Stage 3 anther cultured for 72 h at 28 C. x

7 Pollen ultrastructure in anther cultures. I 475

8 476 J. M. Dunwell and N. Sunderland

9 Pollen ultrastructure in anther cultures. I 477 electron-translucent matrix. Possibly the normal type of sporophytic wall with a fibrillar component develops later. This lack of a fibrillar layer in the dividing wall of the first embryogenic division contrasts with the situation in N. tabacum in which a fibrillar layer develops not only in the dividing wall but also in the intine (Dunwell & Sunderland, 1975). In none of the sections examined was evidence of cytoplasmic degradation found akin to that which occurs in N. tabacum prior to the division of the vegetative cell (Dunwell & Sunderland, 19746). Lysosomes, one of the key features in TV. tabacum, are not present in the embryogenic Datura pollen nor is there any apparent structural regression in the plastids or mitochondria. On the contrary, initiation of embryogenesis in D. innoxia is accompanied by an increase in the electron density of both organelles. A feature suggestive of catabolic events is the deposition of highly electron-dense material on the tonoplast (Figs. 2, 6, 7). Similar deposits occur in the generative cell if a vacuole is present. The deposit, which has similar osmiophilic properties to lipid, increases between 24 and 48 h, and becomes associated with aggregates of small vesicles. The association of osmiophilic deposits with the tonoplast and their increase in size with time suggests that they might be concerned in the dissolution of membrane components during the change from one programme to the other. Similar deposits are not seen in the tonoplast in in vivo pollen. Changes also occur in the generative cell as the vegetative cell divides. Conversely to the in vivo situation (Fig. 3), ribosome density increases to a level greater than that in the vegetative cell (Figs. 7, 8). The generative-cell wall becomes constricted in places and in sectional view assumes a beaded appearance (Fig. 8). In such walls, traces of an electron-dense layer resembling a middle lamella can sometimes be seen (Fig. 8)- DISCUSSION The results indicate that the transformation of the presumptive vegetative cell to function as an embryo mother cell is a less complex process in Datura innoxia than in Nicotiana tabacum. This arises largely from the fact that in D. innoxia the new developmental programme is initiated while the cell is in an undifferentiated state; there does not, in fact, appear to be any postmitotic rise in the RNA and protein content of the vegetative cell as occurs in vivo. It seems likely therefore that the signal for the gene Fig. 7. Section of a pollen proembryo in D. innoxia from the same anther as the one illustrated in Fig. 6. The whole of one of the vegetative daughter-cells is shown. Note the prominent nucleolus and vacuole («). It is assumed that in this grain the cell plate cut off a portion of the vacuole of the mother cell in association with a short phragmo8ome and joined the intine some distance from the generative-cell wall. A vacuole has developed in the generative cell which may account for the higher ribosomal concentration in the cytoplasm. Osmiophilic material coats the tonoplast in both cells. Stage 3 anther cultured for 72 h at 28 C. x Fig. 8. Portion of the generative-cell wall from the embryogenic grain illustrated in Fig. 5. Traces of an electron-dense layer of unknown composition can be seen in the wall. This cell is vacuolate like the one in Fig. 7 and shows a similar high ribosomal concentration. Osmiophilic material coats the tonoplast in both cells, x

10 478 J. M. Dunwell and N. Sunderland inactivation/activation process involved in the switch of programme is perceived during late anaphase-telophase or at the latest immediately after the completion of cytokinesis. In this connexion, it is pertinent that embryogenesis is induced equally well if the anthers are cultured just before, as in the present investigation, or just after the microspore division (Sunderland et al. 1974). The pollen rapidly loses its embryogenie potential thereafter, and while sporadic embryo formation does occur in more advanced anthers, this is due to lagging grains that are still in a relatively undifferentiated state at the time of culture. It follows that the signal for the changed programme is probably neither perceived nor effected until after formation of the 2 gametophytic cells. In N. tabacum, activation of the vegetative cell does not appear to take place until a slightly later stage when the cell is occupied by relatively large amounts of gametophytic cytoplasm; even to the extent of complete resorption of the vacuole (Dunwell & Sunderland, 1974a). The cell, it may be emphasized, cannot express its embryogenic potential in this state; it does so several days later after degradation of much of the gametophytic cytoplasm. The question at once arises as to why the vegetative cell does not function in N. tabacum as it does in D. innoxia and divide before postmitotic RNA synthesis has commenced. To answer this adequately, information is required on the timing and rates of the various synthetic processes in vivo. Experiments on Tradescantia have shown that gametophytic rrna synthesis begins in the microspore just prior to its division and continues in the vegetative cell after the microspore division (for review see Mascarenhas, 1975). Judged by the relative staining capacity of the protein and RNA in the Datura and Nicotiana pollens, before and after the microspore division, and on the time taken for the vacuole to be resorbed and the cell to be completely occupied by cytoplasm, it appears that the premitotic contribution is relatively greater and the postmitotic rate considerably faster in N. tabacum: indeed, the postmitotic rise is so rapid in N. tabacum that by the time the vegetative cell has been laid down it already contains a considerable amount of cytoplasm and resorption of the vacuole is well under way. We do not think, therefore, that there is any fundamental diference between the two species in the timing of the activation process - it is confined to the early G x phase of the vegetative cell cycle in both; but because gametophytic influences are still largely unexpressed in D. innoxia when the signal is perceived the cell can respond immediately to it. A feature in which D. innoxia does differ markedly from N. tabacum is in the plane of the first embryogenic division. As has been seen, in D. innoxia the vegetative cell divides in the same plane as the microspore, and this suggests that the polarity determining the plane of the microspore division is retained during the short intervening interphase. The situation is similar to that described in anomalous in vivo pollen development in which supernumerary divisions of the vegetative cell occur (Sax, 1935; Upcott, 1939; Darlington & Thomas, 1941). In the cultured Datura pollen, however, this polarity is lost after the first division and succeeding divisions occur in different planes so that a spherical proembryo is formed inside the pollen grain (Sunderland et al. 1974). In N. tabacum, on the other hand, the first embryogenic division occurs in different planes, sometimes parallel to, at right angles or even oblique to that of the

11 Pollen ultrastructure in anther cultures. I 479 microspore division. Moreover, the dividing wall frequently bisects the grain into two equal cells and each cell is subsequently completely repopulated with cytoplasm before the next division begins (Dunwell & Sunderland, 1975). In D. innoxia, divisions follow each other rapidly and the large vacuole carried over from the microspore is progressively partitioned and distributed among the daughter cells. Finally, for the vegetative cell to function as an embryo mother cell the generative cell must remain quiescent. Light microscopy has shown that when this occurs (Sunderland et al. 1974) the generative cell degenerates after the first few divisions of the vegetative cell (the so-called A pathway of embryogenesis). It seems highly probable therefore that the generative cell remains attached to the intine during division of the vegetative cell and comes to occupy a position at the periphery of the proembryo and there degenerates. Grains were, however, observed in the 72 h samples in which the generative cell was detached from the intine and the vegetative nucleus still in interphase. These latter grains were interpreted as those in which both gametophytic cells participate in embryogenesis by nuclear fusion. This fusion process provides the theme for the next paper. REFERENCES COLLINS, G. B., DUNWELL, J. M. & SUNDERLAND, N. (1974). Irregular microspore formation in Datura innoxia and its relevance to anther culture. Protoplasma 82, DARLINGTON, C. D. & THOMAS, P. T. (1941). Morbid mitosis and the activity of inert chromosomes in Sorghum. Proc. R. Soc. B 130, DUNWELL, J. M. & SUNDERLAND, N. (1974a). Pollen ultrastructure in anther cultures of Nicotiana tabacum. I. Early stages of culture. J. exp. Bot. 25, DUNWELL, J. M. & SUNDERLAND, N. (1974*). Pollen ultrastructure in anther cultures of Nicotiana tabacum. II. Changes associated with embryogenesis. J. exp. Bot. 25, DUNWELL, J. M. & SUNDERLAND, N. (1975). Pollen ultrastructure in anther cultures of Nicotiana tabacum. III. The first sporophytic division. J. exp. Bot. 26, HOEFERT, L. L. (1969). Ultrastructure of Beta pollen. I. Cytoplasmic constituents. Am. J. Bot. 56, MASCARENHAS, J. P. (1975). The biochemistry of angiosperm pollen development. Bot. Rev. 41, 259-3I4- REYNOLDS, E. S. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, SAX, K. (1935). The effect of temperature on nuclear differentiation in microspore development. J. Arnold Arbor. 16, SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, SUNDERLAND, N. (1974). Anther culture as a means of haploid induction. In Haploids in Higher Plants: Advances and Potential (ed. K.J. Kasha), pp Guelph: University of Guelph. SUNDERLAND, N., COLLINS, G. B. & DUNWELL, J. M. (1974). The role of nuclear fusion in pollen embryogenesis of Datura innoxia Mill. Planta 117, UPCOTT, M. (1939). The external mechanics of the chromosomes. VII. Abnormal mitosis in the pollen grain. Chromosoma I, VAZART, B. (1969). Structure et Evolution de la cellule g6n ratrice du Lin, Linum usitatissimum L., au cours des premiers stades de la maturation du pollen. Revue Cytol. Biol. vig. 32, VAZART, B. (1971a). Premiere division haploide et formation de la cellule g6n6rarrice dans le pollen de Tabac. Annls Univ. A.R.E.R.S. 9, VAZART, B. (19716). Infrastructure de microspores de Nicotiana tabacum L. susceptibles de se developperenembryoides apres excision et mise en culture des antheres. C. r. hebd. Sianc. Acad. Sci., Paris 272,

12 480 J. M. Dunwell and N. Sunder land VAZART, B. (1973 a) Formation d'embryoides a partir de microspores de tabac: Evolution de ^infrastructure des cellules au cours de la premiere semaine de culture des antheres. Soc. bot. Fr. Mhn. Coll. Morphologic pp VAZART, B. (19736). Ultrastructure des microspores de tabac dans les antheres embryogenes. Caryologia 25, {Received 29 March 1976)