Insemination of the archegonium and fertilization in Taxus baccata L
ROGER I. PENNELL* and PETER R. BELL
Department of Botany and Microbiology, University College London, Coiver Street, London W'CIE 6BT, UK
•Present address and correspondence to: John Innes Institutue and AFRC Institute of Plant Science Research, Colney Lane, Norwich NR4 7UH, UK
Summary
A study of fertilization in Taxus baccata in the the formation of numerous points of contact electron microscope has revealed novel features. between the two. The membranes fuse at these Insemination of the archegonium is facilitated by points and pores are rapidly formed. The pro- local perforation of the wall of the young pollen gressive enlargement of these pores ultimately tube. Digestion of the wall begins before the eliminates any partitions and yields the zygotic pollen tube pierces the megaspore membrane but nucleus. There is a possibility that, as in some is not completed until its tip makes contact with other gymnosperms, the plastids and mitochon- the neck cells of the archegonium. As soon as a dria of the zygote come in part from the male pore is formed a single sperm nucleus and some gametophyte, but whether from the remains of cytoplasm of the male gametophyte enter the the spermatogenous cell cytoplasm or from the. archegonium. Which of the paired sperm nuclei pollen tube lumen is not clear. move from the pollen tube into the archegonium appears to be a matter of chance. Close apposition Key words: fertilization, gymnosperm, sperm nucleus, of sperm nucleus and egg nucleus is followed by Taxus baccata.
Introduction differ in their complements of mitochondria and plas- tids. Although the sperms of Taxus are now known to Fertilization of an egg by a non-motile sperm occurs be free nuclei that are morphologically similar (Pennell amongst the vascular plants only in the spermato- & Bell, 1986a), and not strongly dimorphic as pre- phytes, the male gametes being transported to the viously thought (see, e.g., Dupler, 1917), the absence female by a specialized outgrowth of the male gameto- from the pollen tube of prothallial cells and the phyte termed the pollen tube. The pollen tubes of protracted period over which the male gametophyte flowering plants invariably penetrate the embryo sac, develops (Pennell & Bell, 1986a) make the genus ideal often by way of a synergid (Mogensen, 1978), and for the study of fertilization in detail. Here we present liberate within the embryo sac a pair of sperm cells. the results of a structural study designed to clarify the The pollen tubes of gymnosperms fulfil a similar ways in which the sperms of a gymnosperm participate function, but they remain intact as they penetrate the in fertilization, and in which consistent differences in boundary of the female gametophyte and grow towards movement of the two sperms would point to a differ- the archegonia within. In many gymnosperms each ence in competence. pollen tube then inseminates a single archegonium. Since there is no secondary fusion in gymnosperms, half of the sperms are condemned to inactivity. This raises the possibility that in gymnosperms generally the Materials and methods male gametes differ in terms of competence, only one of each pair formed from a spermatogenous cell being Large numbers of ovules were collected from mature female inherently capable of fertilizing an egg. Sperm speci- English yews (Taxus baccata L.) growing close to one or ficity has been demonstrated in the flowering plant more male trees during late June and early July. Thin median Plumbago (Russell, 1983, 1985), where the sperm cells sections of ovules were fixed with glutaraldehydc and osmium Journal of Cell Science 89, 551-559 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 551 tctroxidc, and embedded in Epikote 812 Substitute as de- electron microscope. Evidently the pollen tube wall in scribed by Pennell & Bell (1985«). Ultrathin sections were the region formerly perforated by the worm-like chan- stained with uranyl acetate and lead citrate, and observed nels was now locally discontinuous. Within the pollen with a Siemens Elmiskop 102 transmission electron micro- tube the spermatogenous cell had divided, and the scope. For light microscopy 4ftm sections were examined liberated sperm nuclei lay close to the aperture in the with a Zeiss Photomicroscope II, and those of interest were wall (Fig. IE). No regular differences in the positions remounted according to the method of Woodcock & Bell (1967) for re-sectioning and electron microscopy. of the two sperm nuclei in the pollen tube were observed, nor was there an alignment that suggested that one sperm took precedence over the other in its Results passage into the archegonium. An inseminated pre-karyogamic archegonium was Examination of ovules fixed at the beginning of July observed only once. In this preparation the pollen tube revealed mitotic spermatogenous cells or fully formed clearly lay between the megaspore membrane and the sperm (now known to be free nuclei in Taxus) in the haploid cells of the female gametophyte (Fig. 2A), but presence of eggs or young proembryos. The sperms the area proximal to this site was absent from the were sometimes conspicuous in the pollen tube, and on section. Two nuclei lay in the midst of the archegonium one occasion (out of about 40) a sperm was observed (Fig. 2A). One was circular, about 20 fim in diameter, within the egg cytoplasm. In 10 preparations with and contained a distinct nucleolus. This was identified zygotes or very young proembryos, four pollen tubes as a sperm nucleus. The second was crescentic in each contained a single sperm lying close to the profile, measured approximately 15/.tm X 8/im and archegonium or at the junction of the two gameto- contained no nucleolus but many nuclear bodies phytes. The pollen tube made contact with the micro- (Figs 2A, 3A) of the kind formerly described in Taxus pylar surface of the female gametophyte during June, (Pennell and Bell, 19856). This was identified as the and its tip, or a closely adjacent lateral region, rapidly egg nucleus. Egg nuclei in adjacent virgin archegonia spread over the megaspore membrane. Although the were circular in section but also contained nuclear tube sometimes lay over an area of the gametophyte bodies of similar size and shape to those in the that contained two or more archegonia, it ultimately inseminated egg (Fig. 2A). The sperm lay in the made close contact with only one. The appearance concavity of the egg nucleus so that the envelopes of the within the pollen tube, between the spermatogenous two structures were closely parallel (Figs2A, 3A). cell and the egg cell (up to 100 /.tin beyond the Although the sperm had presumably approached the megaspore membrane), of a mass of osmiophilic cyto- plasm (Fig. 1A) marked the onset of fertilization. This Figs 1A,E, 2A and 3A are light micrographs. The cytoplasm was typically present close to or appressed remainder are electron micrographs. upon the inner surface of the pollen tube wall directly Fig. 1. Structural changes in the pollen tube preceding above the subjacent egg cell (Fig. 1A), but sometimes sperm discharge. A. The pollen tube (pt) is close to the formed a diffuse mass close to the spermatogenous cell. female gametophyte (fg) and a mass of dense cytoplasm It was conspicuously rich in large lipid globules (arrowhead) appears between the spermatogenous cell (sc) (Fig. IB) but also contained a profusion of dictyosomes and egg cell (ec). At this stage the pollen tube has not and ribosomal endoplasmic reticulum (Fig. IB). In breached the megaspore membrane (nun). Bar, SOfim. almost all preparations the pollen tube plasma mem- B. Detail of dense cytoplasm featured in the pollen tube brane in this region was difficult to resolve, but its shown in A. Endoplasmic reticulum (arrowheads), position was demarcated by numerous osmiophilic dictyosomes and large globules of lipid (left) arc abundant globules up to 50 nm in diameter (Fig. 1B,C). Some of in this cytoplasm. The plasma membrane close to this these globules were sometimes associated in the cyto- region is associated with densely staining globules (arrow). plasm with large vesicles (Fig. IB). Local regions of /, pollen tube lumen; w, pollen tube wall. Bar, 0'5jitm. C. The pollen tube plasma membrane adjacent to the distal the pollen tube wall between this mass of cytoplasm mass of osmiophilic cytoplasm (upper) is indistinct, but its and the archegonium below also contained osmiophilic position is made conspicuous by many osmiophilic globules (Fig. ID), situated at the ends of worm-like globules, iv, pollen tube wall. Bar, 0*5 jum. D. Narrow tracks extending into the wall for up to 400 nm from the channels in the pollen tube wall (iv) near the distal plasma membrane (Fig. ID). Aggregates of these aggregate of cytoplasm. Some of these terminate in channels were separated by regions of pollen tube wall osmiophilic globules identical with those formerly that were apparently undisturbed. associated with the plasma membrane (arrowhead). /, pollen tube lumen. Bar, 0-4/*m. E. Paired sperm nuclei (s) Other fixings revealed the onset of sperm discharge. shortly before discharge into the archegonium (not in The region of the pollen tube wall that lay closely over section plane). The pollen tube (pt) is closely appressed to the archegonium became indistinct in the light micro- the female gametophyte (fg) and the boundary between the scope (Fig. IE) and wholly unrecognizable in the two appears to be largely dispersed. Bar, lOf/m.
552 R. 1. Pennell and P. R. Bell k*
Fertilization in Taxus baccata 553 554 /?. /. ana '/, ii. egg nucleus from a micropylar direction, it lay to one 1 or 2nm across. Contact was followed first by the side of the egg nucleus when the two were appressed. annealing of the outer membranes of the two envel- In material containing zygotes and developing proem- opes, and then by the fusion of the inner membranes, bryos a sperm could frequently be observed in the so that a small channel connected adjacent nuclco- pollen tube close to the point of insemination, and in plasms (Fig. 3B). Circular fragments of membrane this position its morphology (Fig. 2B) was little differ- could generally be observed within these bridges. In ent from that preceding insemination, or from that of slightly later stages remnants of membrane could still the sperm that had once accompanied it in the pollen be resolved at these sites (Fig. 3C), but they were no tube. The boundary of this sperm sometimes appeared longer circular in profile. Sometimes the envelope to be disrupting (Fig. 2B). In the micropylar regions of profiles at the point of contact were difficult to inter- all inseminated archegonia the cytoplasm was vacuo- pret, the respective membranes appearing to be in a late, and distinct from that of the chalazal half complex knot before the formation of a pore (Fig. 3D). (Fig. 2A). A mass of osmiophilic cytoplasm was fre- A section of the gametes parallel to the axis of the quently present in its midst, however. This cytoplasm archegonium revealed about 12 points of contact. The contained globules visually identical with polysacchar- numerous fusions formed in this way each rapidly ide-rich 'P-particles' (Fig. 2C) (Heslop-Harrison, expanded laterally, taking in more and more of the 1979) prominent in the earlier development of the male parallel nuclear envelopes until the pair of nuclei were gametophyte (Pennell & Bell, 1986a) and, at high wholly confluent and the intervening cytoplasm was resolution, structures resembling mitochondria and expelled. dictyosomes. When the sperm nucleus lay within the The cytoplasm of the zygote was reminiscent of that concavity of the egg nucleus it was larger than it of the egg from which it developed (Pennell & Bell, appeared in the pollen tube and its diameter was 1987). Structured cytoplasm lay only in the chalazal approximately the same as that of the egg nucleus. The half of the cell and within it the mitochondria, still area of contact amounted to about 50% of the surface circular in profile but with more pronounced internal area of each nucleus, the two envelopes being closely lamellae, were dispersed. In contrast to the cytoplasm parallel and some 500 nm apart. of the egg, that of the zygote contained conspicuous Fusion of the envelopes began at a number of sites proplastids. The membrane-bound osmiophilic glob- clearly separated from one another. Sometimes it ules that were characteristic of the cytoplasm of the egg appeared that the egg nucleus initiated the fusion by were fewer in number and often confined in groups at way of small protrusions from its surface directed the periphery of the cell. A new layer of fibrogranular towards the sperm nucleus (Fig. 3B). These then material appeared between the plasma membrane and stimulated the formation of matching embayments at cell wall, and became more conspicuous as the proem- the points of contact (Fig. 3B). The basal diameter of bryo developed. the protrusions was approximately 25 nm but the tip that made contact with the apposed envelope was only Discussion
Fig. 2. Insemination of the archegonium. A. A pollen tube The low frequency of ovules containing sperm nuclei (pi) overlies the female gametophyte (fg) and has clearly and inseminated archegonia contrasts markedly with penetrated the megaspore membrane (mm). A single sperm the large number in which spermatogenous cells and nucleus (s) has entered an archegonium (left) and is within the concavity formed in the egg nucleus (en). The two proembryos were found. This suggests that the ter- nuclei have apparently rotated anticlockwise through 90° minal stages in the reproductive cycle of Taxus are and become encircled by a concentric zone of cytoplasm. ephemeral. It is likely that the sperms remain in the In the virgin archegonium (right) only the egg cell (ec) is pollen tube for just a few hours, and that insemination conspicuous. The nuclei of both eggs contain nuclear and karyogamy are completed in a similarly short time. bodies, n, nucellus. Square overlay, area depicted in C. It is clear that discharge of the sperm nuclei from the Bar, 50,um. B. Detail of non-functional sperm nucleus after pollen tube depends upon the dissolution of a region of fertilization has taken place. The sperm is little different its wall. The osmiophilic cytoplasm that appears close from that which brought about fertilization and still bears a to and subsequently overlies a comparable area of the conspicuous nucleolus (no). The sperm envelope is locally pollen tube wall contains endoplasmic reticulum and inflated (arrowheads). Bar, 1 nm. C. Detail of aggregate of dictyosomes, both organelles involved in protein syn- osmiophilic cytoplasm typically present in micropylar thesis and targeting. The osmiophilic globules that regions of inseminated eggs and overlain by square in A. Bodies identical with P-particles formerly present in the develop at the plasma membrane subsequently appear vegetative cell of the male gametophyte are present within to traverse the membrane and enter the pollen tube this cytoplasm (#) and are enclosed by an osmiophilic wall as discrete bodies, recalling events reported during mass, which may also contain markers of spermatogenous the movement of pro-orbicular lipid in the tapetum of cell cytoplasm. Bar, 1 fim. Taxus (Pennell & Bell, 19866). The channels that arc
Fertilisation in Taxus baccata 555 V..
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556 R /. and P. R. Bell formed by their motion within the wall are not them- In flowering plants in which the sperm cells remain selves bounded by membrane, but seem to represent intact during insemination of the embryo sac (e.g. localized regions of dissolution. It is conceivable that Plumbago) the plasma membrane of one sperm cell extensive lytic activity of this kind could weaken an fuses with that of the egg and the sperm nucleus enters area of the wall to make it incapable of withstanding the egg cytoplasm (Russell, 1983). The situation in turgor, and lead to its explosive disruption within the Taxus is very different from that in Plumbago, how- archegonial neck. Indeed, in the flowering plants it is ever, and more akin to that in Gossypium, where the the formation by lysis of a small pore, either apically or sperm cells are stripped of their cytoplasm in passing subapically in the wall of the tube (Jensen & Fischer, through the synergid (Jensen & Fischer, 1968). In 1968; Russell, 1982) that permits sperm discharge. In these plants only free nuclei are delivered to the egg cell Taxus the release of the sperms into the archegonium is surface, and hence the process cannot involve the delayed until the tip of the pollen tube has breached the fusion of gametic plasma membranes. It is conceivable megaspore membrane, but the means by which it does that in Taxus the plasma membrane at the tip of the this remain unclear. The complex profiles that develop pollen tube (still intact despite the dissolution of the in this region prevent critical analysis, but suggest that wall) fuses with the egg cell plasma membrane, creat- the steady passage of the tube through the nucellus is ing a cytoplasmic pathway between the pollen tube halted at this interface. The resistance afforded by this lumen and the egg cytoplasm through which gametic barrier could conceivably account for the terminal discharge could occur. In Taxus the presence of P- meanderings of the tube. particles in the neck regions of inseminated arehegonia The mature archegonium of Taxus bears only a short confirms that some vegetative cell cytoplasm ac- neck. In the zooidogamous gymnosperms the fertiliz- companies the sperm nucleus into the egg. The pres- ation fluid above the arehegonia apparently causes the ence of inflated mitochondria and osmiophilic bodies neck cells to swell and separate from one another, derived from lysosomes within the ejected cytoplasm providing access to the swimming sperm (Larson, also indicates its dual origin within the male gameto- 1926; Norstog, 1972). There is no fertilization chamber phyte, since both organelles are characteristic of the in Taxus and no fertilization fluid, and it seems spermatogenous cell (Pennell & Bell, 1986tf). unlikely that the neck cells of the archegonium separate The subsequent movement of the sperm nucleus in this manner. Although it was never possible to towards the egg nucleus takes place in response to observe in detail the means by which the sperms were unknown forces. It seems unlikely that cytoplasmic carried into the archegonium, the convoluted profiles streaming of the 'reverse-fountain' type (Berthold, in this region suggest that each pollen tube forces its 1888) is responsible. It is, however, conceivable that way between the neck cells, and that these in conse- the microtubules associated with the outer membrane quence become disrupted and degenerate. This ap- of the Taxus egg nucleus (Pennell & Bell, 1987), and in pears to take place in all conifers whose arehegonia are other genera with the subsequent zygote nucleus isolated from one another within the gametophyte (Camefort, 1968), participate in the active movement (Singh, 1978). of the sperm, as do the microtubules of the sperm aster of the urchin Lytechinus (Schatten, 1982). The fusion of the two gametic nuclei by way of the Fig. 3. Karyogamy. A-D. Prepared from the 4^
Fertilization in Taxus baccata 557 (tlaupt, 1941) and Abies (Camefort, 1969) it is facili- pollen tube apparently overlies more than one arche- tated by the cup-shaped form of the egg nucleus at this gonium. Although the possibility then exists that both time. The uniformity of these findings implies that sperms may then be functional, fertilizing adjacent localized protrusions are generally responsible for in- eggs, there are no reports of pollen tubes developing itial contact between fusing gametes, although it is two pores. conceivable that points of adhesion between closely parallel membranes become distorted by shrinkage during fixation and give rise to similar profiles. References The molecular interactions that occur between fus- ing higher plant cells at sites such as these remain ADAIR, W. S., HWANG, C. & GOODENOUGH, U. W. (1983). unclear. Cell surface glycoproteins are now known to Identification and visualization of the sexual agglutinin be involved in gamete recognition and agglutination in from mating-type plus flagellar membranes of Chlamydomonas (Adair et al. 1983; Samson et al. Chlamydomonas. Cell 33, 183-193. 1987), and have been implicated in cell recognition in BAIRD, A. M. (1953). The life history of Callitris. flowering plants generally (Clarke & Knox, 1978). Phytomorphology 3, 258-284. Although there are grounds for implicating stylar BERTHOLD, G. (1888). Studien iiber Pivtoplasmamechanik. glycoproteins in incompatible recognition in Nicotiana Leipzig: Arthur Felix. (Clarke et al. 1985), there is no evidence that similar BRENNAN, M. & DOYLE, J. (1956). The gametophytes and molecules in the plasma membranes of flowering plants embryogeny of Athmtaxis. Scient. Pmc. R. Dubl. Soc. 27, 193-257. participate in gametic interactions. Similarly, fusions CAMEFORT, H. (1967). Fecondation et formation d'un between nuclear envelopes during karyogamy has not neocytoplasme chez le Larix decidua Mill. (Larix yet been examined biochemically. europea D. C). C. r. hebd. Seanc. Acad. Sci., Paris, ser. The apparent reappearance of plastids in the zygote D 265, 1784-1787. (they are not visible in the mature egg; Pennell & Bell, CAMEFORT, H. (1968). Cytologie de la fecondation et de la 1987) suggests very strongly that many, if not all, are of proembryogenese chez quelque gymnospermes. Bull. paternal origin. They may come from the cytoplasm of Soc. bot. Fr. 115, 137-160. either the vegetative cell or the ruptured spermatogen- CAMEFORT, H. (1969). Fecondation et proembryogenese ous cell. Paternal or biparental plastid inheritance has chez les Abifitacees (notion de neocytoplasme). Revue Cytol. Biol. veg. 32, 253-271. been proposed in a number of gymnosperms (see, e.g., CHESNOY, L. (1969a). Sur l'origine du cytoplasme des Chesnoy, 1973; Chesnoy & Thomas, 1971; Willemse, embryons chez le Biota orientalis Endl. (Cupressacees). 1974), and is known to occur in some flowering plants C. r. hebd. Seanc. Acad. Sci., Paris, ser. D 268, (e.g. Plumbago; Russell, 1983). Inheritance of mito- 1921-1924. chondria amongst the gymnosperms is maternal CHESNOY, L. (19696). Sur la participation du gamete male (Moussel & Moussel, 1973), biparental (Chesnoy & a la constitution du cytoplasme de l'embryon chez le Thomas, 1971) or paternal (Chesnoy, 1969a,6; Ches- Biota orientalis Endl. Revue Cytol. Biol. veg. 32, noy & Thomas, 1971; Willemse, 1974). Comparison of 273-294. the mitochondrial populations of the egg (Pennell & CHESNOY, L. (1973). Sur l'origine paternelle des organites Bell, 1987) and zygote of Taxus suggest that inheri- du proembryon du Chamaecyparis lawsoniana A. Murr. tance may also be biparental in this genus, but the close (Cupressacees). Caryologia 25 (Suppl.), 223-232. similarity of the male and female mitochondria make it CHESNOY, L. & THOMAS, M. J. (1971). Electron difficult to be certain. A clear picture of the manner of microscope studies on gametogenesis and fertilization in gymnosperms. Phytomorphology 21, 50—63. inheritance of both mitochondria and plastids must CLARKE, A. E., ANDERSON, M. A., BACIC, T., HARRIS, P. await a reliable means of marking these organelles. J. & MAU, S.-L. (1985). Molecular basis of cell The present study throws no light upon the question recognition during fertilization in higher plants. In The of sperm competence in gymnosperms. Although the Cell Surface in Plant Growth and Development (ed. K. paired sperm nuclei of Taxus are closely similar in Roberts, A. W. B. Johnston, C. W. Lloyd, P. Shaw & morphology (Pennell & Bell, 1986«), it seems unlikely H. VV. Woolhouse), pp. 261-285. J. Cell Sci. Suppl. 2. that the inactive nucleus could ever in nature fertilize Cambridge: The Company of Biologists Limited. another egg, since the pollen tube becomes firmly CLARKE, A. E. & KNOX, R. B. (1978). Cell recognition in associated with only one archegonium. In the similar flowering plants. Q. Rev. Biol. 53, 3-28. situations in Pinus (Willemse & Linskens, 1969) and DUPLER, A. W. (1917). The gametophytes of Taxus canadensis Marsh. Bot. Gaz. 64, 115-140. Agathis (Kaur & Bhatnagar, 1984), the inactive sperm HAUPT, A. W. (1941). Oogenesis and fertilization in Pinus cells can be observed at the apex of the egg while lambertiana and P. monophylla. Bot. Gaz. 102, 482-498. karyogamy is in progress. Nevertheless, in Callitris HESLOP-HARRISON, J. (1979). Aspects of the structure, (Baird, 1953), Athrotaxis (Brennan & Doyle, 1956) and cytochemistry and germination of the pollen of rye other conifers that bear archegonial complexes, the (Secale cereale L.). Ann. Bot. 44, Suppl. 1.
558 R. I. Pennell and P. R. Bell JENSEN, W. A. (1964). Observations on the fusion of nuclei RUSSELL, S. D. (1982). Fertilization in Plumbago in plants. J. Cell Biol. 23, 669-672. zeylanica: entry and discharge of the pollen tube in the JENSEN, W. A. & FISCHER, D. B. (1968). Cotton embryo sac. Can.jf. Bot. 60, 2219-2230. embryogenesis: double fertilization. Pliytomorphology 17, RUSSELL, S. D. (1983). Fertilization in Plumbago 261-269. zeylanica: gametic fusion and fate of the male KAUR, D. & BHATNAGAR, S. P. (1984). Fertilization and cytoplasm. Ain.J. Bot. 70, 416-434. formation of neocytoplasm in Agathis mbusta. RUSSELL, S. D. (1985). Preferential fertilization in Pliytomorphology 34, 56-60. Plumbago: ultrastructural evidence for gamete-level LARSON, A. A. (1926). A contribution to the life history of recognition in an angiosperm. Proc. natn. Acad. Sci. Bowenia. Trans. R. Soc. Edinb. 54, 357-394. U.S.A. 82, 6129-6134. MOGENSEN, H. (1978). Pollen tube-synergid interactions SAMSON, M. R., KLIS, F. M., HOMAN, W. L., VAN in Pmboscidea louisianica (Martineaceae). Am. J. Bot. EGMOND, P., MUSGRAVE, A. & VAN DEN ENDE, H. 65, 953-964. (1987). Composition and properties of the sexual MOUSSEL, B. & MOUSSEL, C. (1973). Evolution agglutinins of the flagellated green alga Chlamvdomonas rhythmique de l'appareil de Golgi et du plasmalemme en eugametos. Planta 170, 314-321. liason avec la croissance de la paroi enveloppant le SCHATTEN, G. (1982). Motility during fertilization. Int. prothalle femelle cenocytique de VEphedra distacliya L. Rev. Cytol. 79, 59-163. Caryologia 25 (Suppl.), 97-108. NORSTOG, K. (1972). Role of archegonial neck cells of SINGH, H. (1978). Embryology of Gymnospenns. Zamia and other cycads. Pliytomorphology 22, 125-130. Encyclopedia of Plant Anatomy, vol. 10 (2). Berlin, PENNELL, R. I. & BELL, P. R. (1985a)- Microsporogenesis Stuttgart: Gebruder Borntraeger. in Taxus baccala L: the development of the WILLEMSE, M. T. M. (1974). Megagametogenesis and archaesporium. Ann. Bot. 56, 415-427. formation of neocytoplasm in Pin us svlvestiis L. In PENNELL, R. I, & BELL, P. R. (19856). The use of gold- Fertilization in Higher Plants (ed. H. F. Linskens), labelled ribonuclease as a probe for RNA in nuclear pp. 97-102. Amsterdam, Oxford: North-Holland; New bodies and cytoplasmic inclusions in plant cells. Eur. York: American Elsevier. jf. Cell Biol. 38, 181-184. WILLEMSE, M. T. M. & LINSKENS, H. F. (1969). PENNELL, R. I. & BELL, P. R. (1986a). The development Developpement du microgametophyte chez le Pinus of the male gametophyte and spermatogenesis in Taxus sylveslris entre la meiose et la fdcondation. Ret ue Cytol. baccata L. Proc. R. Soc. Loud. B 228, 85-96. Biol. veg. 32, 121-128. PENNELL, R. I. & BELL, P. R. (19866). Microsporogenesis WOODCOCK, C. L. F. & BELL, P. R. (1967). A method for in Taxus baccata L.: the formation of the tetrad and mounting A(.l resin sections routinely for ultra-thin development of the microspores. Ann. Bot. 57, 545-555. sectioning, jfl R. unerase. Soc. 87, 485-487. PENNELL, R. I. & BELL, P. R. (1987). Megasporogenesis and the subsequent cell lineage within the ovule of {Received 18 November 1987 -Accepted 21 December Taxus baccata L. Ann. Bot. 59, 693-704. 1987)
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