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.