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Comparative Morphological, Anatomicaland Embryological Studies ofPrumnopitys taxifolia and P. ferruginea(Podocarpaceae),and the hydrodynamics of theirsaccate pollen grainsVOLUME IIILLUSTRATIONSSchool of Biological SciencesUniversity of AucklandThesis submitted in fulfillment ofthe requirements for the degree ofDoctor of Philosophy in BotanYJoshua SalterApril 2004

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Figure 2.1: Locations of collection sites lor Prumnopitys taxifolia andP. ferruginea in the North lsland, New Zealand.Pre-dispersal embryo development: Locations close to Auckland that were usedfor frequent collections of developing strobili:P. taxifolia: Locations 11 and 14.P. ferruginea: Locations 9 and 12.At most other sites, only one or two collections were made when opportunities arose.Post-dispersal embryo development: Seeds for planting and exhuming werecollected from beneath trees in the following locations:P. taxifolia: Locations 1, 5 and 11.P. ferruginea: Locations 2, 3, 6, 8, I and 19.Key to locations1 Foley's Bush, Kaitaia, Northland2 Puketi Forest, Northland3 Waipoua Forest and McGregor Reserve, Northland4 Marlborough Rd, near Waipoua Forest, Northland5 Frank Hudson's farm and environs. near Warkworth6 Parry Kauri Park, Warkworth7 QEll reserve, upper Puhoi ValleyI Waitakere Ranges9 Auckland University and Auckland Domain, Auckland City10 Awhitu Peninsula11 Auckland Regional Council Botanic Gardens and Totara Park12 Kirk's Bush, Papakura, South Auckland13 Western side of Hunua Ranges14 Southern end of Hunua Ranges15 Mamaku Plateau, central North lsland16 RangitotoLodge, Rangitoto Range, central North lsland17 Waimana River, northern Urewera National Park18 Whirinaki Forest, and western Urewera National Park19 Owhango Reserve, central North lsland20 Te Porere Redoubt, central North lsland21 Northern boundary of Mount Egmont National Park22 Tree Trunk Gorge, Tongariro River, Kaimanawa Range23 Little Bush, Hawkes Bay24 Northern end of Aorangi Mountalns, southern North lsland

o P. taxifoliao P. ferruginea_ 5-. -6I7 -----,138-1410-'11'12'0156D2Qo22NORTHISLANDANFig.2.1

Figure 3.1: Pollen cone morphology and arrangement in Prumnopitys taxifolia.A : Young fertile branch or spike approximately 14 mm long bearing pollen coneprimordia (arrowheads) in the axils of the leaf-like bracts, Unlike the new vegetativeshoots (arrow), the emerging spikes are glaucous and erect. Photographed earlysummer (December). Scale = 5 mm.B : Fertile branch or spike approx. 50 mm long bearing young pollen cones in axils ofleaf-like bracts (arrowheads). Cones are approx. 3 mm long x 1 mm diam.Photographed in autumn (March).C : Branched pollen cones can occur. In this specimen there are five branched cones(arrowhead) and several abnormally short cones (arrow).D : Mature pollen cones just prior to pollen dispersal. Leaf-like bracts have been shed.Pollen cones have become crowded towards apex of spike (arrow), due to reducedinternode growth. Cones up to 15 mm long and approx. 2 mm diam.E : Pollen cone and spike lengths vary with the individual. These were photographedin late winter (August) on a smalltree propagated by a cutting from adult foliage.F : Young pollen cone in early spring (September). The developing microsporangiaare concealed by the overlapping scale tips (arrowheads). Scale = 1 mm.G : Mature pollen cone in late spring (November). On each microsporophyll the twofree sporangia (arrowheads) are greatly expanded, while the scale tips are unchanged,Scale = 1 mm.

FFig.3.1

Figure 3.2: Young pollen cone of Pramno.pltys taxitolla'Gone in medlan LS and splke axis in TS. Oomposite image of liight micrographs d waxembeddedsp,ecimen, New r,nicrospornpirylls (arrorvheads) are fbnningatthe cone apexSporangial tissue (anow) differentiates on the aba

Fig. 3.2

Figure 3.3: Early initiation and development of the microsporophyll andmicrosporangium in Prumnopitys taxifoliaA to F : Five microsporophylls, from the youngest at the apex (A) to the oldest near thecone base (E and F).A : Microsporophyll primordia (arrowheads) at apex of the young pollen cone shown inFigure 3.2. Scale = 50 pmB : Sporangial initials (arrowhead) differentiate on the abaxialface of microsporophyllsclose to the cone apex. Scale = 50 pm.C : The developing archesporium comprises a distinct epidermis and a hypodermalzone several cells deep. The tapetum (t) and sporogenous tissue (s) are beginning todifferentiate. Scale = 100lrm.D : Developing archesporium (arrowhead) on a scale slightly more mature than thatshown in C. Scale = 100 pm.E : In a scale more mature than that of D, the tapetum (t) and sporogenous tissue (s)are now clearly defined on the abaxial side. A sunken stoma (st) is also visible in theabaxial epidermis. Most of the scale is composed of photosynthetic mesophyll cells(me), with their peripheralchloroplasts stained grey. Scale = 100 pm.F : Detail of the sporangium shown in E. The sporogenous cells (s) now form aroughly spherical mass surrounded by the developing tapetum (t), A band of small darkcells between the tapetum and epidermis indicates the posiiion of the developingstomium or dehiscence zone (dz). Scale = 50 pm.

[..#Hll,';',i. IFig. 3.3

Figure 3.4: Developing microsporangia in the young pollen cone of Prumnopitystaxifolia.A : Growing sporogenous tissue (s) in a scale more developed than that of Figs. 3.3 Eand F. The cells of the surrounding sporangial tissues (tapetum (t), parietal layers (pl)and dehiscence zone (dz) are much smaller than the photosynthetic mesophyll cells(me). Scale = 50 pm.B : Tangentialsection of a microsporophyll shows the two free sporangia (s)developing. Scale = 50 pm.C : Slightly oblique non-median LS of a young cone showing fertile scales (fs) aboveand sterile scales (ss) below. Scale = 100 pm.D : Enlarged view of the lowermost fedile scale of the cone in C. The large granularnuclei (sn) of the sporogenous cells are just visible in the darkly stained cytoplasm. Thesingle layer of tapetal cells (t) is now distinct from the parietal layers (pl). Thedehiscence zone (dz). Scale = 10 pm.E : The tissues of the sporangium (sp) continue to grow and differentiate while themesophyll (me) of the scale tip undergoes little further change. Three sunken stomata(st) lie in the abaxial epidermis. Scale = 100 Fm.F : Detail of the sporangium shown in E. The zone of sporogenous cells (s) in thissection is about 35 pm diam. The exolhecium (ex) is beginning to differentiate aroundthe sporangium. The stomium or dehiscence zone (dz) forms a conspicuous band ofsmall dark cells in the exothecium (ex) and parietal layers (pl). Scale = 50 pm.

Fig. 3.4

Figure 3.5: Scanning electron micrographs of microsporophylls from pollencones ol Prumnopltys taxitolia.A: lmmature microsporophyll, adaxial and basalview. The dehiscence zone orstomium is visible as a transverse groove around each sporangium (arrowhead).Scale = 200 pm.B : lmmature microsporophyll, abaxial view showing two rows of sunken stomata.Scale tip towards bottom of picture. Scale = 200 pm.C : Mature microsporophyll, abaxialview, showing two free sporangia. The stomia arenot visible from this angle. Scale = 500 !rm.D : Mature microsporophyll, adaxialand basalview. The stomium forms a deepgroove around the inner face of each free sporangium. The exothecial cells tend to lie inrows parallelto the stomium. Scale = 500 pm.E : Part of a pollen cone, viewed from below, showing the position of the stomia inrelation to the cone axis. Scale = 1 mm.F: Enlarged view of E, showing that the stomium extends to the widest part of eachsporangium (arrowhead). Scale = 500 pm.

.'TA,{-t,I-t-_--lr:Tfii: i, 1,..1;1';"II"'':E.ETFidb\r-nE.' ,Fig. 3.5

Figure 3.5: Developing microsporangia and vasculature of the microsporophyllin Prumnopitys taxitolia.A : Young pollen cone in tangential section. Two microsporophylls, in tangential LS,show the two free sporangia and the single large resin canal (rc) which accompanies thevascular strand. Other microsporophylls, sectioned obliquely, show the diversity of celltypes within the mesophyll. Scale = 200 pm.B : Enlarged view of the uppermost microsporophyll shown in A. The single vascularbundle (vb) is accompanied by a resin canal (rc) (with the epithelium damaged duringsectioning). The expanding sporogenous tissue (s) is now up to 100 pm. diam. Scale =100 pm.C and D : Two serial sections of the microsporophyll shown in B, cut successivelycloser to the cone axis. In C, the stomial initials (dz) can be seen as a band oftransversely elongated small dark cells in the left-hand sporangium. In D, the stomialinitials of the right-hand sporangium are visible. Scale = 100 pm.E : A microsporophyll in median LS. The resin canal (rc) turns towards one of thesporangia, ending near the parietal layers (pl). Just beyond the bend of the resin canal,the vascular bundle (vb) terminates in a zone of transfusion tissue (tt). (See also Plate3.7). Scale = 100 pm.F : Electron micrograph of the vascular bundle. The xylem (x) consists of just 5-7tracheids with spiralthickenings of the cell walls. The phloem cells (ph) are morenumerous and ordered in rows. The resin canal (rc) is lined with an epithelium (et).Scale = 20 pm.

Fig. 3.6

Figure 3.7: (Prumnopitys taxifolial Transfusion tissue in a youngmicrosporophyll. Mature microsporophylls at the time of pollendispersal.A : Electron micrograph of transfusion tissue in the leaf-like portion of a youngmicrosporophyll. The tracheids (tr), which intermingle with parenchyma cells, have verythin primary cellwalls and irregular spiral secondary thickenings (arrowheads) in variousorientations. Scale = 5 Fm.B : Light micrograph of transfusion tissue in the leaf-like podion of a youngmicrosporophyll. Tracheids with lignified spiralthickenings (arrowheads) intermingle withthe photosynthetic parenchyma. In this resin-embedded specimen stained withmethylene blue, primary cell walls are purple, lignified secondary thickenings areturquoise blue, and nuclei and chloroplasts are dark blue. Scale = 10 pm.C : Mature microsporophylls in LS and tangential LS, showing the prominentexothecium (ex) around each sporangium. The lower sporangium has dehisced (dz), butthe upper sporangia are filled with bisaccate pollen grains. Resin-embedded specimenstained with methylene blue. Scale = 100 Fm.D : Mature sporangium in tangential LS through the sporangia, showing that theexothecium (ex) almost entirely surrounds each sporangium except for a narrow stripcarrying the vascular bundle (vb) and resin canal (rc). Scale = 100 pm.E : Mature microsporophyll in LS through one sporangium, but to one side of the leaflikescale tip. In median section the scale tip would be almost twice the length it appearshere. The exothecium is inlerrupted by the stomium or dehiscence zone (dz) on theinner side of the sporangium, facing the cone axis. Scale = 100 pm.F : Enlarged view of the exothecium shown in E. ln the exothecial cells, the anticlinalwalls appear rigid while the external periclinal walls appear flexible. The stomiumconsists of three or four small cells similar to exothecial cells. Scale = 100 pm.

Figure 3.8: Sporangial wall of the mature pollen cone in Prumnopitys taxifolia.(Resin-embedded specimen sectioned for both LM and TEM).A : Enlarged view of the upper undehisced microsporophyll in Fig 3.7 C, stained withmelhylene blue. The exothecium (ex) with its thickened anticlinalwalls extends aroundall external faces of each free sporangium. The remains of the parietal layers (pl) andthe tapetum (t) are also visible. Scale = 50 pm.B : Electron micrograph of the exothecium (ex), showing that the thickenings on theanticlinal walls (arrowhead) are composed of many fine laminations. There is no cleardistinction between primary and secondary wall in this preparation. Scale = 10 pm.C and D : Enlarged view of the lower dehisced sporangium shown in Fig. 3.7 C,showing the exothecium and upper (G) and lower (D) halves of the stomium. Theprimary walls of each exothecial cell are stained purple, and secondary thickening is pinkand blue, grading to darker blue layers towards the cell interior. The anticlinal walls areat least four times thicker than the periclinal walls. The stomial cells (sto) also showrelatively thickened anticlinal walls. Also visible are the remains of the parietal layers(pl). Scale = '10 pm.E : A tangential section of the sporangial wall gives a TS of the exothecial cells,showing that all anticlinal walls (arrowheads) have secondary thickening (bluish) laid onthe purple primary walls. Scale = 10 pm.

Fig. 3.8

Figure 3.9: (Prumnopitys taxifolial Mitotic sporogenous cells in young pollencones. Decussate microspore tetrads and free microspores fromnearly mature pollen cones.A : Electron micrograph of sporogenous cells (at the stage shown in Fig. 3.6), withunusually pale cell walls (arrowheads indicate boundary of sporogenous tissue). lt isunclear whether this is a developmental feature or a fixation artifact. Scale = 10 pm.B : Electron micrograph of sporogenous cells. Despite failure of osmium tetroxidefixation (resulting in poor preservation of most cytoplasmic details), the chromatin withinthe nuclei has been preserved. Most of the sporogenous cells have greatly enlargednuclei (n), and one cell is at metaphase with several condensed chromosomes(arrowheads). Scale = 10 Fm.C : Light micrograph of resin-embedded specimen stained with methylene blue. Twosporogenous cells are at metaphase with condensed chromosomes (arrowheads). Thethree transversely elongated cells at left represent the developing stomium ordehiscence zone (dz). Scale = 10 !rm.D : Young microspores dissected from fresh sporangia and stained with orcein. Onemicrospore (*) is in polar view, showing the bilateral symmetry of the bisaccate spore.The others are in lateral view. The thick wall at the proximal pole of these microsporeshas a shallow ridge (arrowhead), and the sacci are still uninflated (arrows), indicatingthat these microspores are newly released from tetrads. Several nuclei of tapetal cellsare present at left. Scale = 10 pm.E and F : Two tetrads and a single microspore, dissected from fresh sporangia,stained with orcein, and photographed at two different focal planes to show thedecussate arrangement of the tetrads. The proximal pole of the single microspore isindicated (arrowhead). In tetrad 2 (ld?) shown in E, one pair of microspores lies in thesame focal plane with the proximalfaces at approx. 30'to the horizontal edge of thepicture. In F, in the same tetrad (td2) seen in a lower focal plane, the second pair ofmicrospores lies at 90' to the first pair. In tetrad 1 (td1), in E & F, the right-hand pair ofmicrospores is seen end on, at g0'to the left-hand pair. From the shape of the proximalwalls of the microspores in this tetrad, the shallow ridge of the proximalfaces of the freemicrospores shown in D can be seen to result from the constraints of developing in adecussate tetrad. The sacci develop at the ends of each elongated microspore, more orless at the eight corners of the cube formed by the decussate tetrad (arrows). Scale = 10Irm.

cEFFig. 3.9

Figure 3.10: Development of the microspore wall occurs in association withbreak-down of the tapetum ln Prumnopitys taxifolia.A : Expanding microspores with well-developed sacci pack the sporangium. Theremains ol the tapetum (t), stained black, line the sporangial chamber. Shrinkage of thesporangium and the necrotic layers of parietal cells (pl) may be an artifact of preparationin this specimen. Alternatively, this scale was already damaged or dying prior tocollection of the cone. Scale = 50 pm.B : Enlarged detail of A. The nuclei of these microspores appear to be preparing formitosis. The microspore walls are relatively pale and the tapetal remnants (t) are verydarkly stained, in comparison with lower sporangia on the same cone (see C & D below).Scale = 10 pm.C : A lower microsporophyll on the same cone as the scale shown in A & B. Theexpanding sporangium is now the same length as the leaf-like scale tip. Scale = 100pm.D : Enlarged detail of C. The microspore nuclei appear to be preparing for mitosis, asin B. However, the black-stained matter within the degenerated tapetum (t) is greatlyreduced, and the microspore walls are darker than those in B. The parietal layers (pl)are becoming compressed by expansion of the sporangium. The exothecium is not yetmature, as shown by the partial collapse of anticlinal walls (arrowhead). Scale = 100pm.E : Light micrograph of resin-embedded specimen stained with methylene blue. In thedeveloping mlcrospores, the outermost ektexine (blue tectum and turquoise reticulatemuri in the sdEci) can be distinguished from the endexine (dark blue) surrounding thecorpus (c) of each microspore. A blue-staining substance in the tapetal remains appearsto be accumulating near the surfaces of two adjacent microspores (arrowheads). Scale =10 pm.

Fig. 3.10

Figure 3.1 1: (Prumnopitys taxifolia) Features of the developing exine and intineof young microspores, and the nearly mature sacci of pollen grainsA, B, C and D : Electron micrographs of specimens collected in autumn (March).A: Young microspore with nucleus (n) at the proximal pole, and a large vacuole (v).The electron-dense exine, comprising the tectate ektexine (ekt) and endexine (end), iswell-developed. The electron-transparent intine (int) is just beginning to form on theinner surface of the endexine. Part of the ektexine detaches to form the saccus (l*).Scale = 5 pm.B : Microspore wall at the distal end. The ektexine is absent or very thin in this region,which is the germinalfurrow or leptoma. The endexine is composed of an outerlaminated zone (oen) and an inner finely granular zone (ien). Minute hair-like structuresproject at the surface (arrowheads). Also visible are the intine (int) and the large vacuole(v). Scale=1pm.C : Microspore wall at the distal root of the saccus. The tectum (tec) of the ektexineforms the saccus wall. lnside the saccus ({t), remnants of the foot layer (ft) of theektexine form an irregular granular layer on the endexine, Refer to B for details of thewall at the distal side of the saccus (anowheads).Scale = 1 pm.D : Microspore wall at the proximal end. The tectum (tec), bacula (bac) and foot layer(ft) of the ektexine are thickest at the proximal end of the microspore. The outerlaminated layer of the endexine (oen) is much thinner here than at the distal end, whilethe inner endexine (ien) has a similar thickness all round the corpus. The developingintine (int) is visible as an electron-transparent layer at the inner surface of the endexine.Also visible are the nucleus (n) and vacuole (v). Scale = 1 pm.E and F : Electron micrographs of a specimen collected in spring (October) shortlybefore pollen dispersal. The parietal layers (pl) are now crushed. The tapetal cell wallshave broken down, and tapetal cytoplasm (tc) now forms a continuous layer around thesporangial chamber. Small areas of stacked membranes and other unidentifiableentities in this layer may be degenerating organelles of the tapetal cells. Near thesurfaces of the sacci (*) lie blobs of electron-dense material (arrowheads) of similarconsistency to the material of the sacci. Micropunctae (arrows) approx. 0.25 pm diampenetrate the tectum of the saccus. Scale = 5 pm.

1ḏF'.'':fir'!':*tL 't-', ':q;:..15{.-

Figure 3.12: Germination of the microspore and development of the youngmicrogametophyte up to dispersalstage in Prumnopitys taxifolia.The observed stages have been numbered (1)to (8) in developmental sequence (seetext for details). Starch grains (sg) are abundant in the tube cell cytoplasm until the freenuclear stage (E, F)A : One microspore in prometaphase just prior to germination (1) . Onemicrogametophyte (4) shows the mitotic spindle of the dividing antheridial initial (ais)during formation of the tube nucleus (tn) and antheridial cell (ac). Anothermicrogametophyte (5) shows a fully formed antheridial cell and tube cell. The twoprothallial cells (p1 & p2) lie over the proximal pole. Scale = 10 pm.B : One microgametophyte (2)wilh first prothallialcell (p1) and centralcell (cc). Twogametophytes (3) with antheridial initial (ai) and two prothallialtiers (p1 & p2). ln bothgrains the first prothallial tier now has two cells (p1 x 2). Scale = 10 pm.C : Two microgametophytes at similar stages to grains (4)and (5)in A. The cellwallsof each tier are clearly visible (5/, showing the lens-shaped first prothallial cell (p1) andthe dome-shaped prothallial cell (p2) and antheridial cell (ac) overlying it. Scale = 10pm.D : One microgametophyte (6) atter the transverse division of the antheridial cell toform the body cell (bc) and sterile cell (sc). The two prothallial tiers have also divided toform at least two cells in each tier (p1 x 2, P2 x 4?). Scale - 10 pm.E : One microgametophyte (7)in polar view showing four nuclei in the first prothallialtier (p1 x 4). In two other grains (8),the cellwalls have broken down, and free nuclei (fn)lie in the tube cell cytoplasm. Scale = 10 pm.F : Two gametophytes (8) after cell wall breakdown, with body cell (bc) surrounded byfree nuclei in the tube cell cytoplasm. Scale = 10 pm.

aci-'A?'{!:"Gbcf 'r.-J,(B)EFFig. 3.12

Figure 3.13: The exine, intine and body cell wall of the mature microgametophytein Prumnopitys taxifolia.Electron micrographs of pollen grains just before dispersal, in cones collected inNovember (see Figs. 3.7 C and 3.8 A). Despite failure of the osmium tetroxide fixation(resulting in the damaged appearance of the cytoplasm), the nuclei (especially thechromatin) and the wall layers are clearly distinguishable.A : Two pollen grains with body cells (bc) lying near the proximal pole of the grain.Part of a saccus is visible in the ektexine (ekt). Inside the darkly stained endexine (end),the much paler intine (int) is now about 1 pm thick. ln the upper grain, three thin irregularwavy strands pass from the intine to the body cell wall (arrowheads).Scale = 5 pm.B : Enlarged view of part of Fig 3.13A. The wavy irregular strand (arrowhead) forms aconnexion between the intine (int) and the wall (bcw) of the body cell (bc).Scale = 0.2 pm.C : Pollen grain with body cell (bc) close to the proximal pole. Two wavy strands(arrowheads) extend between intine and body cell wall. The intine has two layers, amore granular outer layer (oi) and an electron-transparent inner layer (ii). Scale = 5 pm.D : View of pad of a body cell and adjacent intine (int). An irregular mass whichconnects the body cell wall (bcw) to the intine, appears to be formed from two coalescingwavy strands (arrowhead) emanating from the intine. Also visible is the body cellnucleus (bcn). Scale =2pm.E : Pollen grain in non-median LS with no free nuclei or body cell visible in the section.Several irregular tapering extensions of intine project into the cytoplasm of the grain(arrowheads). The two nearest the proximal pole are connected to each other by a wavystrand (arrow). Scale = 5 pm.F : Pollen grain in oblique TS, with body cell (bc) and six free nuclei (fn) visible.Several irregular tapering extensions of intine (arrowheads) project into the cytoplasm,mainly on the side nearest to the body cell. Scale = 5 Fm.

EFFig. 3.13

Figure 3.14: Cellular and free-nuclear microgametophytes in Prumnopitystaxifolia.A: Cellular microgametophytes within a sporangium (wax-embedded section of a conecollected in November). Numerous starch grains (sg) are present in the tube cell (tc)cytoplasm only. Scale = 50 pm.B : Free-nuclear microgametophytes within a sporangium (wax-embedded seetion of alower scale on the same cone as shown in A). The body cell (bc) and the prothallial andsterile nuclei (fn) now lie free in the tube cell cytoplasm. No starch grains are visible.Scale = 50 pm.C and D : Pollen grains just before dehydration and dispersal, stained with orcein.Scale = 10 pm.C : lntact pollen grains with body cell (bc) and eight free nuclei (fn) clearly visible.D : Squashed pollen grain with body cell (bc) and six free nuclei (fn) outside the grain,and three still inside.

'".$'fcile?P(froe{DObc(nOeG#t-,r;'r'"! lBDFig. 3.14

Figure 3.15: Germinated pollen grains in the micropyles of two young ovules ofPru m nopitys taxifol ia.These specimens, despite abnormalities in the ovules attributable to the effects of gallmidge larvae (see Chapter 4), show severalfeatures typical of pollen grains aftergermination.A : Pollen grain at surface of nucellus (nuc). The body cell (bc) lies close to the wall atthe proximal pole of the grain. The blue-stained ektexine (ekt) and endexine (end) andmauve intine (int) are clearly distinct. (Note that the position of the grain with proximalpole facing the nucellus, and germinal furrow or leptoma facing away, is unusual (seeChapter 9). Adjacent sections show that the pollen tube emerged from one end of theleptoma and turned towards the nucellus.) Scale = 10 pm.B : A pollen grain which has germinated at a distance from the nucellus. The pollentube wall is formed by growth of the intine through the ruptured exine. The pollen tube ofthis specimen was approximately 300 pm long (only half of it visible in this section). Fourfree nuclei lie in the tube connected by a strand of cytoplasm (arrowheads). The bodycell (bc) remains in the grain near the proximal wall. Scale = 50 pffi.C : Enlarged view of the pollen grain shown in B. At higher magnification the body cell(bc) appears to be attached to the intine (int) by two or three strands (arrowhead) of amauve-stained substance similar to the intine. The numerous dark grey oval bodies inthe grain and tube cytoplasm may be starch grains. Scale = 10 pm.

Fig. 3.15

Figure 3.16: Development of the male gametophyte up to entry of the body celltothe pollen tube, within the ovule of Prumnopitys taxifolia.A : Pollen grain germinating at the surface of the nucellus cone (nuc). The pollen tubeis just beginning to penelrate the nucellus tissue. Several free nuclei (fn) are visiblewithin the grain. Scale = 50 pm.B : Pollen grain with a short pollen tube, which has penetrated to two or three cells'depth in the nucellus (arrowhead at tip of pollen tube). The body cell (bc) lies in thegrain. Two nuclei are visible within the tube (arrows). Scale = 50 pm.C : Branched pollen tube in tip of nucellus. Four free nuclei (arrowheads) lie in themain tube surrounded by numerous starch grains. (See D for pollen grain in adjacentsection). Scale = 50 pm.D : Pollen grain (pg) belonging to the pollen tube in C (seen in an adjacent section).The body cell remains in the pollen grain. Dark irregular strands in the grain body areeither artifacts of fixation or damage to the specimen prior to fixation. However, despitepoor preservalion, the large dark-stained nucleolus of the body cell nucleus (arrowhead)is clearly visible. Scale = 10 pm.E : The body cell nucleus (bcn) entering the pollen tube. The large granular nucleus ispreceded by a mass of green-stained cytoplasm. Scale = 10 pm.F : Five free nuclei (arrowheads) lie in the pollen tube surrounded by starch grains.Behind these lies the body cell nucleus (bcn) which has just entered the pollen tube atthe trailing end of the green-stained mass of cytoplasm. Scale = 50 pm.

D.'r-^- 'i ''lJ: ::ti-laf'Fig. 3.16

Figure 3.17: Pollen tube and body cell progress through the nucellus cone inPru mnopitys taxifolia.A: The pollen tube, having traversed the dense tissue (dt) of the nucellus cone, isabout to enter the spongy tissue (spt) at the base of the cone adjacent to the gynosporewall (gw). The body cell nucleus (bcn) has reached halfway through the nucellus cone.Scale = 100 pm.B : The spherical body cell nucleus (bcn) has a conspicuous nucleolus with a singlenucleolar vacuole (nv), and blocks of condensed chromatin (stained black). Adjacent tothe body cell are numerous starch grains (sg). Scale = 10 pm.C, D and E : Three views, a few sections apart, showing a pollen tube approaching anarchegonium (A1). The tip of the pollen tube has broken through the gynospore wall(gw) and is halfway through the female gametophyte tissue overlying the archegonium(arrowhead at tip of tube) (D). The body cell nucleus (bcn) lags behind, lying at thetransition between the dense tissue (dt) and spongy tissue (spt) of the nucellus cone (E).This archegonium (A1), stillwith a central cell nucleus (ccn), is not yet ready forfertilization (C). Another archegonium (A2) is visible in E. Scale in C & D = 100 pm;scale in E = 200 pm.F: Within the body cell nucleus (bcn), the chromatin is partly condensed and thenucleolus is smaller than that in B. Scale = 10 pm.

11 'l.tiq. .-\ -rf.- ^ .:l i., rt* t,.1F i.;'.'"j.ft--?l'i'ti';+'spt ?.r?:lrqt: _iJ l-,tiI s't :1.i '-]tft^i: 'J;-'- 't -ti F t', o-. 1 i. l ij.g-. l';--itqw-t/ItaarI'Itc:. ,l ,lE-Fig. 3.17

Figure 3.18: The body cell nucleus reaches the female gametophyte inPru m nopitys taxifolia.A, B and C : Three views, a few sections apart, showing two pollen tubes approachingtwo archegonia (A3 & 44) in a group of four at egg nucleus stage. One pollen tube (pt1)has reached the base of the nucellus cone (A) and its body cell nucleus (bcn1) lies justwithin the spongy tissue of the nucellus (C). A second pollen tube (pt2) is passingbetween the nucellus and the female gametophyte, close to archegonium A3 (A). Thetip of pt2 appears to press against the gynospore wall overlying the passage to the neckcells (nc3). The body cell nucleus of the second pollen tube (bcn2), with a largenucleolus, is visible in an adjacent section along with two free nuclei (fn) (B). The fourarchegonia in this ovule contained fully mature large egg nuclei (en),Scale in A & C = 100 ;rm; scale in B = 10 pm.D , E and F : Three views of a pollen tube close to an archegonium at egg nucleusstage. The body cell nucleus (bcn) lies at the gynospore wall (gw) directly outside thepassage to the neck cells (nc) with the tube nucleus (tn) nearby (E). Two other freenuclei (fn) are visible in an adjacent seclion (D). The large nucleolus of the body cellappears slightly translucenl (F). The archegonia in this ovule contained immature eggnuclei (en) still undergoing enlargement, Scale in E = 100 pm; scale in D & F = 10 pm.

Aa ttta.lit'so!a"'aar ?! rraaa**!.-(ltttIIt\ll.țpt2'FtL,tllr'a.1lsfir'Bbcn2\tBItaaD,)l;fl:D rtf-taI\.io j'' r.,f_. {.'l;t,r-',il:,'\cE^tlt\bcn I]./ . jLillr\E .*.JE\r;\ l.\...'\ ,rt ,.\.l a.;*L.aFig. 3.18

Figure 3.19: Targetting of archegonia by the pollen tube; and arrival of the malegametes at the neck cells, in Prumnopitys taxitolia.A: The passage left by the pollen tube (pt2) shown in Fig 3.18 B, showing that achange in its direction occurred as it passed from the dense tissue (dt) to the spongytissue (spt) in the nucellus cone (nuc). Scale = 100 pm.B : The empty pollen grain (pg) which gave rise to the pollen tube shown in Figs 3.18 Band 3.19 A. Scale = 100 pm.C and D : Two views, a few sections apad, showing the paths taken by two pollentubes (pt1 and pt2) to reach two archegonia (A1 and A2). ln C the lower pollen tube(pt2), which originated from the pollen grain pg2, changed direction at the surface of thegametophyte and grew towards the archegonium A2. The upper pollen tube in C (pt1),which originated from the pollen grain pg1 (in D) changed direction within the nucellusand grew towards the archegonium A1 (section not shown). The ruptured neck cells(nc1 and nc2) indicate that both archegonia were fertilized. Scale = 100 pm.E : A pollen tube at the neck cells of a mature archegonium. The body cell has dividedto produce two unequal male gametes (fm = functional male and nm = non-functionalmale) (see Fig 3.20 A). The neck cells, which appear almost ruptured, lie in a hollow ofthe egg cytoplasm, possibly due to the presence of the pollen tube. The egg nucleus(en) is fully enlarged and mature. Scale = 100 pm.F: Another pollen tube (pt) at the neck cells of a mature archegonium. The body cellhas divided to produce two unequal gametes (see Fig 3.20 B). In this specimen the neckcells do not lie in the hollow at the surface of the egg cytoplasm. The egg nucleus (en)here is slightly smaller than that shown in E. Scale = 100 Fm.

l. '1ii, t /'?. j:t-.- I q-, ..i ;'' ., ^.'' . a aa,^a6Ipgc,-FFig. 3.19

Figure 3.20: The male gametes in Prumnopitys taxifolia.A: Detail of Fig 3.19 E showing the two unequal male gametes. The two gametesshare the same cytoplasm. The larger presumably functional gamete (fm) is nearlysurrounded by the dense green metabolically active cytoplasm. The smaller nonfunctionalgamete (nfm) lies at the margin, The enlarged nucleoli contain numerousnucleolar vacuoles (nv). Severalfree nuclei (fn) are visible near the gametes, and starchgrains (sg) are present in the tube cell cytoplasm and in the neck cells. Scale = 10 pm.B : Detail of Fig 3.19 F, showing the two unequal male gametes. The pollen tubecytoplasm (pt) has spread out over the neck cells (nc) which are in a different focalplane, lying on the convex bulge of the archegonialwall (aw) (compare with Fig 3.20 A).The larger presumably functional male gamete (fm) is completely surrounded by a densezone of metabolically aetive cytoplasm, and the smaller non-functional gamete (nfm) hasbeen excluded. The enlarged nucleoli contain numerous nucleolar vacuoles (nv).Starch grains (sg) lie in the tube cell cytoplasm. ec = egg cytoplasm. Scale = 10 pm.

ttOtarla. .'l't'Ia,a-awalraȯ,totaa'a't;At I'tlra.r'.- f|aa.fI-,rr-asgecn":-"*:-B-Fig. 3.20

Figure 4.1: Morphology and development of the female strobilus inP ru mnopitys taxifol ia.A : Young female strobilijust after pollination (October, Auckland Regional Council BotanicalGardens). The slender glaucous upright spikes (arrows) are 25 to 50 mm long.B : Two young strobili near the apex of a leafy shoot. Whatever their location on the axis, thespikes turn to grow vertically upwards. The upper spike is 6 mm long, with four ovules, the lowerspike 12 mm long with five or six ovules. Fertile bracts become distinguishable from sterile onesas their bases enlarge to accommodate the developing ovule (arrowheads).C : Two young ovules at pollination. The glaucous bloom on the epimatium (e), fertile bract (fb)and cone axis (ca) is due to a waxy coating, which is absent on the wettable groove (wg). Twopollen grains (arrows) lie just beneath the inverted micropyle, adhering to the still moist wettablegroove. At this stage the ovules are 1-1.5 mm long.D : Part of a female strobilus, showing post-pollination exlension growth. The ovules (ov) areabout 2.5 mm long, and may not have been pollinated, but can continue growth for severalweeks before aborting. The wettable grooves (wg), although now dry, are still a distinctive featureon the glaucous cone axis (ca).E : Part of a strobilus collected in February at Totara Park, Auckland. The upper ovule isgrowing normally, and is at the free nuclear gametophyte stage, with nipple (np) clearly evident.The ovular complex is 5 mm long. The lower ovule is growing abnormally, due to the presence ofa gall midge larva (see Section 4.2.3). The now dry wettable grooves (wg) have become brown.F : Abaxial view of abnormal ovule with two nipples (np), The fertile bract has abscised, andboth wettable grooves (arrowheads) are visible. The nipple of a normal ovule is often bilobed(see Fig. 4,1 G). This ovule may have developed two separated lobes of one nipple, or two entirenipples, The ovule length including nipple is about 4 mm.G : Three female strobili about 6 cm long, collected late November from the ground in WhirinakiForest in the North lsland. On the lefthand spike there are 6 healthy ovules and two ovulesdeformed by gall midge predation (arrow). The righthand and middle spikes each have 5 healthyovules and 2 or 3 bracVovule scars (bos). Note the bilobed nipples (np).H : Two ovules (ov) collected in early November. The ovule in the foreground is about 6 mmdiameter. At this stage (some 5-7 weeks before fertilization) the archegonia may be less than halftheir mature length (see Fig. a.9). (np = nipple; ca = cone axis).J : Unusual one year old female strobilus (ca1) bearing three ovules at the free nucleargametophyte stage. A young female strobilus (ca2) (now about 10 mm long) has formed in theaxil of a leafy bract of the older strobilus. The apical arrested bract has broken off (arrowhead).K : Four ripening ovules on a drooping flexible cone axis (ca). An arrowhead indicates thebroken tip at the apex. On this spike several ovules aborted early in development, but only onebracVovule scar is visible (bos). Surface blemishes are caused by insect larvae tunnelling in theepimatium. The embryos are unaffected within the hard seed coat.L : One ripe ovule approx. 10 mm diameter. A waxy bloom on the epimatium (e) is still evident,especially on the nipple. On this spike all other ovules aborted (arrow). The spike apex is belowthe picture.

Fig. 4.1

Figure 42 : Earl development of tlte female strobilus ln Frumnopttys tuttoltaA : A young female slrobilus (in slightly obllque LS) and a vegetative bud (bV) (inmedhn L-S). The lirst apperdages to torm on the cone ads (ca) are the sJerile scales(ss) follovrred by sweral sterile bracts (sb) and lastly the fertile bracts (fh), in the axils ofwhich the -o-vularprirnodia (op) appear. Seoetory

'l \A-Fig. 4.2

Figure 4.3 : Initiation of fertile bracts, and differentiation of the tissues of theovular complex, ln Prumnopitys taxifolia.A : Two fertile bract primordia (arrowheads) developing on the conical apex of thecone axis (ca). Older fertile bracts (fb) envelop the apex. Scale bar = 50 pm.B : An ovular complex in median LS, in the axil of a fertile bract (fb). The primordiumof the nucellus (n) forms a bulge on the adaxialface of the epimatium (e) adjacent to thecone axis (ca). Scale bar = 50 pm.C : An ovular complex slightly more advanced than that in 4.2B. The integument (i) isnow forming as a torus-shaped swelling between the nucellus (n) and the epimatium (e).Growth occurs obliquely downwards towards the cone axis (ca). The developingvascular bundle (vb) is also visible. Scale bar = 50 pm.D : Whole ovular complex between fertile bract (fb) and cone axis (ca). Differentialabaxial and adaxial growth of the epimatium (e) tilts the integument and nucellus (n)towards an invefied orientation. Scale bar = 100 pm.E and F : Two views of a developing ovular complex.E : Whole ovular complex, showing elongation of the epimatium at the distal end toform the nipple (np). Scale bar = 100 pm.F : Part of the ovular complex in 4.2E, to show the developing epimatium (e),integument (i) and nucellus (n) on the adaxialface. The nucellus now consists of aroughly spherical zone of cells (indicated by arrowheads). At the center of the nucelluslies a cell which may be the archesporial initial (ar). The wettable groove (wg) at thisstage forms a cleft between the base of the epimatium and a bulge in the cone axis.Scale bar = 50 pm.

:i{Li ,.i:i;.i",'.]',':r-' ?:) i '( '| -' j:',*ir,'t -r..)i'j"J -. 'i--,il.z'=I li i14:.1-t i ,';-t .i"Ei::ff:i i$'il;J{.i.il;-'r.*?:"ii'i'ihi -rlit-r. - a ; -'l:Ali',rr#t,- .llt'ii,i\il' "=,: .f,,-fttit4':*;ii; Frt-:e,vi,$iilggg$ff.**'Fdil:Jiii'il,Yfrrf$$..li$;Lffi,rii1;1il6q fi"...^\ti-:'ift*RRi*,\i\r"lFFig, 4.3

Figure 4.4 : The ovular complex at the stage of megasporocyte development inP ru mnopitys taxifol ia.A : Whole ovular complex, in median LS. The ovule, consisting of the nucellusenclosed by the integument (i), is now almost entirely surrounded by the epimatium (e).The nipple (np) is at the chalazal end of the ovular complex. The vascular bundle (vb)and secretory canal (sc) are visible in the fertile bract (fb) and cone axis (ca). Thewettable groove (wg) is beginning to widen. Scale bar = 100 pm,B : Detail of the ovular complex shown in Fig. 4.4 A. Downward extension of theepimatium (e) and integument (i) is beginning to form the micropyle (m). Themegasporocyte (mc) has differentiated (see also Fi1.4.12 G and H), and one of thesurrounding archesporial cells is dividing (arrowhead). The external part of the nucellusis enlarging to form the nucellus cone (nuc). Scale bar = 50 pm.C : Whole ovular complex at a more advanced stage than that in Fig. 4.4 A. Themegasporocyte (mc) is clearly visible in the denser tissue of the nucellus. The smallcells of the integument (i) are now more easily distinguished from the epimatium (e).The micropyle (m) and wettable groove (wg) are also visible. Scale bar = 100 pm.D : Detail of the ovular complex shown in Fig. 4.4 C. The megasporocyte (mc) issurrounded by developing tapetal cells (t), one of which is in telophase (arrowhead). Theexternal part of the integument (i) is now about 4 cells thick and the micropyle (m) ismore pronounced. Scale bar = 50 pm.E : Part of an ovule at a similar stage to that in Fig. 4.4 A and B, showing one cell inmetaphase (arrowhead) in the integument (i). Patl of the nucellus cone (nuc) is visible.Scale bar = 20 pm.

! ;:ii,+rht'r#!*:::,Lt';gjlg-r$_H.tfl,ir "--i,ffi#;:li{$B' .t'm,i..::ill*1,'i'.,iI r.'?'lfli:;r-, .fo!Z+tli.,-, i?f , , -.'tA#,i1;'ji;ir-ti r.',,$#mi}iuH+3; -:' O!a)!\.,\roFig. 4.4

Figure 4.5: (Prumnopitys taxifolial By pollination time, the various tissues ofthe ovular complex are welldifferentiated,A : Whole ovular complex (ovc), in median LS (collected early November,Mangatawhiri, Hunua Ranges), showing the open micropyle (m) of a recenlly pollinatedovule. The wettable groove (wg) has widened due to growth of the fertile bract (fb)where it meets the cone axis (ca). Scale bar = 100 pm.B : Enlarged view of the specimen shown in Fig. 4.5 A. The tissues of the epimatiuminclude the epidermis (ep) with stomata (st) especially numerous at the nipple (np),hypodermis (hyp) and mesophyll (me). Tanniniferous cells (tnc) are grouped at thechalaza (ch) and form a layer separating epimatium from integument (i). Within theintegument, the sporangium or nucellus contains the megasporocyte (mc) in a tapetum(t), surrounded by the paler parietal layers (pl). The nucellus cone (nuc), free from theintegument, is at the upper end of the micropyle (m). Some cells at its exposed tip havebroken down (see Fig. 4.6 D). An ungerminated pollen grain (pg) at the mouth of themicropyle may have been dislodged from the nucellus cone during preparation. Thewettable groove (wg) and part of the fertile bract (fb) are visible. Scale bar = 50 pm.C : Whole ovular complex (ovc), collected shortly after pollination time (early November,Totara Park, Auckland), but with no pollen grains in the micropyle (m). lt is not knownwhether the necrotic nucellus cone (nuc) degenerated before or after production of apollination drop. The megasporocyte (mc) is stillvisible. The vascular bundle (vb)accompanied by a secretory canal (sc) ends as transfusion tissue (tt) in the nipple. Theseparate vascular bundle to the fertile bract (fb) is not visible in this section. Scale bar =100 pm.D : Detail of the specimen shown in Fig. 4.5 C. On the inner epidermis of theintegument (i) at the upper end of the micropyle (m) is a group of small darkly stainedcells, which may be unelongated closing cells (clc). The outer epidermis of themicropylar tube has several enlarged tanniniferous cells (tnc). Part of the degeneratednucellus cone (nuc) is visible. Scale bar = 50 pm.

A.:.:)t..r'.'.'l':-tit.f"' "ii' - -..,-J'!i a:..ii, '. at. t -)'.r' ,.-.. t"' i-il.'l't,-'-". itif-sr' "' ;1.'-'l.'f-"''rraa, lrt'..,*."a{" ti f;'t.t ta.,_:! l'fr a'}FI Ia-t-'t.tltit,",...rt,i\.i,;'."r:.ii:"'tidf'.;,j.-$-:-:}r::frl-'.-',- jr'i;. ,'.t:i:W:. ,,i\itdffiffitr:;ri.'[r!:'d l, j;rr.t*" 1 ='i'jQ. "J'*3,t ttt=-'I *:= ,n"J::f"=!in-pl - --'l-. rar Ir.. t'l'r-,ta|'l r1mct .rrat.,v'3r;JL a'.. i.il tj t''t r.'tat'.r .';. 'EFTJS; :' ..rr'- -d-alllfl\ #frfi?.'r4tJ. l1Ps f^flf,If;:''s ,,i' ws.d{f;f #.';i- ..^-i,o 'it{'e ,i)".7'. l.l i' :rt^..1'.'-rr;:'. t-*1.'.:; j;: J{.:., !;; :i, ..tq{ri'.' -'-t-.,' --' . t " rru''.. .J-fr,lI.tIIt'/iFig. 4.5

Figure 4.6:The nucellus cone before and after pollination inPrumnopitys taxifol i a.A, B, C and D : Details of the nucellus cone (nuc) in four different specimens collectedat pollination time, arranged in sequence to show progressive changes to the cells at thenucellus tip. Parts of the integument (i) and micropyle (m) are visible.A : Most of the cells of this nucellus cone (nuc) are typical in having densely stainingcytoplasm and relatively large, granular nuclei (n). A few cells contain smallinconspicuous vacuoles (v), In the cells at the more or less convex tip of the nucelluscone, the cytoplasm is slightly withdrawn from the cellwalls (arrowhead). Scale bar =2Qpm.B : In the tip of this nucellus cone (nuc) the green-stained cytoplasm of the first tworows of cells has retracted from the cell walls (arrowhead). Scale bar = 20 pm.C : In this nucellus cone (nuc) the cytoplasm of the cells at the tip has become roundedand contracted, appearing to lie free in the cell lumen. The dense cytoplasm is stainedgreen, a sign that it is still metabolically active. The external cell walls are rounded andintact (arrowhead). One interpretation is that production of the pollination drop isimminent. Scale bar = 20 pm.D : Detail of the specimen in Fig. 4.5 A and B, showing the entire nucellus, comprisingnucellus cone (nuc), parietal layers (pl), tapetum (t) and megasporocyte (mc). Severalcells at the tip of the nucellus cone are contracted and green, while others are blackenedand dead (arrowhead), and a pollen grain (pg) is present at the entrance to the micropyle(m), indicating that production of a pollination drop had already occurred at the time offixation. Scale bar = 50 pm.E : Part of an ovule collected shortly after pollination. A germinated pollen grain (pg)lies in the micropyle (m) with its pollen tube beginning to enter the nucellus cone (nuc).About four rows of cells have degenerated (arrowhead) at the tip of the nucellus. Themicropyle appears closed due to the slightly oblique plane of the section and a bend inthe micropylar tube, but no sign of elongation of closing cells was found. The presenceof fungal hyphae (anow) entering the micropyle is unusual. Scale bar = 50 pm.

| !'i lrr fi,t trttll,- +.B-I$t t.:inuc r olll,f ta?;,,(,{,|-l ,?Jt.ttrt"$ F Jl *Grr Blr 1lid:tTri;ffl#rt. ;r] 'l l'mt:111'lHiji':?Hr'*:*";;ft rT3J'fE Ijr;i'r'f?t.* flc-m

Figure 4.7: Post-pollination development in the ovular complex ofPrumnopitys taxifol ia.A : Whole ovular complex at megaspore tetrad (tet) stage (see Fig. 4.13 D). Themicropyle (m) has closed and two pollen grains are visible in the pollen chamber (arrow).The elongated nipple (np) is now about 1 mm long. Scale bar = 500 pm.B : Part of an ovular complex, collected at megaspore tetrad stage, showing theelongated closing cells (clc) which form from epidermal cells of the inlegument (i). Apollen grain (pg) lies in the small pollen chamber, and its pollen tube has begun topenetrate the collapsed cells at the tip of the nucellus cone (nuc). Fungal spores (arrow)are clustered outside the micropyle, and hyphae have entered the pollen chamber.Scale bar = 50 pm.C and D : Two views of the same specimen.C : Whole ovular complex with a newly formed free-nuclear gametophyte (fng) (seeFig. 4.14 B). The pollen chamber (pc) is visible above the closed micropyle (m). Scalebar = 500 pm.D : Enlarged detail of part of the ovule shown in Fig. 4.7 C. A pollen tube (pt)penetrates the nucellus cone (nuc) from a pollen grain (pg) lying in the pollen chamber.Elongation of the closing cells (clc) has crushed several other pollen grains lodged in themicropyle (m). Scale bar = 50 pm.

Fig. 4.7

Figure 4.8: Transverse sections of the ovular complex at free-nucleargametophyte stage in Prumnopitys taxifolia.A, B, C, D and E : Successive sections of the same specimen.A : Whole ovular complex (ovc), fertile bract (fb) and part of the cone axis (ca),sectioned at the level of the micropylar tube (mt). In the epimatium the main tissues incentripetal order are : epidermis (ep), photosynthetic hypodermis (hyp), photosyntheticmesophyll (me) with scattered sclereids (scl), two vascular bundles with xylem (x),phloem (ph) and secretory cells (sc), and a zone of tanniniferous cells (tnc) whichpartially surround the circular aperture. Inside this is the micropylar tube with elongatedclosing cells (clc). In the fertile bract the main tissues are epidermis (ep), photosyntheticmesophyll (me), secretory canal (sc), phloem (ph) and xylem (x). Scale bar = 100 pm.B : Section of the whole ovular complex, through the middle of a young free-nuclearfemale gametophyte (fng) of about 150 pm diameter. ln the epimatium (e) the ascendingvascular bundles (vb) on the dorsal side are further apart, and photosynthetic mesophyll(me) now forms a ring around the central ovule. (The descending vascular bundles onthe ventral side are too small to see at this magnification.) A distinct layer of darkstainingtanniniferous cells (tnc) marks the inner boundary of the epimatium. Theintegument (i) forms a more or less uniform layer all round except for two lines of smallercells which will later become the dehiscence zone (dz) of the seed coat. Inside theintegument lie the parietal layers (pl) of the 'sporangial wall', the tapetum (t), and thefree-nuclear gametophyte with one of its four nuclei visible (arrow). Scale bar = 500 pm.C : Whole ovular complex, sectioned tangentially to the chalazal end of the nucellus.The tapetum (t), at the centre of the parietal layers (pl), is shown enlarged in Fig. 4.8 E.The integument (i) and tissues of the epimatium (e) are as in Fig. 4.8 B. Scale bar = 500pm.D : Enlarged detail of the micropylar tube (mt), from a section taken slightly above thatshown in Fig. 4.8 A. The closing cells (clc) occur in three longitudinal zones of theepidermis lining the micropyle. In this section, the tanniniferous cells (tnc) extend allround the circular aperture in the epimatium. Scale bar = 50 pm.E : Enlarged detail of the centre of the section shown in Fig. 4.8 C. At the centre aretapetal cells (t), each with a round granular nucleus and a large vacuole (v) towards oneend of the cell. The cells of the surrounding parietal layers (pl) tend to have smallernuclei elongated in the same direction as their cells. Scale bar = 50 pm.

BffiSlricFig. 4.8

Figure 4.9: Tissues of the one year old ovular complex in Prumnopitys taxifolia.An ovular complex of at least 3.5 mm diam. and over 6 mm long including nipple(removed to facilitate fixation), in median LS.The female gametophyte (fg) (albeit collapsed in this specimen) is now fully cellular, andarchegonia (A) are about half of their mature length. The tissues in centripetal order are:outer epimatium comprising epidermis (ep), hypodermis (hyp), mesophyll (me); innerepimatium comprising a broad layer of pale cells with scattered tanniniferous cells (tnc),which become more concentrated at the irregular epimatium/integument boundary (eib),vascular bundles (vb) and secretory canals (sc),The integument (i) is relatively featureless except for the dehiscence zone (dz). ln thenucellus, the parietal layers (pl) of the sporangial wall are becoming compressed, whilethe nucellus cone (nuc) retains its form. The cellular gametophyte has a prominentgynospore wall (gw) and two archegonia about 450 pm long (see Fig.4.17 F to L fordetails). Scale bar = 500 pm.

Fig. 4.9

Figure 4.10: (Prumnopitys taxifolial The tissues of the nucellus cone, and twodeveloping archegonia in a one year old ovule.A : Nucellus cone of a one year old ovule, in median LS (resin-embedded specimenstained with methylene blue). The thick-walled cells of the dense tissue zone (dtz) havedarkly stained cytoplasm containing small vacuoles (v), and numerous starch grains (notvisible in this image). Cellular break down (arrowheads) occurs in the vicinity of a pollentube (pt). The cells of the spongy zone (sz) at the broad base of the nucellus cone aremuch larger, with thin walls, large vacuoles (v) and very little cytoplasm containing a fewscattered starch grains. A few pink-stained starch grains (sg) are visible in the pollentube. Scale bar = 100 pm.B : Electron micrograph of an epidermal cell from the broad base of a nucellus cone.Darkly stained cytoplasm surrounds vacuoles (v) containing what appears to beflocculent material and with electron-dense matter (arrowheads) around their margins. Athick cuticle (c) covers the external wall, Also shown are cells of the spongy zone (sz),with large vacuoles (v) and intercellular spaces between the thin cell walls (arrow).Starch grains (sg) appear as round white bodies with dark lines crossing lhem,suggesting that they have electron-refractive properties. Scale bar = 5 pm.C : Electron micrograph of cells from the dense tissue zone of a nucellus cone. Thelarge nuclei (n) contain eleclron-dense patches of chromatin. Several small vacuoles (v)and starch grains (sg) lie in the darkly stained cytoplasm. The paler secondarythickening of the cell wall (sw) is clearly visible overlying the darker primary cell wall (pw).Scalebar=5p1p.D and E : Two views of the same section from a wax-embedded specimenD : Light micrograph of part of a one year old ovule, showing the nucellus cone andpart of the sporangial wall composed of increasingly compressed parietal layers (pl). Anepidermis of tanniniferous cells (arrowhead) overlies the external surface of thesporangial wall and the spongy zone (sz) of the nucellus cone. The dense tissue zone(dtz) is penetrated by a pollen tube (pt), In the female gametophyte (fg) are twoarchegonia (Al and A2) at the'frothy'stage of development, with highly vacuolatecontents, darkly staining jacket cells (jc) and a central cell nucleus (ccn) near the neckcells (nc). The thick gynospore wall (gw) becomes thinner in the vicinity of the neckcells. Scale bar = 100 pm.E : Light micrograph of the same section as in Fig. 4.10 D, viewed with pseudo phasecontrast lighting, in which starch grains glow brightly due to their refractive properties.The dense tissue zone (dtz) can thus be distinguished from the spongy zone (sz), Otherstarch-rich structures such as the neck cells (nc) and the pollen tube (pt) are highlighted.The remaining tissues which have few or no starch grains appear dark, with reddishnuclei and cytoplasm. Also prominent is the thick cuticle (c) of the nucellus cone, whichappears much thicker than it is, and the archegonial wall (aw). Scale bar = 100 pm.

Fig. 4.10

Figure 4.11: (Prumnopitys taxifolia) One year old ovular complex, with nearlymature archegonia.This specimen is at a more advanced stage than that in Fig. 4.9. The two nearly fullsizedarchegonia (only A1 visible) now reach to the centre of the female gametophyte(fg) and have been overgrown by gametophyte tissue. Although still highly vacuolatewith a small central cell nucleus, dense green cytoplasm (arrowhead) has begun todevelop at the chalazal end of the archegonium. (For details of the archegonium seeFig.4.20 A and B).When factors such as growth, individual variation, and variation in staining are taken intoaccount, the tissues of the epimatium (e) are seen to be essentially the same as those inFig. 4.9. ln the integument, three different layers are becoming visible. These are: theoutermost layer (oli) one to a few cells thick, adjacent to the epimatium; the middle layer(mli) which will harden to form the sclerotesta of the seed; the inner layer (ili) which willbecome compressed to form a thin endotesta.The remains of the tapetum (t) and crushed parietal layers (pl) can be seen where thegynospore wall (gw) has shrunk away from the nucellus, The tanniniferous cells of theepidermis (ep) on the external surface of the nucellus are clearly visible. In thisspecimen, the nipple (np) is intact, and the micropylar tube (mt) lies in a false micropyle(arrow) created by overgrowth of the epimatium. Scale bar = 500 pm.

'/'t-.{{t v|

Figure 4.12: Development of the megasporocyte in Prumnopitys taxifolia.A to M : Ten specimens arranged in approximate developmental sequence, showingthe megasporocyte at the centre of a developing tapetum. All ovules with micropylar endoriented directly (A, B, C, D, E, J, K, L) or obliquely (F, G, H, M) towards lhe bottom ofthe picture. All scale bars = 10 pm.A : One cell (anow) enlarges at the centre of the archesporial cells.B : Occasionally two cells enlarge (arrows). The surrounding 3-4 layers will developinto the tapetum.G : The large nucleus of the megasporocyte develops a prominent nucleolus (arrow).In this specimen the cells of the nucellus cone (nuc) were necrotic.D and E : Two views of the same megasporocyte in two focal planes, to show twonucleoli (arrows). Half of an anaphase cell (arrowhead) is visible in the tapetum.F : The megasporocyte (arrow) continues to enlarge, especially lengthwise. (Fromspecimen shown in Fig. 4.4C,D)G and H : Two views of the same megasporocyte in two focal planes. ln this case thenucleus had four nucleoli (arrows), two visible in G, and two in H. A tapetal cell intelophase is also visible in H (arrowhead). (From specimen shown in Fig. 4.4 A, B)J : Before meiosis, the large nucleus (arrow) moves to the micropylar end of themegasporocyte, and the chromatin coalesces into a single mass.K : Megasporocyte (arrow) with the nucleus at leptotene in preparation for meiosis.L : Megasporocyte (arrow) with the nucleus at zygotene in preparation for meiosis.Two mitotic tapetal cells are visible, one in prometaphase, the other in telophase(arrowheads). (From specimen shown in Fig. 4.5 A, B and 4.6 D)M : Megasporocyte (arrow) with the nucleus at pachytene in preparation for meiosis.

; q;"';fi*sjfE-!lffiffii..tQrtTl-. '-iiifi:i'.;,iP)!.-;.dit1;i?r'{jill**f^t,;iil;ll!11 ?lrD;#cF-ltrgplS:^r.;l;frF..'t t lt+rtfi-irid.:j.trfjii'.:f;tii;'.Et'#ffiKJ'-r?.li?lil ti*L -+ .t*o ".a"rl ,--l--"g'4.,tt*"g'Q ?I re ^E 'r-l-tMt t flt.'lr a I- O*.-l-L t,Fig. 4.12

Figure 4.13: Megaspore tetrads in Prumnopitys taxitolia.A to M : Eight different megaspore tetrads. Whether linear or non-linear, the tetradtends to retain the elongated spindle shape of the original megasporocyte within thetapetum (t). Allspecimens oriented with micropylar end towards bottom of the picture.Some show part of the nucellus cone (nuc). All scale bars = 10 pm.A and B : One specimen in two focal planes. In A the two uppermost cells areseparated by an oblique wall (arrow), showing that the tetrad is not strictly linear. In Bthe overall spindle shape of the tetrad is clear (arrowheads).C : A linear tetrad (arrowheads). One cell (arrowhead) at the chalazal end, which islarger than the others, may be the functional megaspore.D : A linear tetrad, very shrunk, but with one cell at the chalazal end larger than theothers. (From the specimen shown in Fig. 4.7 A).E and F : One specimen in two focal planes. In E two cells (t1, t2) of a non-lineartetrad can be seen, and in F the other two cells (13, t4) are visible (arrows).G and H : Another specimen of a non-linear tetrad in two focal planes, showing twocells in G (t1, t3) and two in H (t2, t4).J : A shrunken tetrad (arrowheads). As in Fig,4.13 D, the uppermost cell, which hasbecome separated from the lower three, may have become the functional megaspore.K and L : One specimen in lwo focal planes. In K two nuclei are distinct (arrowheads)and a third is equivocal (arrow with asterisk). In L another cell is visible (arrowhead).This specimen may thus be either a linear tetrad or a linear triad.M : A non-linear non-cellular triad (arrowheads), which may have resulted from failureof the largest nucleus (arrow) to undergo meiosis ll. Several cells of the tapetum alsoappear abnormal.

o. irt"::i: I*.';1. rlrl .;- ::.;$';#TF{\-Fr --* tt 'ftfi*!t,vilrii:.i :1'4f:2rr-. -'ii;.,#fiHi't.i*?' ;.Ttu.it- .-t-,/- a t-' .aii.t,:: -.-!-i ;.3J'.'..n,-*,. -.ti:.-. * '_ t^r'.. t.. .i i -3. :i.rE#-"til;.1:;t',.':' .:. '-.. : r.{;i :it.'.....1i r.i J.- ..:::" rf :::lir-'. l-l'r"'; :.1i:";.?.1-': -i#ii':'=$i;-E:'-.T?Eii.^.i,' {lcq ..!':':- i'-i,1 ;. *';Itttr#,,.,i:;i".Jiriiii: ;i.:. ];ia.?- d ti.' '.t.rrit*:.fw;ffil"Iiglo.,1a^L. ^lFilEt'!!,.,7aa;nllliii,j, 'i;r:;!r r '!%.KLo. , .Tl'J..f. ri':;.i.t - r{',-.2. ' ' f | .5 t0riOIoIra n.Itrgrlb * f-jrI>licP-,t7*.l {'vI t,trO'O o'rll-Mq;!'IOt9OJ*rllOl.r'lf.a;E%r*l\_r.aoaFig. 4.13

Figure 4.14: Development of the female free-nuclear gametophyte in Prumnopitystaxifolia.A : Functional megaspore (fms) enlarging in the tapetum (t). Several tapetal cells havedegenerated (arrow). Scale bar = 10 Fm.B : Either a very large functional megaspore or a very small (2-nucleate at most) freenuclear gametophyte. Only one nucleus (n) was observed in the thin band of cytoplasmwhich surrounds the centralvacuole (v). In the tapetum (t), the innermost cells havedegenerated (arrows). Scale bar = 10 pm.C : Parl of an ovule showing an expanding free nuclear gametophyte (fng)approximately 230 pm diameter, with 37 nuclei. Only four nuclei are visible. Also visibleare parts of the nucellus cone (nuc) and integument (i). The parietal layers (pl) arebecoming compressed and the cells of the tapetum (t) have a loose disruptedappearance. Scale bar = 50 1rm.D : Part of an ovule showing a free nuclear gametophyte (fng) 237 pm diameter withonly nine of 153 nuclei visible. Around this is the tapetum (t) and compressed parietallayers (pl) which grade into the spongy zone (sz) of the nucellus cone (nuc), and part ofthe integument (i). A pollen tube (pt) is visible in the dense tissue zone, but the pollengrain and the tip of the nucellus cone have been broken off during preparation (arrow).Scale bar = 100 pm.E : Part of an ovule with very shrunk free nuclear gametophyte (fng) which wasoriginally about 600 pm diameter. Remnants of the lapetum (t) adhere to the collapsedgynospore wall. The nucellus is now flask-shaped due to growth of the parietal layers(pl) of the sporangial wall. A pollen tube (pt) is visible in the nucellus cone (nuc). Part ofthe integument (i) was removed. Scale bar = 100 pm.F : Part of an ovule with a 600 pm diameter free nuclear gametophyte (fng) puncturedduring preparation. The gynospore wall (gw) appears to have flowed through the brokensporangial wall, and detached tapetal cells (t) lie in the empty chamber. Scale bar = 100um.

d*iui:tii*.,t l*t i,i a ':'r . t" iit..'it -tf ,ti ::-a-rna- rng rns-|'..jit -':" i { ,'/+. l.''l ii,',.ri"; 'l ,l!t'.i;,#$1fc'lt,i.-T'ilo{.iit;ia

Figure 4.15: (Prumnopitys taxifolia) Features of the gynospore wall andsporangial wall surrounding the expanding cellular femalegametophyte.A : Part of an ovular complex, showing epimatium (e), integument (i) and flask-shapednucellus with spherical sporangial chamber containing a collapsed female gametophyte(fg). Although the female gametophyte is now cellular and about 900 pm diameter,archegonia have not yet begun to form. Note that, although the nucellus cone (nuc) hasnot changed, growth in the parietal layers (pl), while enlarging the sporangial chamber,has resulted in extension of that part of the nucellus which is free from the integument(arrows). Scale bar = 500 pm.B : Pafi of an ovule containing a cellular female gametophyte (fg). The thickgynospore wall (gw) is thinner near the two developing archegonia (A1 and A2). A detailof the parietal layers (pl) is shown in Fig. 4.15 C. Two pollen grains (pg) lie in the pollenchamber at the tip of the nucellus (nuc). Most of the integument (i) has been removed.Scale bar = 100 pm.C : Part of the sporangialwall of the ovule shown in Fig. 4.15 B. Tanniniferous cells ofthe nucellar epidermis (ep) are visible in the bottom left corner. lnside this are theparietal layers (pl) which become increasingly compressed and collapsed. The tapetum(t) also appears collapsed. The gynospore wall (gw) at top right encloses the cellularfemale gametophyte (fg). Scale bar = 10 pm.D : Detail of the gynospore wall from the specimen shown in Fig, 4.9 and 4.17 F to L.Two layers are visible: a smooth inner layer or nexine (nx) and a pilose outer layer orsexine (sx). Scale bar = 10 pm.E : Part of the sporangial wall of an ovule with full-sized archegonia (see Fig. 4.22 and4.23) showing cells of the female gametophyte (fg), gynospore wall (gw), collapsedtapetal remains (t) parietal layers (pl), tanniniferous cells of the epidermis (ep) and thickcuticle (c). Scattered starch grains (sg) are visible in the parietal layers, some trappedbetween the walls of the compressed cells (arrows). Scale bar = 10 pm.F : Electron micrograph of the gynospore wall, showing that the sexine (sx) iscomposed of electron-dense bacula (anows) which irregularly coalesce, and that thenexine (nx) lies on the cell wall (cw) of the gametophyte cells (fg). At2-g pm thick, thispart of the wall is only half of the maximum thickness of 5-6 pm. The electron-densebodies (arrow) may be a staining artifact. Scale bar = 2 pm.G : Electron micrograph of the gynospore wall in the vicinity of the archegonia. Thesexine (sx) is much thinner and more disorganized than that shown in Fig. 4.15 F, whilethe nexine (nx) appears very similar. In the female gametophyte cell (fg) lies a granularnucleus (n) and four plastids ({F). The electron-dense matter (arrow) may be a stainingartifact. Scale bar - 2 pm.

)U ,r \L-"\ "'€P ],rr 'blJlt*G'=-'-Fig. 4.15

Figure 4.16: Occasionally two or even three female gametophytes develop in theovule of Prumnopitys taxifolia.A, B and C : One of only two specimens observed containing two femalegametophytes.A : Part of the ovule in median LS, showing two separate cellular approximatelyhemispherical gametophytes (G1 and G2) with their centers aligned on the longitudinalaxis of the ovule. In each gametophyte the center of alveolation is visible as a zone ofsmaller cells (arrows) at its center. The two gametophytes, which are separated by athin transverse line (arrowhead) are enveloped by a single thick gynospore wall (gw).The cell walls in the middle layer of the integument (mli) have begun to thicken. Scalebar - 500 pm.B : Another section of the specimen in A. The gametophyte at the micropylar end (G1)contains an immature archegonium (A1), while the one at the chalazal end (G2) doesnot. Scale bar = 500 pm.C : Detail of the junction between the two gametophytes (G'l and G2). Eachgametophyte appears to be surrounded by a thin brown layer which between them formsthe thin separating line shown in A (arrowheads). Another similar layer, overlaid by athicker darker layer (arrow), clearly encloses the two gametophytes on the surfaces incontact with the tapetum. These features suggest that the gametophyte and the tapetumboth have a role in forming the gynospore wall, Scale bar = '10 pm.D, E and F : One specimen containing three female gametophytes.D : Part of the ovule in oblique LS/TS, showing three cellular gametophytes (G1, G2and G3) of unequal sizes but oriented so that all three have contact with the nucelluscone (nuc). Three large archegonia with mature egg nuclei are developing ingametophyte G1 (A1, A2 and A3). Scale bar = 100 pm.E : Another section of the same ovule showing that all three gametophytes (G1, G2and G3) contain archegonia (A1 to A7). In G1 (as shown in D) the archegonia (41, A2and A3) are at egg nucleus stage. In G2, 44 has been fertilized (later sections show afree nuclear proembryo), and A5 is degenerating. In G3, 46 and A7 share a singlejacket layer and have not developed beyond the 'frothy' stage. Scale bar = 500 pm.F : Detail of the junction between gametophytes G2 and G3. As in Fig. 4.16 C, eachgametophyte appears lo be surrounded in its own thin wall (arrowhead), and the thickerdarker layer lies only on the external surfaces of the gametophytes which are in contactwith the tapetum (arrow). Scale bar = 50 pm.

t\1rG2 I.l{\-'{; -,jGiJ {.D(Gz clG2rt't.t'tt-aEFFig. 4.16

Figure 4.17: ln Prumnopitys taxifolia. archegonia develop from archegonialinitials at the surface ol the female gametophyte.A to E : An ovule collected in October in Auckland, with two newly formed archegonia in a 900pm diameter female gametophyte (fg). Due to collapse of the gametophyte in this specimen, thearchegonia were unmeasurable.A : An archegonium (A'l) is developing at the surface of the gametophyte (lg), just inside thegynospore wall (gw) and above the base of the nucellus cone (nuc). Scale bar = 50 pm.B : A second archegonium (A2) is developing closer to the center of the nucellus cone (nuc)base. Scale bar = 50 pm.C : Enlarged view of the archegonium A2 shown in Fig. 4.17 B. The central cell is growing byexpansion of its large vacuole (v). The central cell nucleus (ccn) lies close to the neck cell initial(nci). The neck cell cap (ncc) is already visible adjacent to the gynospore wall (gw). Scale bar =10 pm.D : Between the two young archegonia, developing jacket cells fic) form orderly rows. Despitethe severely collapsed condition, one cell in prometaphase (arrowhead) and another inmetaphase (arrow) are visible. Scale bar = 10 pm.E : Enlarged view of the archegonium 41 shown in Fig. 4.17 A. The central cell vacuole (v),centralcell nucleus (ccn) and neck cell initial (nci) are more distinct. Degenerated tapetal cells (t)lie near the gynospore wall (gw). Scale bar = 10 pm.F to L : Details of two archegonia developing in the ovule shown in Fig. 4.9 and 4.15 D.F : Part of the ovule, with an archegonium (A1) about 450 pm long, with its neck cells located atthe sudace of the female gametophyte (fg), adjacent to the gynospore wall (gw) and just abovethe base of the nucellus cone (nuc). The shape of the elongating central cell is governed by theoriginal pattern of alveolation. Scale bar = 100 pm.G : Mitotic gametophyte cells (arrows) near the developing jacket cells (jc). Scale bar = 50 pm.H : A section between the two archegonia, showing that the orderly rows of jacket cells (c) andthe narrow tip of the second archegonium (A2) are aligned with the rows of cells in the femalegametophyte (fg). Scale bar = 50 pm.J : Enlarged view of the archegonium (A1) in Fig. 4.17 F, showing the neck cells (nc) and theenlarging highly vacuolated central cell enclosed by densely staining jacket cells (c). Scale bar =50 pm.K : A section adjacent to that in Fig. 4.17 J, showing the central cell nucleus (ccn) lying close tothe neck cells (nc). Scale bar = 10 Fm.L : Enlarged detail of the section shown in J. The convex thickening of the neck cell cap (ncc)appears to continue between the neck cells (nc). Scale bar = 10 pm.

{#:i ,,tI t-fl/\+L-#,J-"fli*!t\,ilrr.jD+l , o/ ,'.., "':F-lc-.d:'iA1tG'rl'ffi- CCn,1,'I,G-KA'tA1HJLFig. 4.17

Figure 4.18: Features of the central cell in the growing archegonium inPrumnopitys taxifol ia.A and B : Two views of archegonia in the ovule shown in Fig. 4.10 D and E.A : Two developing archegonia (A1 and A2). The central cell appears frothy due to itshighly vacuolated cytoplasm. The area within the square is shown enlarged in 4.18 B.The centraf cell nucleus (ccn) lies close to the neck cells (nc). (See detail in Fi1.4.24 A).A passage through the overgrown gametophyte tissue is visible (ncp) between the neckcells and the surface of the gametophyte. The gynospore wall (gw) is thinner in thisregion. Scale bar = 50 pm.B : Enlarged view of the area within the square shown in 4.18 A. The vacuoles (v) inthe central cell are separated by very small areas of cytoplasm. The wall of the centralcell is visible as a thin light brown wavy line (arrowheads) alongside the straight walls(arrows) of the jacket cells (c). Scale bar = 10 pm.C, D and E : Three successive sections of two archegonia (Al and A2), from a oneyear old ovule (in oblique LS).C : The cytoplasm in the wider part of the central cell (as in A1) is still filled withvacuoles (v), but at the narrow chalazal end (as in A2) the vacuoles have been replacedby densely staining cytoplasm. Scale bar = 100 pm,D : One large vacuole (v) is usually present near the center of the widest part of thearchegonium (as in 41). The transition between vacuolated and non-vacuolatecytoplasm is visible in A2 (arrow). Scale bar = 100 pm.E : The central cell nucleus (ccn) remains close to the neck cells, and a small area ofdenser cytoplasm, termed here the reception zone, develops nearby. (See detail in Fig.4.248). Scale bar = 100 pm.

fg,|A/bI/EuiFig. 4.18B

Figure 4.19: Characteristics of the jacket cells and neck cells of the archegoniumin Prumnopitys taxifolia.A : Two archegonia (A1 and A2) with highly vacuolated central cells and welldeveloped jacket cells (jc). (Specimen resin-embedded and section stained withmethylene blue. Note that this section is slightly oblique, and that the true tips of thearchegonia lie in an adjacent section.) One jacket cell (arrow) near the tip ofarchegonium Al is undergoing mitosis. Scale bar = 100 pm.B : Enlarged detail from Fig. 4.19 A, showing the jacket cells (jc) near the tip ofarchegonium 41. ln one cell, three nuclei (arrows) are in metaphase. Two of the nucleihave very clear chromosomes, and counts give numbers close to the haploid number ineach nucleus (19 in P. taxifolia). The cytoplasm in the jacket cells is packed with largenumbers of tiny vacuoles (arrowheads), distinguishing them from the cells of the femalegametophyte (fg). Also visible are the larger vacuoles (v) of the central cell, and theblue-stained cell wall (ccw), the thickness of which is partly due to the oblique sectioning.Scale bar = 10 pm.C : Part of an ovule with two archegonia at egg cell stage (See Fig. 4.26B, C and D fordetails). One end of this section passes between the two archegonia ({t) and showsmature jacket cells in transverse section at the micropylar end. The other end of thesection passes through the narrow tip at the chalazal end of one archegonium (A),showing jacket cells (jc) in longitudinal section. The multinucleate jacket cells are mostclearly visible in TS, and less so in LS (arrows), Some adjacent cells of the femalegametophyte (fg) are also multinucleate (arrowheads). Scale bar = 100 pm.D : Enlarged view of part of Fig. 4.19 C, showing jacket cells (jc) in TS, containing two,four or eight nuclei (anows) in the densely stained cytoplasm. Multinucleate cells(arrowheads) in the female gametophyte (lg) tissue have similar numbers of nuclei.Scale bar = 50 pm.E : Part of an ovule in TS. This section, passing obliquely through the micropylar endof the female gametophyte (fg) near two archegonia, shows the neck cells (nc) of onearchegonium and the passage (ncp) through the gametophyte tissue to the neck cells ofthe other archegonium. Scale bar = 50 pm.F: Enlarged view of the neck cells (nc) shown in Fig. 4.19 E. In this neck cell rosette,12-13 cells are visible and 3-4 cells do not reach the centre (anows). The sectionpasses obliquely through the neck cell tier and through the brownish wall ({e) of thecentral cell beyond. The center of the rosette is filled with a similar substance. Starchgrains (sg) are abundant in the neck cells and absent in the surrounding cells of thefemale gametophyte (fg). Scale bar = 10 pm.

AEF*b?tFig,4.19

Figure 4.20: The archegonium reaches fullsize during the central cellstage inP ru mnop itys taxifol ia.A : Longitudinal section of an immature archegonium from the whole ovular complexshown in Fig 4.11. The archegonium, 1152 pm in length, is at or near full size. In thecentral cell, three cytoplasmic zones are visible: a highly vacuolate zone (v), a zone ofdense cytoplasm free of vacuoles in the narrow tip ({t), and a marginal layer, heretermed the fibrillate zone (fz), in which cytoplasmic strands (arrows) extend from thecytoplasm in the middle to the central cell wall on the inner face of the jacket cells (jc).The central cell nucleus (ccn) lies near the neck cells (nc), accompanied by a smallreception zone (rz) which is more visible in an adjacent section. (See Fig. 4.18 E and4.24B for another example.) Scale bar = 50 pm.B: The archegonium shown in Fig. 4.20 A, as seen under pseudo phase contrast.Starch grains (white) are visible in the nucellus cone (nuc) and the neck cells (nc), butare lacking elsewhere in the archegonium (A) or female gametophyte (fg). The centralcell wall (ccw) and gynospore wall (gw) are also visible. Scale bar = 100 pm.C: The archegonium shown in Fig. 4.20 D, as seen under pseudo phase contrast. Inaddition to the starch grains visible in the nucellus cone (nuc) and neck cells (nc), thereare now numerous starch grains (sg) in the female gametophyte cells (fg) around thearchegonium (A). The archegonial cytoplasm is filled with yellow granules which areabsent in the vacuoles (v) and reception zone (rz), and very sparse in the narrow tip(arrow) at the chalazal end of the archegonium. Scale bar = 100 pm.D: An archegonium at a laler stage than that in Fig. 4.20 A. lts small central cellnucleus (not seen in this section) is shown in Fig. 4.24 F. The central cell is now filledwith densely staining cytoplasm, containing fewer vacuoles (v) than that in Fig. 4.20 A).The reception zone (rz) has enlarged. Black bodies (arrows) occur throughout thecytoplasm except at the narrow tip (*) and in the reception zone. The central cell wall(ccw) is visible as an undulating line between the fibrillate zone (fz) and the jacket cells(jc). Scale bar = 50 pm.

..lt|". \t,4.a -'.t, t-C|lr !f, 'i.tr'.".A-DFig. 4.20

Figure 4.21: The developing central cell nucleus is associated with a receptionzone in Prumnopitys taxifolia.A: Part of an ovule with three full-sized archegonia (part of A1 and A2 shown). Inarchegonium A1, the reception zone (rz) is now much larger than the central cell nucleus(ccn) (cf. Fig. 4.18 E and 4.20 A). Also visible are vacuoles (v), jacket cells (jc), andneck cells (nc). Scale bar = 100 Fm.B: Part of the same ovule as that shown in Fig. 4.21 A. Archegonia A2 and A3 areseen in full length. The normally straight archegonia are curved due to collapse of thefemale gametophyte (fg). The reception zone (rz) of archegonium A2 is visible close tothe central cell nucleus (ccn). (For details of the nucleus see Fig. 4.24 G). Also visibleare the vacuoles (v), jacket cells fic), neck cells (nc) and the vacuole-free cytoplasm (*)at the narrow chalazal ends of the archegonia. Scale bar = 100 pm.C: Enlarged view of part of archegonium 41, shown in Fig. 4.21 A. The relativelyhomogeneous reception zone (rz) is surrounded by cytoplasm containing numerousblack spots and streaks. A narrow extension of the reception zone is in contact with thecentral cell nucleus (ccn). The prominent neck cells (nc) are surrounded by femalegametophyte (fg) tissue. Also visible is the cap of secondary thickening (arrowhead) onthe neck cells, and the thickened wall of the central cell (ccw). In the central cell nucleusthe chromatin (arrow) is beginning to condense in preparation for mitosis. Scale bar = 20pm.

Fig. 4.21

Figure 4.22: (Prumnopitys taxifolial Details of the cytoplasmic zones and the neck cellsof the archegonium, just prior to division of the central cell nucleus to formthe egg nucleus.A to M : Successive transverse sections of a group of three archegonia, from the narrow tips to the neckcells, showing the various cytoplasmic zones of the archegonium and the surrounding female gametophytecells.A : TS of the narow tip at the chalazal end ol one archegonium (A1). The central zone of cytoplasm issurrounded by the fibrillate zone (fz), composed of radiating strands of cytoplasm extending from the centreto a thin peripheral cytoplasmic layer. The single layer of jacket cells (jc) is visible. Scale bar = 10 !rm.B : All three archegonia (A1 , A2, A3) a short distance from the narrow tips. A few black bodies lie in thecytoplasm, while the fibrillate zone (lz\ is pale. Starch grains in the surrounding female gametophyte cellsare not easy to see at this magnification. Scale bar = 50 pm.C : The same section as that in B, seen in pseudo phase contrast. The starch grains (sg) are clearlyvisible in the surrounding female gametophyte. Strands in the fibrillate zone show red, as do the jacket cellsand the nuclei of the female gametophyte cells. Scale bar = 100 Fm.D : Archegonia A1 , A2 and A3, sectioned slightly further from the tip, still in the non-vacuolate zone ofcytoplasm. Some black bodies have coalesced (arrow). The fibrillate zone (fz) is quite pale, while lhe jacketcells (jc) are distinct. Archegonia A'l and A3 are separated by a single jacket layer (arrowhead). Scale bar =50 pm.E : Archegonia A1 , M and A3 sectioned a little further f rom the tip than in D, showing the vacuolale zone(v). Coalescing black bodies (arrows) are common. The fibrillate zone (fz) has dwindled in archegonia A2and A3, and has disappeared in 41 . Scale bar = 50 pm.F : A section adjacent to that in E, seen in pseudo phase contrast. Slarch grains (sg) are less abundant inthe surrounding female gametophyte (fg). Yellow granules occur throughout the central cell cytoplasm,except in the vacuoles (v). Red strands of cytoplasm can still be seen in the fibrillate zone (tz), and jacketcells (jc). The central cell wall (ccw) is clearly visible. Scale bar = 100 pm.G : Archegonia 41 , A2 and A3, sectioned at their broadest part, showing central cell nuclei (ccn) in 41 andA3, and reception zones (z) in A2 and A3 (See also Fig. 4.23 A). Black bodies and vacuoles arenumerous. In A1 and A3, the central cell nucleus appears in a more lateral position than usual, withreception zones elongated on a nearly horizontal axis. Also shown are the gynospore wall (gw) and theparietal layers (pl). Scale bar = 100 pm.H : TS a few sections beyond that in G. Archegonia A2 and A3 are still visible but all that now shows of 41are a few jacket cells (jc), part of the neck cells (arrow) and part of the central cell wall (arrowhead). Alsovisible are the gynospore wall (gw) and the parietal layers (pl). Scale bar = 100 pm.J : The same section as that in H, seen in pseudo phase contrast. Starch grains are lacking in lhe femalegametophyte tissue, but their presence highlights the position of the neck cells (nc) of archegonium Al .Yellow granules fill the central cell cytoplasm of A2 and A3, except in the vacuoles (v). The jacket cells (c)appear red. Scale bar = 100 pm.K : Archegonium A2, sectioned at the level of the central cell nucleus (ccn) (For details see Fig. 4.23 B).The section passes through the female gametophyte tissue (fg) which has overgrown the neck cells ofarchegonia 41 and A3. Also visible are the gynospore wall (gw) and the parietal layers (pl). Scale bar = 100Fm.L and M : Two adjacent slightly oblique sections of the neck cells to archegonium A2, showing a single tierof 12 cells in the rosette. ln L, the section passes through part of the cenlral cell wall (ccw) between theneck cells (nc) and the cytoplasm of archegonium A2. ln M, the section passes through part ol the thickenedcap (ncc) overlying the neck cells. Scale bars = 10 Fm.

i; i t,)DtA..4 r!j'.0Bj; S ujft ;. n,*lr, ,''l' ,;.T I -.e"'- :'t a- .'rrrrir,l'tttzDEGHs. -o;;.it"le%*ilIncc i,'.: 3.?-tIKM-Fig. 4.22

Figure 4.23: (Prumnopitys taxifolia) Details of archegonia just prior to division ofthe central cell nucleus to form the egg nucleus and the ventralcanal nucleus.A: Enlarged view of the section in Fig. 4.22G, showing archegonia 41, A2 and A3.The central cell nuclei (ccn) in A1 and A3 are in prophase prior to mitosis. Both A2 andA3 have a large central reception zone (rz). The reception zone of archegonium 41occurs in an adjacent section (see Fig. 4.24H\. Archegonial lengths, estimated fromcounts of serial sections, were 41 = 1248 pm; A2 - 1296 pm; A3 = 1176 ;rm. See alsoTable 4.2. Scale bar = 50 pm.B : Enlarged view of the archegonium A2, shown in Fig. 4,22K. The central cellnucleus (ccn) is larger than those in archegonia 41 and A3 (shown in A), and is at a laterstage of prophase prior to mitosis. Fig.4.24 J shows this nucleus in a different focalplane. The uneven layer of jacket cells fic) is typical of the micropylar end of thearchegonium. One cell with at least six nuclei (arrow ) is visible. Near the neck cells thejacket layer gives way to paler cells of the female gametophyte (fg). The obliquelysectioned central cell wall (ccw) appears as a pale brown boundary zone. Scale bar =20 pm.

a!I|l.IA1.a.':t_.s. o!-- e-t.!.t'. Ut\?l e- a-aFt.ol.IaIt.*Ef;;1ef!ccn:'nt*dalt'\i3d trlA3aaF.t ot, aIe,aa'at|la'i .,1 Jfr,i Fig. 4.23

Figure 4.24: Stages in the maturation of the centralcell nucleusin Prumnopitystaxifolia.A to J : Thirty-three central cell nuclei and nucleoli were measured in 15 specimens, numberedStage 1 to Stage 15 in order of increasing maturity (see Table 4.2 and histograms in Fig. 4.25).Nine of them from seven different specimens are shown here, representing examples of Stages 4,6, 7, 10, 13, 14 and 15, as indicated in the histograms. Allscale bars = 10 pm.A : Stage 4, A relatively large nucleus (ccn) with a vacuolate nucleolus (nol), beside the neckcells (nc), in a young archegonium 91 2 pm long, still at the f rothy stage (see Fig. 4. 1 0 D). Noreception zone has yet formed. The neck cell cap (anow) is clearly visible.B : Stage 6. Nucleus (ccn) with a vacuolate nucleolus (nol) in a full-sized but highly vacuolatearchegonium 1280 pm long (see Fig. 4.18 E). A reception zone (rz) is developing close to thenucleus.C and D : Stage 7. Two of the nuclei from three full-sized archegonia with highly vacuolatecytoplasm (a collapsed specimen in TS). In C, the nucleus (ccn) and vacuolate nucleus (nol)appear normal. ln D, the somewhat shrunk nucleus (ccn) is dwarfed by its enormous nucleolus(nol) in which the vacuoles appear to be swollen almost to bursting. Due to its size, this nucleolusis shown in Fig. 4.25 B as a broken column. The nucleus of the third archegonium resembled thatshown in C.E : Stage 10. Nucleus (ccn) with a vacuolate nucleolus (nol), in a f ull-sized archegonium 1 150pm long with cytoplasmic zones developing (see Fig. 4.11 and 4.20 A). The nucleolus appearshollow due to one vacuole being larger than the rest. The neck cells (nc) and cap (arrow) arevisible. The approximate location of a developing reception zone (rz) in an adjacent section isindicated with a dotted line.F : Stage 13. Nucleus (ccn) with a vacuolate nucleolus (nol) in a f ull-sized archegonium 1344pm long. The mature zoned cytoplasm is filled with black bodies. As in E, the nucleolus appearshollow. The reception zone, larger than that in B and E, is in an adjacent section (see Fig. 4.20D).G : Stage 14. Nucleus (ccn) with a small non-vacuolate nucleolus (nol) beside the neck cells(nc) in a nearly mature archegonium 1050 pm long (see Fi1.4.21 B), Chromatin (arrow) isbeginning to condense in preparation for mitosis. The pear-shaped reception zone (rz) extendsfrom the central cell nucleus towards the centre of the widest part of the archegonium. Blackbodies are abundant in the surrounding cytoplasm.H and J : Stage 15. Central cell nuclei from archegonia 41 and A2, two of the three archegoniain TS shown in Fig. 4.23 A and B.H : Prophase nucleus (ccn) with a non-vacuolate nucleolus (nol) in archegonium A1. Thissection, adjacent to that shown in Fig. 4.23 A, shows the reception zone (rz).J : Prophase nucleus (ccn) with a non-vacuolate nucleolus (nol) in archegonium 42, as shownin Fig. 4.23 B but with nucleus viewed in different focal plane. Despite shrinkage, the originaloutline of the nucleus has been retained in the cytoplasm. At 40 pm diam. and 35 x 103 pmt, thiswas the largest central cell nucleus observed.

ftY'-, - Yt.)T.t't'.l-.Al1ilr'i.2 t,_CCnW'?tt- ?T'r**-ti artBorlI-c/\/\/-rZ"1"--#F-"'{slrtd'-ncoITJrDEFFig. 4.24

Figure 4.25: Volumes of thirty-three central cell nuclei and nucleoli from fifteenovules at different developmental stages in Prumnopitys taxifolia.A and B : Central cell nuclei (ccn) and nucleoliwere measured in 33 archegonia from15 ovules (12 with 2 archegonia, 3 with 3 archegonia), collected from two individualtrees(9 ovules from one tree, 6 from the other). Average diameters were obtained from twomeasurements on shortest and longest axes of the ccn (see Table 4.2). Volumes (V =ol"nr31of each ccn and nucleolus have been plotted as histograms (A: ccn volumes; B:nucleolar volumes) to show changes in size during development and maturation of theccn.The volume data has been arranged to reflect the chronological sequence ofdevelopment, from Stage 1 (youngest archegonia observed) to Stage 15 (archegoniawith ccn in prophase). The developmental sequence was initially determined byarchegonial length, and, in archegonia over 1000 pm long (assumed to be full-sized), bythe appearance of the central cell cytoplasm, and, in full-sized archegonia with maturecytoplasm, by the appearance of the ccn. Note that no particular time interval is impliedby this succession of stages.Small triangles (v) indicate the nine central cell nuclei shown in Fig. 4.24 A to J. Stage6 was the earliest stage at which archegonia were full-sized.A : Volumes of 33 ccn plotted over archegonial developmental stages. In full-sizedarchegonia (from Stage 6 onwards) the ccn showed an average volume of 10.4 x 103pmt. Stage 15, with 3 nuclei in prophase, and Stage 8, with shrunken nuclei, wereexcluded.B : Volumes of 33 nucleoli, from the ccn represented in A, plotted over archegonialdevelopmental stages. One unusually large nucleus from the Stage 7 specimen (seeFig.4.24 D) is plotted as a broken column.

Changes in central cell nucleus in Prumnopitys taxifoliaAN3530EaXq)EoZ3201qt0A04 s lu 7 a e 10 11 12 t3 14 1sIArchegonial stages (timeintervalvaries)Changes in nucleolus of central cell nucleus in P. taxifolia3'100|I3000 l h700600E 500(D 4Cl0Lf300200100Bos4slo7IArchegonial stagesB I 10 11 12(time intervalvaries)Fig. 4.25

Figure 4.26: ln Prumnopitys taxifolia. the reception zone of the central cellbecomes the perinuclear zone of the egg cell, enlarging toaccommodate the expanding egg nucleus. The egg cytoplasmappears similar to that of the mature central cell.A : Micropylar end of an archegonium showing a young egg nucleus (en), 65 x 103pms, at the centre of the perinuclear zone (pz), surrounded by the egg cytoplasm whichcontains numerous vacuoles (v) and black bodies (arrow). Also visible are neck cells(nc), female gametophyte tissue (fg) and the gynospore wall (gw). Scale bar = 50 pm.B, C and D : Three views of an ovule with recently formed egg cells in two archegonia(A1, A2).B : A detail of the archegonium A1 in C, showing the young egg nucleus (en) in theperinuclear zone (pz). The ventral canal nucleus (vcn) lies at the periphery at a distancefrom the neck cells (nc). Except for a few vacuoles (v), black bodies fill the cytoplasm allround the perinuclear zone. Scale bar = 50 pm.C : Whole archegonium (A1) with young egg nucleus (en) (shown enlarged in B). Theegg cytoplasm has numerous black bodies and very few vacuoles (v). A neck cellpassage (ncp) is visible in the female gametophyte tissue (fg) between neck cells (nc)and gynospore wall (gw). Scale bar = 100 pm.D : Part of the second archegonium (A2) containing a young egg nucleus (en) centralin the perinuclear zone (pz), with its nucleolus (nol) visible. Also shown is a large centralvacuole (v). In the nucellus cone (nuc) a pollen tube (pt) is not quite half way to thefemale gametophyte (fg). Scale bar - 100 pm.E and F : Two views of an ovule with two archegonia (A1, A2) containing greatlyenlarged mature egg nuclei (en).E : The large egg nuclei (en) in A1 and A2, estimated at 1875 x 103 pm3and 1499 x 103pm3 respectively, are over 23 times larger than the egg nucleus in Fig. 4.26 A (see Table4.2). A narrow perinuclear zone (pz) separates the nuclei from the egg cytoplasm.Scale bar = 100 pm.F : The same archegonia as in E, seen in pseudo phase contrast lighting. The eggnuclei (en) appear dark and featureless, within the even darker perinuclear zone (pz).Except for its vacuoles (v), the rest of the egg cytoplasm is filled with yellow granules.Starch granules (sg) in the female gametophyte tissue (fg) are most abundant towardsthe chalazal end of the archegonia. Also visible is the egg cell wall (ecw). Scale bar =100 um.

laa"i- t,.A'nc'-#!tI .fg oa.'.,. .. - g*t'' .-:...a.cD-ItEFig. 4.26

Figure 4.27: (Prumnopitys taxifolia) In the mature egg nucleus, the nucleolus isrelatively small, and usually lies towards the side furthest from theneck cells.A, B, C and D : Part of an ovule with two archegonia (A1, A2) containing mature eggcells. (jc = jacket cells, fg = female gametophyte, gw = gynospore wall)A, B and C : Different sections of archegonium A2. ln A, a pollen tube (pt) has brokenthrough the gynospore wall (gw) and has reached the neck cells (nc). The egg nucleus(en) appears small in tangential section. In B, the ventral canal nucleus (vcn) lies closeto the egg cell wall (ecw), and part of the pollen tube (pt) is visible outside lhe wall. In C,the egg nucleus (en) is in median section. The conspicuous nucleolus (nol) is thuslocated centrally but away from the micropylar end of the egg nucleus. ln this specimen,the black bodies (arrow) tend to coalesce into linear structures aligned around lhevacuoles (v). The perinuclear zone (pz) appears more dense than the surroundingcytoplasm. Scale bars = 50 pm.D : A median section of archegonium A1 from the same ovule, showing a highlygranular egg nucleus (en) and prominent nucleolus (nol) at the furthest side from theneck cells (nc). The black bodies (arrow) tend to be aligned in a pattern radiating fromthe perinuclear zone (pz). Scale bar = 50 pm.

cD'lal- -a.*nca)aFig. 4.27

Figure 4.28: Up to four archegonia containing mature egg nuclei have beenobserved in ovules of Prumnopitys taxitolia.A, B, C and D : Part of an ovule with four archegonia (A1, A2, A3, A4), ranging from1440 to 1520 pm long. The egg nuclei (en) of archegonia 41 (in A) and 44 (in D) arefully rounded, measuring 1 87 x 103 trrms and 239 x 1 03 pm3 respectively. In the other twoarchegonia A2 (in B) and A3 (in C) the egg nuclei appear shrunken, possibly a fixationartifact. All have a darkly staining perinuclear zone (pz) and numerous vacuoles (v) inthe surrounding egg cytoplasm. In B, severaljacket cells fic) are in mitosis (arrows)between archegonia A1, A2 and A3. In C, the ventral canal nucleus (vcn) ofarchegonium A4 is visible. Two pollen tubes (pt1 and pt2) are approaching archegoniaA (in C) and A3 (in D), but neither has penetrated the gynospore wall (gw). The neckcell passage (ncp) to archegonium 44 is especially clear in D. The egg cell wall (ecw)shows as a yellow-brown boundary to the egg cytoplasm. Scale bar = 50 pm.

:aAaaatal aaaa' fg,'aa a 's'IaaaacDgw-' !r o tr.a-aFig. 4.28

Figure 4.29: The cell wall of the mature egg cell in Prumnopitys taxifoliaischaracterised by clusters of diversely shaped wall projections.A and B : Enlarged details of the two archegonia at central cell stage shown in Fig.4.20, viewed under phase contrast. (fg = female gametophyt€, V = vacuole, jc = jacketcells)A : ln a full-sized archegonium with immature cytoplasm (see Fig. 4.2O A) the centralcell wall (ccw) is noticeably thicker than the other cell walls. Scale bar = 50 pm.B : ln a central cellwith mature cytoplasm (see Fig. 4.20 D), the central cell wall (ccw)has become thicker than that in A. Scale bar = 50 pm.C, D and E : Details of the cell walls enclosing the mature egg cells shown in Fig. 4.28.C : Between the jacket cells fic) of two archegonia (A2, A3), a narrow strip ofarchegonium 41 is visible, cut tangentially along the egg cell wall (ecw) and showing wallprojections (wp) in TS. The egg cytoplasm of archegonium A2 has been torn away fromthe jacket layer (fle) but a small porlion is visible with the egg cell wall intact (arrow).Scale bar - 20 pm.D : Portion of archegonium A2 which was torn away from the jacket layer shown in C.Flame-like and peg-like wall projections (wp) protrude from the inner face of the egg cellwall (ecw) into the egg cytoplasm (ec). The prominent black body is typical of manyspecimens of mature archegonia. Scale bar = 20 pm.E : Jacket layers (jc) of archegonia A3 and A4, separated by a layer of femalegametophyte cells (fg). The egg cell walls (ecw) of both archegonia have numerous wallprojections (wp) protruding into the egg cytoplasm (ec). Scale bar = 20 pm.F : The middle portions of two archegonia (A1, A2) from the ovule shown in Fig. 4.30.Archegonium A2, with a pollen tube outside the neck cells, is close to fertilization. ln thisspecimen, the wall projections (wp), many of them branched (arrows), occur in irregularclusters, and protrude some 10 pm into the egg cytoplasm (ec). Scale bar = 50 pm.

EFig. 4.29

Figure 4.30: Some characteristics of the female gametophyte and the egg celljust before lertilization in Prumnopitys taxifolia.A, B and C : Three views of an ovule in which a pollen tube has reached one of thetwo archegonia. (ec = egg cytoplasm, pz = perinuclear zone)A : The micropylar end of archegonium 41, showing two unequal male gametes (mg)outside the neck cells (nc) (see Chapter 3 for details). The egg nucleus (en) is 572 x 1Q3pm3. An even larger egg nucleus (not shown) was observed in the second archegonium,with no pollen tube close by. Scale bar = 59 p6.B : Enlarged view of the egg nucleus (en) shown in A and C. The nucleolus (nol) liesat the end most distant from the neck cells, close to the nuclear membrane (nm). Theperinuclear zone (pz) stains darker than the surrounding egg cytoplasm (ec). A fewblack spots (arrow) are present in the egg nucleus, generally smaller than the numerousblack bodies in the cytoplasm. Scale bar = 10 Fm.C : Part of an ovule with two archegonia 41 and A2 (both containing large egg nuclei),viewed in pseudo phase contrast. As shown in A, the pollen tube outside archegoniumA2 contains two unequal male gametes (mg), and the egg nucleus (en) (shown in A andB) is close to fertilization. The nuclei of the male gametes appear as yellow circles underthis lighting. Archegonium A2 is visible for only about half of its full length; the narrowtips of both archegonia 41 and A2 extend into the centre of the female gametophyte (fg),where starch grains (sg) are most abundant. Except for the egg nucleus in itsperinuclear zone (pz), the egg cytoplasm resembles that of the central cell (see Fig.4.2OC). The neck cells (nc) and the starch grains (arrow) in the pollen tube nearby arehighlighted. Also visible is the passage of the pollen tube (pt) through the starch-filledcells of the nucellus cone (nuc) and through the neck cell passage (ncp). The egg cellwall (ecw) appears as a bright white irregular outline to the egg cell (shown enlarged inFig. 4.29 F). Scale bar = 100 pm.

d. jc-'-' , -Jj\ rt,l'>',sA-a.. 'l I ..'lect'.Ott'.t','.'tl.at'en->a,a'.lnmt\'fo..'+. it'1.'i.:: ..,i :. .i 'aiBFig. 4.30

Figure 4.31: Another example of an archegonium close to fertilization, in an ovuleol Prumnopitys taxifolia.A : The micropylar end of one archegonium (A1). A pollen tube (pt) obscures the neckcells (nc) and contains two unequal male gametes (mg) (see Chapter 3). The eggnucleus (en), at 339 x 103 pm3, is only about 40 "/o of the volume of that in Fig. 4.30),while the ventral canal nucleus (vcn) is relatively large. The perinuclear zone (pz)comprises a darker region of cytoplasm around the egg nucleus. Throughout the eggcytoplasm are rounded 'hollow' bodies stained light brown, with one or more'cavities'which contain variously stained contents. Two of these (anows) are shown in D and E.The black bodies obserued in other archegonia are absent. Scale bar = 50 pm.B : Enlargement of the egg nucleus (en) and venlral canal nucleus (vcn) shown in A.The perinuclear zone (pz) is an ill-defined region of denser cytoplasm. The concavesurface of the egg cytoplasm near the neck cells may be a fixation artifact. The egg cellwall (ecw) has retained its convex form despite withdrawal of the egg cytoplasm(arrowheads). Scale bar = 10 pm.C : A different section of the ovule with archegonia 41 and A2, seen in pseudo phasecontrast, showing the chalazal end of archegonium 41 (the other end shown in A), andthe micropylar end of archegonium A2. Starch grains (sg) are abundant in the femalegametophyte tissue (fg) beyond the tips of the archegonia. Their density has beenexaggerated by shrinkage of the gametophyte. The neck cells (nc) of archegonium A2and lhe nucellus cone (nuc) are also highlighted. The egg cellwall (ecw) forms a distinctwhite outline around both archegonia, except where they meet diagonally at the center.Here the obliquely sectioned egg cell walls show as two broad bands of white spots(arrowheads) inside the jacket layers (c), resulting from the transverse sectioning ofmany wall projections (see also Fig. 4.29 C). The egg cytoplasm appears reddish inplaces, while severalvacuoles (v) have a red glow at their margins. The yellow granulesobserved in other archegonia are absent. In archegonium A2 the egg nucleus (en) isvisible in tangential section, within the darker perinuclear zone (pz). In archegonium 41a similarly darker zone is visible towards the chalazal end (not fully visible in thissection). Scale bar = 100 pm.D and E : Two views of the same area of egg cytoplasm in different focal planes,showing two of the round 'hollow' bodies (arrows) from archegonium 41 (arrows in Fig.4.31 A). The upper one has at least three'cavities' stained dark green and one stainedpale brown. The lower one contains a single large 'cavity' which appears to have formedfrom at least two smaller ones. Other minute 'cavities' are also visible in both bodies.Scale bars = 10 pm.

ic{'a''I s'y.-lrIr_mgA-*P.\nmrFII'l'rtecwTsBDEFig. 4.31

Figure 4.32: ln Prumnopitys taxifolia. unfertilized egg nuclei continue to enlarge,despite the fertilization of adjacent archegonia.A and B : Part of an ovule containing one fertilized and two unfertilized archegonia.(fg = female gametophyte, gw = gynospore wall, jc - jacket cells, nc = leck cells).A : Non-median oblique LS of the micropylar ends of archegonia A1, A2 and A3. Themiddle archegonium A2 has been fertilized (a four-nucleate proembryo at the tip is notshown). Archegonia 41 and A3 are unfertilized. The granular egg nucleus (en) in 41has collapsed due to puncluring of the archegonium during preparation, but theperinuclear zone (pz) is still visible. Scale bar = 100 pm.B : Another section close to that shown in A. The large egg nucleus (en) inarchegonium A3 is 1231 x 103 pms, with a narrow perinuclear zone (pz). Egg cytoplasm(ec) has leaked from the ruptured archegonium A1 into the female gametophyte tissue(fg). The two unfertilized egg cells contain numerous vacuoles (v), but in the fertilizedarchegonium the cytoplasm at this end has become disorganized. Scale bar = 100 pm.C and D : Part of an ovule containing one fertilized and one unfertilized archegonium.(fg = female gametophyte, lfw = gynospore wall, jc - jacket cells, nc = o€ck cells, nuc =nucellus cone).C : Oblique LS of the micropylar ends of archegonia 41 and A2. Archegonium 41 hasbeen fertilized (a cellular proembryo on elongating suspensor is not shown) and itscytoplasm has a typically disorganised appearance. In archegonium A2 the egg nucleus(en) is 1406 x 103 pm3 and the ventral canal nucleus (vcn) is spherical. The perinuclearzone (pz) is visible as a region devoid of black bodies around the egg nucleus. Scalebar - '100 pm.D : Another section close to that shown in C. ln the fertilized archegonium 41 thecytoplasm at this end of the former egg cell has rounded up. The egg cytoplasm andperinuclear zone (pz) appear normal around the unfertilized egg nucleus (en). Scale bar= 100 pm.

cDFig. 4.32

Figure 4.33: Electron micrographs of the jacket cells, the egg cell wall, and theperinuclear zone, ln Prumnopitys taxifolia.A, B and C : Three views of part of an archegonium at egg cell stage.A : The primary wall of the egg cell (ew) is unevenly thickened, while the contiguous jacket cellwall fiw) is the same thickness as the other cell walls of jacket cells and female gametophyte cells(cw). A vacuole (v) and a 'hollow' osmiophilic body (ob) are present in the egg cytoplasm (ec).Several smaller bodies (arrowheads) are also visible. The darkly stained cytoplasm of the jacketcells contains numerous small vacuoles (v). An arrow indicates the region of egg cell wall shownenlarged in B and C. Scale bar = 5 Fm,B : Detail from the area indicated by the arrow in A, showing part of the osmiophilic body (ob)and egg cell wall (ew). Seven electron-dense bodies (arrow) form an array inside the'cavity' ofthe osmiophilic body. The egg cytoplasm (ec) has various membrane systems (arrowheads).Several vesicles (vs) lie near the egg cell wall. Groups of plasmodesmata occur in pit-fields (pf)between thickened regions of the egg cell wall. Scale bar = 1 pm.C : Enlarged view of a pit-field with two complete plasmodesmata (pd) visible. The positions oftwo others (arrows) are visible at the edges of the pit-field. In the egg cytoplasm (ec), threevesicles (vs) lie close to an invagination (le) of the plasmalemma (p). The material within theinvagination differs from the granular layers of the egg cell wall (ew) and jacket cell wall (jw), andis similar to the contents of the vesicles. The dark appearance of the jacket cells at lowermagnifications is due in part to the presence of numerous organelles (arrowheads). Scale bar =0.2 pm.D and E : Two views of an archegonium from an ovule at egg cell stage, with unusualjacketcells.D : The cytoplasm of the jacket cell (c) consists of numerous irregular organelles or membranesystems in an electron-transparent matrix. A small zone of light material (arrow) in the eggcytoplasm (ec) appears to have entered via a damaged pit-field in the jacket cell/egg cell wall(ew). An intact pit-field (pf) lies nearby. Numerous membranes and small osmiophilic bodies arepresent in the egg cytoplasm. Scale bar = 5 pm,E : Enlarged view of the intact pit-field (pf) shown in D. Four plasmodesmata (arrowheads) arevisible, passing between jacket cell (c) and egg cell (ec). The thickened egg cell wall (ew) andthin jacket cell wall (jw) are visible, In the jacket cell are remnants of membrane systems andseveral structures that may be mitochondria (mt). The plasmalemma (p) of the jacket cellappears to be disrupted. Scale bar = 0.5 Fm.F : Part of a jacket cell (c) and egg cell, showing the thickened egg cell wall (ew) and thinjacket cellwall (w). The egg cytoplasm (ec) has several solid osmiophilic bodies (ob) and variousmembrane systems (arrowheads). The jacket cell nucleus (n) has a vacuolate nucleolus (nol).Scalebar=2Vm.G : Part of a jacket cell fic) and egg cell. Numerous small vacuoles (v) are present in the jacketcell cytoplasm, and the jacket cell nucleus has a vacuolate nucleolus (nol). A cluster of irregularelectron-transparent outgrowths (arrow) projects from the thickened egg cell wall (ew) into the eggcytoplasm (ec). Scale bar = 5 Fm.H and J : Two views of an egg nucleus (en), showing that the perinuclear zone (pz) consists ofa region of densely packed mitochondria (mt). The radius of the egg nucleus was estimated to be37pm, givingavolume ot212x 103pm3. Scalebars: H=2 pm,J= 1 pm.

Fig. 4.33

Figure 4.34: Electron-micrographs of some features of the egg cytoplasm andegg nucleoplasm in Prumnopitys taxifolia.A, B, C and D : Four views of the egg cytoplasm near an egg nucleus.A : Despite numerous sectioning marks, several features of the egg cytoplasm arecleady visible. ln the egg cytoplasm (ec), between a vacuole (v) and the perinuclearzone (pz) surrounding the egg nucleus (en), lies a large'hollow' osmiophilic body (ob)and several smaller osmiophilic bodies among various membrane systems (arrowheads).Scalebar=5pm.B : Enlargement of the 'hollow' osmiophilic body shown in A. The two larger'cavities'have a membrane (arrows) around their inner surfaces. The two smaller'cavities'contain irregular masses of a darker material. The body appears to be composed of amass of dark granules. Between this and the large vacuole (v) lies a small vacuolesurrounded by several loosely concentric membranes (arrow). Scale bar = 2 lrm.C : View of the egg cytoplasm just above the osmiophilic body (ob) in B, showing agroup of sinuous parallel membranes (arrowhead) and another group of concentricmembranes (anow). The perinuclear zone (pz) and nuclear membrane (nm) are alsovisible. Scale bar = 2 pm.D : Enlarged view of the group of concentric membranes in C, showing that they aredouble membranes (arrowheads). Scale bar = 0.5 pm.E : Detail of the cytoplasm in an archegonium either at central cell or egg cell stage.Various membrane systems (arrowheads), vesicles (vs) and dark granular osmiophilicbodies (ob) occupy the space between vacuoles (v). A double membrane system(arrow) follows the curve of one vacuole. Scale bar = 2pm.F : Another detail of the cytoplasm in the same specimen as is shown in E. A Golgibody (gb) is visible among various membrane slructures (arrowheads). Cytoplasmicmaterial (arrowhead) appears to have entered the vacuole (v). Scale bar = 1 Fm.G : Some vacuoles (v), contained irregular membrane structures (arrowheads). Scalebar = 2 pm.H and J : Two views of part of an egg nucleus.H : Despite the poor quality of this image, it has been included to provide the locationof the structure shown in J. A black spot (arrow) lies close to the nuclear membrane(nm) in the egg nucleus (en). The perinuclear zone (pz) and various membranesystems (arrowheads) and an osmiophilic body (ob) in the egg cytoplasm (ec) are alsovisible. Scale bar = 10 pm.J : At higher magnification, the black spot shown in H can be seen to comprise a zoneof irregular osmiophilic granular matter enclosing a zone of granules in highly regularparallel rows (arrow). Scale bar = 0.2 pm.

GFig. 4.34

Figure 5.1: lntact and penetrated neck cells ln Prumnopitys taxifolia.A : Intact neck cells (nc), in LS, from an archegonium containing a mature eggnucleus (en) and normal egg cytoplasm (ec). A thick neck cell cap (ncc) overlies theneck cell rosette. Also visible is the neck cell passage (ncp) and an unbrokengynospore wall (gw) around the female gametophyte (fg). Scale bar = 50 pm.B and C : Parts of two archegonia (A2 & A3) from a specimen with four archegonia.(see also Fig. 5.6). A proembryo was growing from archegonium A3.B : In archegonium A2, the egg cytoplasm (ec) is degenerating, but the neck cells(nc) and gynospore wall (gw) are intact. Scale bars = 50 pm.C : Archegonium A3 has disrupted neck cells (nc). Pollen tube cytoplasm (pt) liesbetween the neck cells and at the broken gynospore wall (gw). Several small nuclei(n) are visible in the degenerating egg cytoplasm (ec), Scale bar = 50 pm.D and E : Neck cells of two archegonia (A1 & A2), in TS, from a specimen with aproembryo developing from archegonium Al (see also Fig.5.17).D : Intact neck cells (nc) to archegonium A2 (see Fig. 5.17 L & N). Scale bar = '10pm.E : Penetrated neck cells (nc) to archegonium A1. Pollen tube cytoplasm (pt)occupies the centre of the former rosette (see also Fig. 5.17 M). Scale bar = 10 pm.F and G : Neck cells of two archegonia (A1 & A2), in TS, from a specimen with aproembryo growing from archegonium 41 (see also Fig. 5.18). Starch grains (sg) arehighlighted by phase contrast lighting.F : Intact neck cells (nc) to archegonium A2 (see Fig. 5.18 D). Scale bar = 10 pm.G : Penetrated neck cells (nc)to archegonium 41 (see Fig.5.18 D). Pollen tubecytoplasm (pt) lies at the centre and periphery of the neck cells. Scale bar = 10 pm.

lfnco()l7L{?,rIDIa.1,anc\?e-| ',t,I11' - ' 't u't):'a- 1,,(p: 'rF)l--^' 1.'IGFig. 5.1

Figure 5.2 Material from the pollen tube enters the egg cell via the disruptedneck cells, in Prumnopitys taxifolia.A : Two archegonia (A1 & A2) in oblique LS. Black-stained pollen tube cytoplasm(pt) enters archegonium 41 between the neck cells (nc). A cellular proembryooriginating from this archegonium was observed in another section. Scale bar = 100pm.B : Neck cells of a fertilised archegonium (A1), in LS, from a specimen with threearchegonia. (Detail of Fig. 5.5 B). Archegonium A1 contained a four-nucleateproembryo (see Fig. 5.4 F - H). The other two archegonia were unfertilized (see Fig.5.7 A - C). A spread out pollen tube (pt) overlies the neck cells. A small (possiblymale) nucleus (n) is close to the neck cells in the remains of the egg cytoplasm (ec).Scale bar = 10 pm.C : Part of a fertilized archegonium (A2), in LS, from a specimen with twoarchegonia. (Detail of Fig. 5.5 A). Archegonium A2 contained a four-nucleateproembryo (see Fig. 5.3 F - H). Archegonium 41 was unfertilized (see Fig. 5.8 A -C). Pollen tube cytoplasm (pt) lies outside the disrupted neck cells (nc). In the eggcytoplasm (ec), a group of starch grains (arrowheads) close to the neck cellsresembles starch grains (sg) in the pollen tube cytoplasm. Scale bar = 10 pm.D and E : Two views of a female gametophyte (fg) with two archegonia.Archegonium A1 contains a four-nucleate proembryo. Archegonium A2 (not visible)was unfertilized (see Fig. 5.7 D - G).D : Archegonium 41 in LS. Pollen tube remains (pt) fill the neck cell passage fromthe gynospore wall (gw) to the neck cells (nc). A four-nucleate proembryo(arrowhead) lies in the dense zone (dz) (see also Fig. 5.4 B). The rest of the eggcytoplasm (ec) is degenerating. Scale bar = 100 pm.E : The same archegonium as in D, seen in pseudo phase contrast lighting. Starchgrains (sg) are abundant in the female gametophyte. Also highlighted are the eggcell wall (ecw) and starch grains, visible as a white line, passing between the neckcells (nc). Scale bar = 100 pm.F : Enlarged detail of the neck cells (nc) in D, showing starch grains (arrowhead) ofmale origin passing into the egg cytoplasm (ec)from the pollen tube (pt). Starchgrains (sg) are also visible in the surrounding tissues of the female gametophyte (fg).Scale bar = 10 pm.

-.t'nc ,/-Pt #/dl"fg\.'\'-/sg,f'AB(/-)l^.1-J,:l. .r, .,' J{-' '-},. ,.i f.t ..! i.' .,I.:'' iAl );.' u r.gwDt iFFig. 5.2

Figure 5.3: ln Prumnopitys taxifolia,the first and second divisions of the newsporophyte take place soon after fertilization, while adjacentunfertilized egg nuclei begin to degenerate.A to E : Different views of three archegonia (A'1, A2 & A3) in oblique TS.Archegonium A2 was fertilized and contains a two-nucleate proembryo.A : A large egg nucleus (en) in archegonium A3. The perinuclear zone (pz) variesin thickness. Also visible are parts of archegonia 41 and 42, the disrupted neck cellsof A2, the gynospore wall (gw) and the nucellus cone (nuc). Scale bar = 100 pm.B : A large egg nucleus (en) in archegonium A1. The perinuclear zone (pz) variesin thickness. The jacket cells (jc) still form a distinct layer. Scale bar = 100 pm.G : The three archegonia, A1 A2 and A3, sectioned halfway between their broadand narrow ends. A two-nucleate proembryo (arrowhead) is visible in archegoniumA2. Scale bar = 100 pm.D : Enlarged view of the egg nucleus (en) in archegonium A1, in a different sectionfrom that shown in B. The perinuclear zone (pz) is interrupted by an encroachingvacuole (v). Scale bar = 59 p6.E : Enlarged view of the two-nucleate proembryo shown in C. The position of thesenuclei (arrowhead) suggests that the first division of the zygote has occurred recentlyon a transverse spindle. Scale bar = 50 pm.F, G and H : Three views of a four-nucleate proembryo migrating to the chalazalend of an archegonium.F : Two archegonia (A1 & A2), in LS. A four-nucleate proembryo (arrowhead) liesat the chalazal end of a large vacuole (v) in archegonium A2. Intact neck cells (nc)on archegonium 41 show that it has not been fertilized (but see Fig. 5.8 A - C foranother section of this unusual archegonium). Scale bar = 100 pm.G and H : Adjacent sections of the four-nucleate proembryo shown in F. Thesomewhat llattened appearance of the nuclei (n1 to n4) may be a fixation artifact, oran effect of expansion of the central vacuole (v) and compression of the surroundingegg cytoplasm (ec). Scale bars = 10 pm.

Fig. 5.3

Figure 5.4: ln Prumnopitys taxifolia,the four-nucleate proembryo has arandom arrangement of nuclei within the dense zone at thechalazal end of the archegonium.A : Four-nucleate proembryo (nl to n4). In this specimen the dense zone (dz)appears unusually pale due to very light staining. Scale bar = 50 pm.B : Four-nucleate proembryo from the specimen shown in Fig. 5.2 D - F. Onlythree nuclei (n1 to n3) are visible in the dense zone (dz). Starch grains (sg) areabundant in the surrounding female gametophyte (fg). Scale bar = 59 p6.C, D and E : Two archegonia in non-adjacent TS. Archegonium 41 contains afour-nucleate proembryo. C: The nucleus (nl ) is nearest the chalazal tip. D: Thenuclei, n2 and n3. E: The nucleus n4 lies towards the limit of the dense zone.Archegonium A2 is unfertilized. Scale bar = 50 pm.F, G and H : Three views of an archegonium containing a four-nucleate proembryo.This specimen contained two other unfertilized archegonia (A2 & A3) (see Figs. 5.7A-c).F : Archegonium A1 in slightly oblique LS. Part of the four-nucleate proembryo(arrowhead) is visible in the dense zone. lrregular masses of egg cytoplasm (ec) lieat the margin of the large central vacuole (v). Some of the jacket cells fic) betweenarchegonia A1 and A2 are unusual (see Fig. 5.5 E). Scale bar = 100 pm.G : The chalazal end of archegonium 41 is shown with the four-nucleate proembryo(arrowhead). Also visible is part of archegonium A2, and the surroundingmultinucleate cells of the jacket layer (jc) and female gametophyte (fg). Scale bar =100 pm.H : Four-nucleate proembryo (n1 to n4). Several cytoplasmic spheres (arrows)have formed at the junction between the dense zone (dz) and the large vacuole (v).Scale bar = 50 pm.

aa.t..:It,..a, .a I 'attr3 r'':.'..aa:. .aa-.;t l. r' /a) . .rfGl; :j ("{;t? ir+ .r.r i i. t.r.''-!c.ar-'at' ' i.' .t a i'.-.- -jnZ'o. l .I l. |laai.ir'\-r"{ Y.- o-n33.ata..oI.i tABD.- t .. -ra t a r.-.i2 t.. ;rl ' 4t. .-.t.Fl{ 5'a .' ?.n'lit.-l -a. l.t,-.- t .'"-E'f to t -','. ,t-,oa.tla'-tar_.:{'l.';f'at t a..an20tJ.--all4oOO-at./a fIatdzroo3t ioi;tn1o Ian3.fQ,a3oCo,t,| .,1'tt tl.o IoaHFig. 5.4

Figure 5.5 After fertilization, the micropylar end of the archegonium beginsto degenerate, in Prumnopitys taxifolia.A : Fertilized archegonium in LS. Part of archegonium A2 from the specimen inFig. 5.3 F. The vacuoles (v) and black spots (arrow) appear to have coalesced. Theremaining egg cytoplasm (ec) is disrupted and patchy. The penetrated neck cells(nc) are visible (see also Fig. 5.2 C). Scale bar = 100 pm.B : The micropylar end of archegonium A1 from the specimen shown in Fig. 5.4 F -H. ln the patchy egg cytoplasm (ec) the vacuoles (v) appear to coalesce into fewerlarger ones. The penetrated neck cells (nc) are also shown in Fig 5.2 B. Scale bar =100 pm.C and D : Jacket cells fic) at the micropylar end of the archegonium shown in Fig.5.4 A.C : A binucleate jacket cell. Part of one of the nuclei (arrowhead) appears to havepassed through a narrow opening into the archegonium (A1). Phase contrast lightingshows that starch grains (sg) are present in nearby cells. Scale bar = 10 pm.D : Nuclear and cytoplasmic material including starch grains (sg) appear to beentering the archegonium (A1) via a rupture in the jacket cells (jc). A small distanceaway a jacket cell nucleus (arrowhead) appears to be passing through thearchegonialwall. Scale bar = 10 pm.E : Unusual nuclei in the jacket layers fic) between a fertilized (A1) and anunfertilized archegonium (see also Figs 5.4 F - H and 5.7 A - C). ln two cases,connexions pass between the nuclei in adjacent cells (arrowheads). The surroundingmultinucleate cells appear normal, and several cells undergoing mitosis wereobserved (arrow). Scale bar = 10 pm.

A1ct.rDFig. 5.5

Figure 5.6: Unfertilized archegonia eventually degenerate like adjacentferti I ized archegon i a, in P ru m n opity s taxif ol i a.A to E : A female gametophyte with four archegonia (A1 - A4), in a non-adjacentseries of sections. Only archegonium A3 was fertilized, with a well advanced cellularproembryo arising from it. All scale bars = 100 pm.A : Part of archegonium 41, with intact neck cells (nc), but no egg nucleus. Arounda large vacuole (v) the degenerating egg cytoplasm contains several medium to smallnuclei (two medium seen here). One may be the ventral canal nucleus (vcn?) andthe others jacket cell nuclei (n).B : Parts of archegonia A1, A2 and A3. Archegonium A2 has intact neck cells (nc),no egg nucleus, and a large vacuole (v) in the degenerating cytoplasm (see also Fig.5.1 B). Numerous smallto medium nuclei (n), some likely to be male nuclei, arepresent in archegonium A3. Also visible is the nucellus cone (nuc).C : Parts of archegonia A1, A2 and A3, showing the penetrated neck cells (nc) ofarchegonium A3. In the degenerating cytoplasm of A3, a dark mass near the neckcells resembles egg nucleoplasm (arrowhead) (see also Fig. 5.1 C), and small nuclei(n) are present. Also visible is a pollen grain (pg), and pollen tube (pt) in the nucelluscone.D : Parts of archegonia 42, Ag and A4. Archegonium A4 has intact neck cells (nc),no egg nucleus, a large vacuole (v) and small nuclei (n) in the degeneratingcytoplasm. ln archegonium A3, the jacket layer (jc) has broken down at themicropylar end. At its chalazal end, several cytoplasmic spheres and other shapedmasses (arrows) lie between the degenerating egg cytoplasm and the suspensor (s)of a developing proembryo.E : Archegonia A3 and A4. The cellular proembryo (arrowhead) arising from A3has a suspensor (s) about 450 pm long (see also Fig. 5.19 A - D). The curve in themidline of the archegonial complex and female gametophyte (fg) is a fixation artefact.F : The same section as in E, seen in pseudo phase contrast lighting. Starchgrains (sg) are present near the embryonic group of the proembryo (anowhead) butmore abundant near the growing suspensor (s), and absent around the archegonia(A3 & A4). Also visible are the large vacuole (v) in archegonium A4, and the dull rednuclei of the female gametophyte cells (fg).G : Enlarged view of the chalazal ends of archegonia M and A3, from the sectionshown in D. Near the ends of the suspensor cells (s) lie three nuclei from the U tier(U) open to archegonium A3. Scale bar = 50 pm.

.t.ao.'l' . ill rl-'o.)- t '!./ \-aa": ':.a--:. .< rlt ..'. )a/ ' t ''"J,Jl(.1- t. -'.tlotdlFig. 5.6

Figure 5.7: Changes in unfertilized archegonia adjacent to fertilizedarchegonia are greatly variable in Prumnopitys taxifolia.A to C : Two unfertilized archegonia (A2 & A3) adjacent to the fertilizedarchegonium (A1 ) shown in Figs. 5.4 F - H and 5.5 B.A : Archegonia A2 and A3 in median LS. The egg cytoplasm (ec) appears normal,but in each archegonium the large egg nucleus (en) lies close to the jacket cells (jc).A ventral canal nucleus (vcn) is visible in archegonium A3. Also shown is thenucellus cone (nuc). Scale bar = 100 pm.B : Enlarged view of archegonia A2 and A3, in a different section from that shownin A. Around each egg nucleus (en), the perinuclear zone is ill-defined, and inarchegonium A2, three vacuoles (v) are in contact with the egg nucleus. The intactneck cells (nc) of A2 are also visible. Scale bar = 50 pm.C : A non-median LS of archegonia A2 and A3, showing a porlion of the perinuclearzone (pz). Some nuclei (arrows) appear to have entered the archegonium from thejacket layer (jc). Also present is a small nucleoid body (arrowhead) which may ormay not be a jacket cell nucleus. At this magnification, the egg cytoplasm (ec) has astreaky appearance. Scale bar = 50 pm.D to G : One unfertilized archegonium (A2) adjacent to the fertilized archegoniumshown in Figs. 5.2D - F and 5.4 B.D : Archegonium A2, with a large vacuole (v) at the middle. Two small nuclei(arrowheads) lie in the disorganised egg cytoplasm. Scale bar = 100 pm.E : Archegonium A2, showing its intact neck cells (nc). Two nuclei (arrows) lieclose to the jacket layer (jc). Scale bar = 100 pm.F : Enlarged view of the small nuclei (n1 & n2) shown at arrowheads in D. Possiblythese are the products of division of the ventral canal nucleus. The egg cell wall(ecw) is clearly visible. Scale bar = 50 Fm.G : Enlarged view of the nuclei (n3 & na) shown at arrows in E. Each nucleus hasa dark spot resembling a nucleolus, and they lie between a vacuole (v) and the jacketcells fic). These are possibly daughter nuclei of a divided egg nucleus. Anothersmall nucleoid body (arrow) is also visible. Scale bar = 50 pm.

e. dEUR-i r =.Fig. 5.7

Figure 5.8: An unusual archegonium in a normalfemale gametophyte, andan eight-nucleate proembryo in an abnormal female gametophyte(P ru m n opitys taxif ol i al.A to C : Three views of an unfertilized archegonium (A1) adjacent to the fertilizedarchegonium (A2) shown in Figs. 5.2 A,5.3 F - H, and 5.5 A. The intact neck cells ofarchegonium A1 are shown in Fig. 5.3 F.A : Archegonium 41 in median LS, in a well preserved female gametophyte (fg)showing very little shrinkage. Although the cytoplasm appears typical of an egg cell,with a vacuolate zone (v) and a dense zone (dz), the egg nucleus is lacking. A smallnucleus (arrowhead) lies close to where the egg nucleus would have been located.Part of the nucellus cone (nuc) and the integument (int) are also visible. Scale bar ='100 pm.B : The same section as in A, seen in pseudo phase contrast lighting. The starchgrains (sg) in the female gametophyte (fg) are most abundant beyond the narrow tipof archegonium 41. The small dull red nucleus (arrowhead) is easily visible in thedark reddish brown cytoplasm. Also visible is the egg cell wall (ecw). Scale bar = 100pm.C : Detail of archegonium 41, from the same section as in A, showing that the smallnucleus (arrowhead) lies at one side of a darker region resembling a perinuclear zone(pz). Due to loss of an unknown number of sections, it was not possible to determinewhether this was actually one nucleus or two, one behind the other. (See Fig. 5.9 Aand B for an example of two nuclei in an unfertilized archegonium.) The cytoplasm(ec) is typical of the vacuolate zone seen in egg cells (see Chapter 4). Scale bar =50 pm.D to F : Three views of a developing megasporangium containing sevenarchegonia (Al to A7) in three female gametophytes (G1, G2 & G3), in oblique TS(see also Chapter 4, Fig. 4.16 D - F).D: Archegonia A1, A2 and A3 (in G1)are at egg cell stage. Archegonia 46 and A7(in G3) are at central cell stage. In gametophyte G2, archegonium A5 has beenfertilized, as indicated by penetrated neck cells (nc). Archegonium 44, with intactneck cells (not shown) and lacking an egg nucleus, is typical of unfertilizedarchegonia adjacent to a fertilized archegonium. Scale bar = 100 pm.E : Archegonia A4 and A5 (in G2), seen in a section close to their chalazal ends.Also visible is part of gametophytes G1 and G3. Scale bar = 100 ptm.F : Enlarged view of archegonia 44 and A5, from the section shown in E. Fournuclei (n5, n6, n7 and n8 of an eight-nucleate proembryo) lie in the dense zone ofarchegonium A5. Scale bar = 50 pm.

-1-'.'...fgG2DG3G2.-G1A5 ;i;':{j lr-.' i':44.lr;i.i,i.:'':.''AE' ' :'f=t.: Ic' ,f . r rr ,Pi\i*{. tl t,,:;''4#t i!; ,''.'1"6#jii;: ,nut'* 'l 't':'o i.r Q .r.'' :!'ItIl '-o ' il,*nr-dr!; Io . .r oof.,,*.,',,ctFFig. 5.8

Figure 5.9: ln Prumnopitys taxifolia,the proembryo undergoes the fourthfree nuclear mitosis in the dense zone. Changes in unfertilizedarchegonia nearby are variable.A to D : Three archegonia (A1, A2 & A3) in oblique non-adjacent LS. ArchegoniumA3 contains an eight-nucleate proembryo in metaphase (see F & G). In thisspecimen the archegonial group is linear rather than triangular. The fertilizedarchegonium (A3) is at one end of the group. Of the two unfertilized archegonia (A1& A2), only A2 is adjacent to archegonium A3. Archegonium 41 contains a large eggnucleus with a complete perinuclear zone (not shown).A : Archegonia 41 and A2. ln archegonium A2, the egg nucleus is lacking but twosmall nuclei (arrowheads) lie at the chalazal side of a large vacuole (v). A ventralcanal nucleus lies close to the intact neck cells in another section (not shown). Scalebar = 100 pm.B : Enlarged view of the two nuclei (arrowheads) shown in A. Scale bar = 50 pm.C : Archegonia A1, A2 and A3. Archegonium 43 has penetrated neck cells (nc)and degenerating cytoplasm (ec). The dense zone (dz) of archegonium 41 has theappearance typical of an egg cell. The cytoplasm of archegonium A2 appears slightlydisorganised. Scale bar = 100 pm.D : Narrow tip of archegonium A3, containing a developing proembryo in the densezone (dz) (see F & G). Cytoplasmic spheres (arrow) can be seen at the lower limit ofthe dense zone. The smaller cells at the midline of the female gametophyte (fg) arevisible, beyond the tip of archegonium A3. Scale bar = 100 pm.E : The same section as in D, seen in pseudo phase contrast lighting to show thegreat abundance of starch grains (sg) in the tissues around the developingproembryo (anowhead) and beyond it along the midline of the female gametophyte(fg). Scale bar = 100 pm.F : Enlarged view of the narrow tip of archegonium A3, from the section shown inD. Four metaphase groups (arrowheads) are just visible in the dense zone (dz).Beyond this zone, the cytoplasm has formed spheres (arrows) and other irregularmasses. Scale bar = 10}rm.G : Proembryo undergoing a fourth free-nuclear division. Composite drawingbased on four serial sections of the tip of archegonium A3. The eight mitotic spindles(ms) appear to be randomly oriented. The stippled area represents the dense zone(dz) and the solid line the egg cell wall (ecw). Scale bar = 10 pm.

fgDFc-ncGFig. 5.9

Figure 5.10: ln Prumnopitys taxifolia, two free nuclear proembryos candevelop in adjacent archegonia.A to Q : Two fertilized archegonia (A1 & A2) in a non-adjacent series of TS, from thechalazal (A) to the micropylar (Q) ends. (See Fig. 5.11 for details of the two developingproembryos) fg = female gametophyte; gw - gynospore wall;jc = jacket cells; pl - parietallayers of the nucellus; ppcl= pseudo phase contrast lightingA : Archegonia A1 and A2, at the level of the dense zone. A group of mitotic chromosomes(arrowhead) is present in A2. Scale bar = 100 pm.B : The same section as in A, seen in ppcl. The mitotic group appears as a dull red spot(arrowhead). Starch grains (sg) are most abundant at this level. Scale bar = 100 pm.C : Enlarged detail of A. The mitotic chromosomes (arrowhead) in A2 are in metaphase.Scale bar = 50 Bm.D : Archegonia A1 and A2, at the level of the large vacuole between dense zone anddegenerating egg cytoplasm. A cytoplasmic sphere (arrow) is seen in A1. Scals [61 = 100pm.E : The same section as in D, seen in ppcl, showing the abundant starch grains (sg) and theegg cell walls (ecw). Scale bar = 100 pm.F : Archegonia A1 and A2 in a section dilferent from that in D. A cytoplasmic sphere(arrow) is present in A2. Scale bar = 50 pm.G : Archegonia 41 and A2, at a point halfway between narrow tips and neck cells, showingthe degenerating egg cell cytoplasm. Scale bar = 100 pm.H : The same section as in G, seen in ppcl. The starch grains (sg) are less dense. The eggcell wall (ecw) is still a prominent feature. Scale bar = 100 pm.J : Enlarged detail of G, showing a small nucleus (n) in A2. Scale bar = 50 pm.K : Archegonia A1 and A2, at a level close to the neck cells. The jacket cell nuclei(arrowheads) are swollen and degenerating. Scale bar = 100 pm.L : The same section as in K, seen in ppcl. The degenerating jacket cell nuclei show up asdull red bodies. No starch grains are present. Scale bar = 100 pm.M : Archegonia 41 and 42 at the level of the former egg nuclei. The cytoplasm appearsturbulent. A small nucleus (n) is visible in A1. Scale bar = 50 pm.N : A section close to the micropylar end of the female gametophyte, through the neck cells(nc) of A1 and the neck cell passage (ncp) of A2, showing that pollen tubes reached botharchegonia. Scale bar = 100 pm.P : The same section as in N, seen in ppcl, highlighting the neck cells (nc) of A1. Thedense pollen tube cytoplasm in the neck cell passage (ncp) outside 42 is also visible. Scalebar = 100 pm.Q : Enlarged detail of the section in N. Dark remnants of pollen tube cytoplasm occupy thecentre of the penetrated neck cells (nc) of A1, and fill the neck cell passage (ncp) of A2.Scale bar = 50 pm.

AcDFGJMNoFig. 5.10

Figure 5.11: ln Prumnopitys taxifolia,lhe sixteen-nucleate proembryo canundergo a tifth free-nuclear division.A to E : Archegonia 41 and A2 from the same specimen as that shown in Fig 5.10.A non-adjacent series of TS, in a chalaza-to-micropyle direction, through the twodeveloping free-nuclear proembryos. All scale bars = 50 pm,A : Archegonia 41 and A2, at a level close to their narrow tips, showing two nucleiin 41, and none visible in A2.B : One nucleus in 41; three metaphase groups of chromosomes (arrowheads) inA2 (see also F).C : Four nuclei in 41; two nuclei and two metaphase groups of chromosomes(arrowheads), on spindles oriented parallel to the plane of the section, in 42.D : Five nuclei in 41; six nuclei in A2.E : Section close to the limit of the dense zone. Two nuclei and a cytoplasmicmass in A1; three nuclei in A2.F : Enlarged view of archegonium A2, from the same section as in B, showing threemetaphase groups, with their spindles oriented more or less perpendicular to theplane of the section.G : Longitudinal reconstruction of the archegonia 41 and A2 shown in Figs. 5.'10and 5.11 A - E, drawn to scale from serial sections. ln each archegonium, a freenuclearproembryo is developing in the dense zone (dz) at the narrow tip. ln 41 thesixteen nuclei of the proembryo (proE 1) are at interphase. In 42 the sixteennucleateproembryo (proE 2) has seven nuclei at interphase, and nine at metaphase,undergoing a fifth free-nuclear division, with the observed orientations of the mitoticspindles (arrowheads) represented diagrammatically. Also shown are variouscytoplasmic spheres (arrows), degenerating egg cytoplasm (ec) containing groups ofblack bodies (bb) and several small nuclei (n), and the neck cells (nc) penetrated bypollen tube cytoplasm.

Aattaattt a , .3ea '''A2 -trt r-',Alt I a '.. t a . t ttt''1.t?.'. a.1 _,'a j D tt 't.a.a"o'" a i t .'arr t --lB^t I' t al t..' . t.(t .' f r. I ta t. a ''a'..r'it--It'o.cDtl.l.ae ., t"t- .- a t r -F^n o . | . . A.1 _:^if* $* . '{.aa *'',,?,. + ''!i . '.t'.t''' |i,-r-it"tI".. I o, a' ' a.tt t" ''I''tj .''GaItr,:tE-ta'{llt..aata'.' a i -a a a','a,lo.f" * t',1] '.iot )'' IaQar. f'r a-tarr1l, t.l.Ia far)t.]It'FII v,-t!A2',*tltlF'r*tf^lIi'ftlFig. 5.11

Figure 5.12: ln Prumnopitys taxifolia, some of the proembryonal nuclei areexcluded from the newly cellular proembryo.A to D : The first of three newly cellular proembryos (proE 1) in a single specimen,in adjacent slightly oblique LS. See also Fig. 5.13 (proE 2), Fig. 5.14 (proE 3) andFig. 5.15. All scale bars = 50 pm.All cells of the embryonal group are binucleate, except for one unusual cell (uc) seenin A and B which has 3-4 nuclei. In A, B and C, about six nuclei excluded from theproembryo lie in a remnant of the dense zone (dz) open to the archegonium (A1).One (arrowhead) is in prometaphase. The closed cell nearby (cl) may belong to thistier of cells. The binucleate cap cell (cc), in C and D, has a thickened wall at its apex.Large and small cytoplasmic spheres (arrows) and wall projections (wp) are alsovisible, in B.

I'LaI.o.,aolt..t\.rrbf t,i";--''r'-'J:,\-&':-- l}rar r'!rLN.. ItI I-ttI |'.2{..Eit'iJr. aa oo ao t ..o a.t-r.a ..'-^ttaa''atlao.tei'.ol1tttr ''O.l.' . a ra.I I a. I r' Il. I a at1o()l... o L aa' ao't*. s'il.ffiil. Erotraor ...a t.ol-'.a Oor. a . f", aata aa a atia-e\t I i.l I d':''I:?, "^-a. r. l. lrmaIaao-ad.aIa.aaa--#:i\r.: Ia.'a()3I'1..6;vi)-oai:ta I aatlr aa aIa'.o :"'' '.-r. $lj . o.N/oI.t't . rl l.a |.aaaaaFig. 5.12

Figure 5.13: Outside the newly cellular proembryo of Prumnopitys taxifolia,some excluded nuclei may continue to divide.A to D : The second of three newly cellular proembryos (proE 2) in a singlespecimen, in adjacent slightly oblique LS. See also Fig. 5.12 (proE 1), Fig. 5.14(proE 3) and Fig. 5.15 (lrom the same section as in A). All scale bars = 50 pm.The embryonal cells are all binucleate, and the cap cell (cc) has a thickened wall atits apex, in A and B. While some cells open to archegonium A2 are degenerating,several excluded nuclei (arrowheads) are in prometaphase at the micropylar end ofthe proembryo, in A to D. Also visible are cytoplasmic spheres (arrows) and wallprojections (wp), the latter most noticeable in A.

o . . i^t,a!^.o.ltaoVaa$ l'l! 'N1ata..-OoOatsr o-j i,1+*"frrr"l'a .taa^aoratta\r 1rlftr;::::.{'* ()f '- - . . . t tt,arQ-a._+Sa',o o . ,a ol ' rt.i, F ?t- -- ':=- t'o."r. ::..t"ar.r. -.o : t .aa'1tl t: L^ _.-..fatlto.'aa-F; YhtsTT:. E' t'.&5uitaata.ara," aa t-', otla.aa.aiaaa!-a ta . ' t fo aa-t-fra.O -iD :!(\tr lr ol|rQ ttaGao I aoul'aOa.at"t//ij.llI tFig. 5.13

Figure 5.14: ln Prumnopitys taxifolia, elongation of the cells in the suspensortier begins within the archegonium.A to D : The third of three newly cellular proembryos (proE 3) in a single specimen,in adjacent slightly oblique LS. See also Fig 5.12 (proE 1), Fig. 5.13 (proE 2) andFig, 5.15. Allscale bars = 50 pm.The cap cell (cc) has a thickened wall at its apex, in A and B. Below the embryonaltier is the suspensor tier, with elongating suspensor cells (s) about 75 pm long, in C.Degenerating remnants of the excluded nuclei (arrowhead) are still present in the tieropen to archegoniunr A3. Cytoplasmic spheres (arrow), wall projections (wp) andpart of ar:chegoniurn A2 are also visible.

".-latt.arI..--.: I Jlte''s' fi,r'i. --" t'f I r i'.aaaat"6'.s-'.rl- n, l''o'tr 3.J'oJla.T tt.I,c'h aai1t -t ' | ,attattrflr,l. r'|l..ter{!t .rra l.i a, I r/rt?''(,'tt'tl\lil rr\-{."It'+- \* tFtt.l r . rrrl. i .. I.t .l-1 .ri'1-.--(,t..tla Itr'Iat-'a_O.'fl ira r0..^a..: ':, t tr io .*'.ilr.'r. at't.".t.: r.- .,rt,oooir{f,,!r,ffiT ;:' to' . ':.\1.. I lr 'r'', , '' i' 'ttrrI'Fig. 5.14

Figure 5.15: ln Prumnopitys taxitolia, the cellular proembryo at the tip of thearchegonium is located deep in the female gametophyte evenbefore growth of the suspensors.A : Female gametophyte (fg) with three archegonia, in nearly median LS througharchegonia 41 and A2 and one proembryo (proE 2), from the same section as shownin Fig. 5.13 A. Each archegonium contains a cellular proembryo in the chalazal tip.See also Fig.5.12 (proE 1), Fig. 5.13 (proE 2) and Fig.5.14 (proE 3). Also visible arethe gynospore wall (gw) and the parietal layers (pl) of the nucellus. Scale bar = 500Fm.B : Archegonium 42 with its conical proembryo (proE 2), from the same section asshown in Fig. 5.13 A. A large vacuole (v) or space separates the developingproembryo from the degenerating egg cytoplasm (ec) at the micropylar end. Thissection passes close to the archegonial wall in the mid region, showing remnants ofegg cytoplasm (ilt) adhering to the wall projections. The jacket layer (jc) is intact atthe chalazal end but has degenerated towards the micropylar ends of the archegonia.Also visible are the penetrated neck cells (nc) of archegonium 41, the femalegametophyte tissue (fg), the gynospore wall (gw) and parietal layers (pl) of thenucellus. Scale bar = 100 pm.C : The same section as in B, viewed with pseudo phase contrast lighting. In thefemale gametophyte (fg), starch grains (sg) are abundant ahead of and near theproembryo (proE 2) and absent around the rest of archegonia Al and A2. Wallprojections (wp) are highlighted where the section parallels the archegonialwall(ecw). At the micropylar or proximal end of the proembryo, one of the excludednuclei (arrowhead) is visible as a dull red body (see also Fig.5.13 A). Also visible arethe penetrated neck cells (nc) of archegonium 41 and the parietal layers (pl) of thenucellus. Scale bar = 100 pm.

Fig. 5.15

Figure 5.16: Five-celled and eight-celled suspensorsin Prumnopitys taxifolia.A : A cellular proembryo with a suspensor (s) over 300 pm long. The conicalembryonal group (e) has 12 cells, and the suspensor about 7 cells (see SpecimenNo.19 in Table 5.1). Simultaneous elongation of the suspensor cells pushes theproembryo into the female gametophyte (fg). Large starch grains (sg) are mostnumerous near the distal end of the proembryo, and less so near the archegonium(A1) at the proximal end. Some gametophyte cells (arrowheads) in contact with theproembryo have black-stained cytoplasm and appear to have collapsed. Scale bar =50 pm.B : Eight-celled suspensor (s) in TS within a female gametophyte (fg). Therelatively dense cytoplasm in the suspensor cells indicates that the section is close tothe proembryo (not shown). Some gametophyte cells (arrowheads) next to thesuspensor appear to have collapsed. Starch grains (sg) are present but notabundant. Scale bar = 50 pm.C and D : Suspensor in TS (D viewed with pseudo phase contrast lighting). Thesame section as in B, showing that starch grains (sg) are confined to a narrow zoneclose to the suspensor (s), and are especially concentrated in the collapsedgametophyte cells (arrowheads). Scale bars = 100 pm.E to J : One of two cellular proembryos in a non-adjacent series of oblique LS. Allscale bars = 50 pm.E : Archegonia 41 and 42 lack cytoplasm, and have disrupted neck cells (nc),indicating that they were both fertilized.F : Two suspensors, one (s1) from archegonium A1 , with 5 - I cells, and another(s2)from A2, with 5 cells. The suspensors are growing and coiling together, within amass of collapsed gametophyte tissue.G to J : The proembryo originating from archegonium A1, in three adjacentsections. The binucleate condition of the embryonal cells is most distinct in J. Theconical embryonal group (e) has 15 cells and the suspensor (s1) 5 - 8 cells,indicating that the originalfree-nuclear proembryo had 20 -23 nuclei at the time ofcell wallformation (see Specimen No.12 in Table 5.1). The five-celled suspensor (s2,1 - 5) from archegonium 42 appears to have displaced the suspensor (s1) belongingto proE1. The proembryo from A2 (not shown) lies ahead of proEl in thegametophyte tissue (fg). Starch grains (sg) are numerous.

g*".*-.;f i.) i. ,.,'.l t'rt " --t ",; y','\;,Eh-,, , '.' :.' '' n"1.;; - - ,:.a'. a ..d'n,{*,]"', -t' .t.lr.t,'n'r, -' l !q ,;: .,r\,. :,. , .. ., .i....,. ,ia:_ !.i;*j:Itail,4-,FIel,fgA,aa t-_l)t t:. r,tf,.l,'ait-'1r'i#,J" All'a.I'n:,:' I,t'.\r'l ll. j.e. l.\I .l , r' " l, Y''r."! . '"',,', u'G5r'4+ar: . l''- ''Er). "s?,,'^,'-ilt"!'l1. l',r , i'4ff,a,, ,,''qtBcaattq'I)..lr.'-fIa.a&HJ. +: ' ,: 'e-l." :'rf '"f- t ,,jir{s3-'' t..l*.t..-ttnlttla'4tl . Y i-alL,. E2- 1'rl'.r-'a?1r'6t. ..t 'sla'r.,r !.--' . .-r, .,':2- 4,,,,. i ., - +la .'Qic g,-etifi'''iz " '"]4ir\3 j,'tr.Fig. 5.16

Figure 5.17: Some proembryos can have a ten-celled suspensor, inPru m nopitys taxifolia.A to N : A developing proembryo within the female gametophyte, in a non-adjacentseries of TS from embryonal group to neck cells. Of three archegonia (A1, A2 & A3),only 41 was fertilized. fg = female gametophyte, gw = gynospore wall, pl = parietallayers of the nucellus, ppcl = pseudo phase contrast lighting. Scale bar for N = 100pm; all other scale bars = 50 pm.A and B : Embryonal group of the proembryo (proE), in two adjacent sections, witha cell count of 8 - '10 cells (see Specimen No.1 1 in Table 1 ).C : The same section as in B, seen in ppcl. Starch grains (sg) are numerous nearthe embryonal cells (proE).D and E : The suspensor (s), in different widely spaced sections, has 10 cells.F : The same section as in E, seen in ppcl. Starch grains (sg) are numerous nearthe suspensor (s) and collapsed gametophyte cells are distinct.G : The 1O-celled suspensor (s), seen in ppcl, at a point halfway along its length.The coiled suspensor is sectioned twice in TS, with a short portion in LS between(arrowhead). Except for one or two isolated cells still containing starch grains (sg),and the collapsed cells around the suspensor (arrow), starch grains are lacking in thesurrounding gametophyte tissues.H : The 1O-celled suspensor (s) close to its proximal end at archegonium A1 , withthe black-stained tips of archegonia A2 and A3, in a mass of collapsed cells whichhave become detached from the rest of the gametophyte.J : The same section as in H, seen in ppcl. Starch grains are absent. Thesuspensor (s) and archegonia A2 and A3 are particularly clear.K : Archegonia A1, A2 and A3, sectioned near their micropylar ends, with blackstainedrelics of cytoplasm remaining. The thick archegonialwalls are composedmainly of jacket cell remnants and the egg cell walls (ecw).L : Archegonia A1 and A3, and the intact neck cells (nc2) of archegonium A2 (seealso Fig.5.1 D).M : Part of archegonium 41 with its penetrated neck cells (nc1) (see also Fig. 5.1E), and part of archegonium A3 with intact neck cells (nc3).N : The whole female gametophyte, from the same section as in L. The intact neckcell rosette (nc2) is clearly visible even at low magnification. Asterisks ({t) indicatethe approximate locations of the neck cells to archegonia 41 (nc1) and A3 (nc3).

, ;_\';i{,.i,PfoE',J t .Ar . l a. I, tt fa r IBIf-.DEHK L MIplNFig. 5.17

Figure 5.18 : ln Prumnopitys taxifolia, the suspensor can have up to 13 cells.A to E : A female gametophyte (fg) with two archegonia (A1 & A2) and oneproembryo arising from A1, in a non-adjacent series of TS from proembryo to neckcells. All scale bars = 50 pm.A: Proembryo (proE)with 6-8 cells in the embryonal group (see Specimen No.16in Table 5.1). Starch grains (sg) are present but not abundant in the gametophytetissues.B : Suspensor (s) with 13 cells, in a section near its distal end. Starch grains (sg)are more abundant than near the embryonal cells shown in A.C : The 13-celled suspensor (s) near its proximal end at archegonium A1. Thenarrow tip of the unfertilized archegonium (A2) lies nearby. Starch grains (sg) areabundant in the surrounding tissues. The cells in contact with the suspensor may bejacket cells of the 'parent' archegonium (jc?).D : The penetrated neck cell (nc1) of archegonium 41 (see also Fig. 5.1 G), and thebroad end of archegonium A2, with degenerating egg cytoplasm (ec) and jacket cells0c).E : The intact neck cells (nc2) of archegonium A2 (see also Fig 5.1 F), and theapproximate location of the neck cell passage (ncp) to archegonium 41.

Aaatl'-".BcD.: "-.\-. ,...'/.'...,.1lr.EI ' I -. --!'.-trtaa-r! iFig. 5.18

Figure 5.19: ln Prumnopitys taxitolia, each suspensor cell is usually acontinuous tube with a single nucleus towards its distal end.A to D : A proembryo in adjacent LS, from the same specimen as shown in Fig.5.6. The conical embryonal group (e) has 18 binucleate cells and the 450 pm longsuspensor (s) has 9 cells (see Specimen No. 8 in Table 5.1). The cap cell (cc) andtwo others have collapsed at the distal end, in A and B. Except for one or two smallcells (arrowheads) at its proximal end, the tubular suspensor cells extend for the fulllength of the suspensor. The single large nucleus (nS) in each suspensor cell usuallylies towards the distal end, in C and D. Nuclei of the U tier (nU) are visible in thecytoplasm of archegonium A3. Collapsed gametophyte cells (arrows) packed withstarch grains (sg) lie close to the suspensor. The narrow tip of an adjacentunfertilized archegonium (A2) is also visible. All scale bars = 50 pm.

...'fd.i:-.l .r- '. '-t ?-t. )* .t'.r-t_S;t'T. -' .t ,i :[. t ':?F - li--t',a..ttt{tI! i o.t ' ;rl."' r los. ,tlt..,rlfltir,\__i'a- ,a aa-tt a?t!aIt-Ir a? , || It o,a,rlrtaa.a 'att'ri ;".. ?aa..'tl.'l'o+at.. ?'t-['k''{,:|,..r .aht .1.:.,.. . :_er - -" -;C)a -' , a . a a O t,..ro.rr?'..' . tT '-" 't/",.r'.-.." ..; ,'- r . ..t']"-'a 'l'--lr-''l ., . rl:r '' ''-=- ''."{r'IE '... '.";,...,, . ,l ,',t."":'o t. '' "t'-. .?r.1..i-.l,';ri v'{tt-*r";,r"rr.-1,=St--..---':l :l.r_ t, 'o a,cra.''..a, clt I. ' l I I f ' !o .(dl*r .:ll..I.\'E...tt1.,.* ' ) 't'l!,+*1. zIF{?'frrtt'aoia',. rot?,V V&. .SN{ .;S*Fig. 5.19

Figure 5.20: The middle of the suspensor begins to develop coils while thedistal end continues to lengthen, ln Prumnopitys taxifolia.A to D : Two proembryos (proE 1 and proE 2) in non-adjacent LS, each with asuspensor about 500 pm long. Both proembryos have an 8-celled suspensor and 8 -9 cells in the embryonalgroup (see Specimen No.7 in Table 5.1).All scale bars = 50 pm.A : The suspensor (s1) and part of the embryonal group (e1) of one proembryo(proE 1). Coiling (arrowheads) has begun in the middle of the suspensor. Collapsedgametophyte cells (arrows) containing starch grains (sg) lie alongside the suspensor,and the degraded remains of the U tier (U) and archegonium 41 are visible at itsproximal end.B : Another section of the suspensor (sl ) and part of the embryonal group (el ) ofproE 1. The nuclei of the suspensor cells (nS) are close to their distal ends. Part ofsuspensor (s2) of proE 2 is visible.G : Both proembryos (proE 1 & proE 2), close together in the gametophyte tissue(fg), showing proE 1 with the cap cell (cc) of the embryonal group and one suspensorcell (s1), and proE 2 with conical embryonal group (e2) and suspensor (s2).D : The second proembryo (proE 2), showing the conical embryonal group (e2), avery large cap cell (cc) and the suspensor (s2)with a large nucleus (nS) in one cell.

t-'/''f.'.tfgoGa^,ltrt.€t.r+tr* ..'td -ProEl {+ri riqi'- /ni:,#I j '*^tiali' fr, - i" lFl ,Si ,*. 'l',{i, 'E:-;:;, . .z )'fi'(liflltltt ctotq:rF'. ;St;'l,'d\', a a, I f tqr)r ': - q(tlJ\-\-A.r rr. a| -rf i,, Ioat. \\t ;t,l,r, Ir t t. :l, 'II\r+; , t].*:'l1fll. (.\ \'t: l' ,tt ' .-.D ? t '-l-\l ^.^*..t ;,.' .iuiffrilp Ir'. . -\'"-r il/ ..-ltAlBcIrt.r,Drro:{i'J'l''fi,tnS l,l+i',1.. $-E--.* slILr1ift proE?U,L'rX,i{,${ ,-.; !q.{ it'':{ v[ ''r"'^- ! -, l'i :'r,j'.i,i'': ^t- :+5,.,6d; '-:Fig. 5.20

Figure 5.21 : ln Prumnopitys taxifolia, some cells may be lost from thesuspensor during the elongation and coiling phase. Meanwhile,archegonia, whether lertilized or not, are reduced to hollow sacs.A to J : A female gametophyte with two archegonia (A1 & A2) and one proembryoarising from A2, in non-adjacent TS from the distal end of the-suspensor to the neckcells. The embryonal group was lost in sectioning. All scale bars = 50 pm.A : The distal end of the suspensor (s) has seven cells. Within the gametophytetissues (fg), starch grains (sg) are numerous.B : Another section of the seven-celled suspensor (s) close to a region of coiling.C : The coiled region at the midpoint of the suspensor (s). The extreme coiling mayexert lateral pressure on the gametophyte tissue (fg) and assist to anchor thesuspensor. In one oblique TS in the coils, the suspensor has seven cells (s7),whereas another part shows eight cells (s8).D : The proximal end of the suspensor (s) has eight cells (see Specimen No.10 inTable 5.1 ). The remains of the unfertilized archegonium (A1) are also visible.E : A section through the U tier (U)just beyond the suspensor, showing that it haseight cells in the same arrangement as the suspensor cells in D. Archegonium 41 isalso visible.F : Archegonia 41 (unfertilized) and A2 (fertilized) both contain remnants of eggcytoplasm (ec).G : Archegonia 41 and A2, sectioned at the broad micropylar ends. Both arereduced to hollow sacs.H : Archegonium A2 near the neck cells. The intact neck cells (nc1) ofarchegonium A1 are visible in the gametophyte tissue (fg).J : The penetrated neck cells (nc2) of archegonium A2. The location of the neckcell passage (ncp) to archegonium 41 is indicated.K : The remains of two archegonia (A1 & A2) in a gametophyte containing onedeveloping proembryo. Despite the collapse at the chalazal ends due to growth ofthe suspensor (s) from archegonium 41, the two archegonia retain their general formas hollow sacs within the female gametophyte (fg). Scale bar = 100 pm.

"= ,.r.J{trl -l . . I.,"rtti .!*...,-;.uf,Fu$;,i. j'..tr{'fi lNrb,$,' .'ia o - :' ,e'!A B cD-.taa,t'-lI l. s-,{Al\Itrt-,.fII\ IEa--l''tFG.IHJFig. 5.21

Figure 5.22: ln Prumnopitys taxifolia,the embryonal group develops into adome-shaped mass. Embryonal tube cells proliferate at itsproximal end to form a secondary suspensor.A, B and C : Three domed embryos (e) in order of increasing size, in differentfemale gametophytes (fg), each with a poorly preserved secondary suspensor (ss) atits proximal end. All scale bars = 50 Fm.D, E and F : Three views of a specimen containing one embryo, in median LS.D : Whole female gametophyte (fg) containing a domed embryo (e) with a massivesecondary suspensor (ss). A trace of the original suspensor (s) is present in thecentral cavity. Also visible are the remains of an archegonium (A), the multinucleategametophyte cells (arrows) and part of the nucellus (nuc).Scale bar - 500 pm.E and F : Enlarged detail of the domed embryo shown in D, in adjacent median LS.At its domed (distal) end the cells divide in all directions. At the proximal end, rows ofembryonal cells (arrowheads) give rise to embryonaltubes which form a secondarysuspensor (ss), Scale bars = 50 pm.

ssI,!'tr#.i{l**bI./:t,ia'rG.?!tDf,-r'T;)Itt'r'.f Al1g;"tB.,Tg,c-aft2aFFig. 5.22

Figure 5.23: ln Prumnopitys taxifolia,the embryo first becomes club-shapedas the root and shoot apices differentiate. When mature, theembryo becomes cylindrical with two short broad cotyledons.A and B : A club-shaped embryo, in adjacent oblique LS. Scale bars = 50 pm.A : The differentiating root apex (ra) lies at the micropylar end of a zone ofmeristematic cells. A shallow peak at the distal end represents the shoot apex (sa).The procambial tissues of the hypocotyl (h) will develop from the region betweenthese apical meristems.B : The root cap (rt) lies below the root apex, in the micropylar direction (arrow-m),the position of the root apex (ra) uncertain due to the oblique plane of this section.C : A mature embryo, dissected from the gametophyte before fixation, in a medianLS on a plane almost parallel to the two cotyledons, thus passing through themargins of both cotyledons (c1 & c2). The shoot apex (sa) forms a shallow moundextending the full width of the embryo (arrowheads) between the cotyledons. Adotted line indicates the shape of the cotyledons. Several other recognisable tissuesare: procambial tissues (pc), root apex (ra), and root cap (rt). The hypocotyl (h), at850 pm long, is unusually short (cf. embryo shown in D). Between the living cells ofthe root cap and the remains of the secondary suspensor (ss) are several rows ofdead cells (arrow). Also visible are the crushed remains of gametophyte cells (;le)trapped between the cotyledons. Scale bar = 100 pm.D : A mature embryo dissected from the gametophyte before fixation, in median LSperpendicular to the cotyledons (c1 & c2). Between shoot apex (sa) and root apex(ra), the hypocotyl (h) is 1 130 pm long. Other recognisable tissues include: root cap(rt), epidermis (ep), cortex (cx), procambium (pc) and pith (pi). Close to the shootapex, procambial tissue turns outwards and enters the bases of the cotyledons ({r).Two mitotic cells were observed, one in the root cap (arrow) (shown in E) and theother in the root apex (arrowhead) (shown in F). Also visible are the remains of thesecondary suspensor (ss). Scale bar = 100 pm.E : Cell at metaphase, from the root cap area (arrow in D). Scale bar = 10 pm.F : Cell at metaphase, from the root apex (arrowhead in D). Scale bar = 10 pm.

\c2\ fr- :k ,,/-> "i' l,.trV.-.r+ 4r,ffi"| ' tt,r"-+sat,'l't.-.\A --sscD-ss.. . I+m(,tttrt; *R'r'6t.J'rt'IIItrtI.ht\,!Itl".l'l*,, ) tlr tr lt,.>ir-+,aaBEF-Fig. 5.23

Figure 5.24: Within the female gametophyte of Prumnopitys taxitolia,thegrowing embryo enlarges mainly in the micropylar direction,crushing the surrounding tissues, and obliterating the suspensorand archegonia.A : A whole female gametophyte (fg) containing an embryo close to germination, ina non-median LS parallelto the cotyledons, and passing through one cotyledon (cot).The hypocotyl (h) is now approximately 1600 pm long. A band of procambial tissue(pc) branches into two strands (anows) close to the level of the shoot apex (sa).Developing secretory canals (sc) lie between the procambium and the cortex (cx).Traces of the suspensor (s) are visible between the root cap (rt) and the space left bythe former archegonia (A). Also visible are crushed gametophyte tissues(arrowheads) and the shrivelled nucellus cone (nuc). Scale bar = 5gg ut.B : Another section from the same embryo as that in A, showing the procambialtissues (pc) at the top of the hypocotyl (h) branching into the two strands (arrows) thatenter the cotyledon (cot). Also visible are the crushed remains of gametophytetissues ({t). Scale bar = 50 pm.

iil{fitiill';:'ltF'l;{Iffi.#ffiilfiIgFig. 5.2;A

Figure 5.25: Transverse sections of the embryo of Prumnopitys taxifolia showthe arrangement of the developing vascular tissues in relation tothe cotyledons.A - H : A mature embryo within the female gametophyte (fg), in a non-adjacentseries of TS. The gametophyte centreline has been drawn on each image to showthat this particular embryo has diagonal orientation with respect to the broad axis (ba)of the gametophyte. Scale bar (A) = 500 pm. Scale bars (B-H) = 100 pm.A : Whole female gametophyte (fg) with the embryo (e), sectioned at the level ofthe upper hypocotyl (see also E). The dark line around the perimeter is allthatremains of the parietal layers (pl) of the nucellus, reduced to a papery'skin'. Notethat the difference between the broad and narrow axes of the gametophyte has beenexaggerated by sectioning pressure. The gametophyte was also damaged duringremoval of the hard seed coat.B : The crushed suspensor (s) lies in the cavity toward the micropylar end of thegametophyte.C : Dead cells are present at the tip of the root cap (rt).D : ln the lower hypocotyl, the developing vascular tissues are radially symmetrical.Recognisable tissues are: epidermis (ep), isolated tanniniferous cells (tc) under theepidermis, cortex (cx), six secretory canals (arrowheads), and pith (pi) at the centre ofthe procambialtissue.E : Detail of the same section as shown in A. In the upper hypocotyl, the vasculartissue arrangement becomes a rectangle defined by the six secretory canals(arrowheads) and two bands of procambial tissues (pc), with pith at the centre.F : Just below the cotyledons, only the four outer secretory canals (arrowheads) arepresent, with four zones of procambialtissue (pc).G : At the base of the cotyledons (cot), the shoot apex (sa) extends the full width ofthe embryo, with zones of meristematic tissue (arrows) at both ends. Each cotyledonhas two vascular bundles (vb), each accompanied by a secretory canal.H : The cells at the tip of the shoot apex (sa) are quiescent. At this level, the twovascular bundles are more clearly defined in each cotyledon (cot). The adaxialfacesof the cotyledons meet above the shoot apex.

Aa-:EDHFig. 5.25

Figure 5.26: One embryo was observed with three cotyledons, but inPrumnopitys taxifolia, two cotyledons are usual, and the first two leaf primordiaarise at opposite ends of the shoot apex.A - D : An embryo with three cotyledons (c1, c2, c3), in a non-adjacent TS series.All scale bars = 50 pm.A : One cotyledon (c3) is smaller than the other two (c1 & c2).B : The three cotyledons (cl, c2, c3) are almost triangular where they meet abovethe shoot apex.C : The quiescent tip of the shoot apex (sa) is rounded between the threecotyledons. Crushed gametophyte tissue (arrow) fills the spaces between them. Thetwo larger cotyledons have two vascular bundles (vb). A single less distinct vascularbundle (arrowhead) is at the centre of the third cotyledon.D : At the base of the cotyledons, the shoot apex (sa) forms a triangle, with whatappears to be meristematic tissue at each corner (arrow).E : An embryo, fixed before germination, in TS. A leaf primordium (lp) has formedto one side of the shoot apex (sa). Also visible are secretory canals (sc) and vascularbundles (vb) in the cotyledons (cot). The surrounding female gametophyte (fg) wasdamaged during removal of the hard seed coat. Scale bar = 100 pm.F and G : The upper portion of an embryo within the female gametophyte (fg), inTS, fixed after germination of the root portion. sc = secretory canal; vb = vascularbundle. Scale bars = 100 pm.F : Two leaf primordia (lp) developing asynchronously at both ends of the shootapex (sa) between the cotyledons (cot).G : A section at the base of the shoot apex (sa) shows that the leaf primordia (lp)arise from opposite ends of the shoot apex, just inside the margins of the cotyledons(cot). Also visible are several meristematic zones (arrowheads) which will give rise tolater leaf primordia.H : Part of an embryo close to germination, in median LS parallel to the cotyledons(cot). Two leaf primordia (lp) are well advanced at opposite ends of the broad shootapex (sa). Numerous periclinally oriented mitotic cells (arrowheads) indicate thatelongation groMh has commenced in the cotyledon. Crushed remains of gametophyte tissue (arrow) overlie the shoot apex. Scale bar = 100 pm.

. - ..:-1P.-- -=sar::;Y ..t . ..-", . :,Af-- ', t .' -c1!-., .t . j ', ,' -\t '.at.t r...''/a':h czZ'':' '1"" ,;'Fr,^ l.-.li:l _ t.' *'E.:_l., )-l3Jbr{-)-_2?{) I1 1BFcDFig. 5.26

Figure 5.27: The dehiscence zone in the immature seed coat, and agerminating embryo, in Prumnopitys taxitolia.A : Part of the integument from the ovule containing the archegonia shown in Fig.5.3 A - E. The walls of the integument cells begin to thicken at this time (aroundfertilization), becoming progressively thicker until the cell lumen is almost completelyoccluded, making the mature seed coat extremely difficult to section. ln thisspecimen the cells are at an early stage of wall thickening, and this section at themicropylar'beak' shows that little or no thickening occurs in the elongated cells alongthe dehiscence zone (arrowheads). Also visible are tanniniferous cells (tc) at the freesurface of the micropylar'beak'. Scale bar - 50 pm.B : A germinating embryo (e)with shoot apex (sa) in median LS inside the femalegametophyte (fg), and emergent root (r) in oblique TS. The curve in the seedling axisis probably due to negative gravitropism in the root, but since this specimengerminated in a plastic bag in a refrigerator, its actual position at the time ofgermination was not certain. An endodermis (en) has already formed between thecortex (cx) and the vascular cylinder. Elongation growth is underway in thecotyledons (cot). Other sections showed that leaf primordia were developing. Alsovisible are isolated tanniniferous cells (tc) beneath the epidermis, secretory canals(sc) between the cortex and procambialtissue (pc), and vascular strands (arrows)passing into the cotyledons. Scale bar = 500 pm.

Fig. 5.27

Figure 5.28: Transition between hypocotyl and root anatomy in thegerminating embryo ol Prumnopitys taxitolia.A and B : A germinating embryo in two non-adjacent sections.Scale bars = 500 pm.A : Shoot apex of the embryo within the female gametophyte (fg), in obliquemedian LS parallel to the cotyledons (cot). The first two leaf primordia (lp1 & lp2) areelongating, and between them are new leaf primordia (arrowheads) on the growingshoot apex. Also visible are two vascular bundles (vb) in the cotyledon, secretorycanals (sc) in the hypocotyl (h), and remnants of the gynospore wall (gw).B : Part of the emergent axis of the embryo, in oblique TS at the point of downwardcurvature. In the right hand end, the anatomy is typical of the hypocotyl, with sixsecretory canals (1 - 6) surrounding two pale bands of fibres/phloem (f/p) (see alsoFig. 5.33 B). The left hand end shows a similar anatomy but with an endodermis (en)between cortex (cx) and vascular cylinder, which is typical of the primary root (seealso Fig. 5.33 C). Transition from hypocotylto primary root thus appears to occur atthe point where the emergent axis first turns downwards.

..; i.hrl .'lft'"-.*...,t_,'.rAB Fig. 5.28

Figure 5.29: In the germinating embryo/seedling of Prumnopitys taxifolia,stomata differentiate on both faces of the growing cotyledons.A to K : Part of an embryo/seedling exhumed during germination. The shoot apex,removed from the gametophyte with one cotyledon attached, is shown in median LS(A). The second cotyledon, which remained inside the gametophyte, is shown in TS(E).A to D : Shoot apex in a non-adjacent series of LS. The cotyledon (cot) is nowabout 1600 pm long, with stomata developing in the epidermis. One is visible in theadaxial epidermis (arrowhead) (A, see also H). The shoot apex (sa) now lies at thetip of a growing plumule (plu), bearing several leaf primordia on its flanks, andoverarched by the first two leaves (lf 1 & lf2). In the hypocotyl are two bands offibre/phloem cells (f/p) and a central zone of xylem cells (x) (spiral thickenings can beseen at higher magnification) (C, D). Scale bars = 100 pm.D, seen in TS within thefemale gametophyte (fg). The tissues include: epidermis (ep), stomata (st), isolatedtanniniferous hypodermal cells (tc), mesophyll (me), two vascular bundles comprisingxylem (x) and fibre/phloem (f/p), accompanied by secretory canals (sc). Scale bar =50 pm.E : Second cotyledon from the specimen shown in A -F, G and H : Three stages in development of guard cells, from the cotyledon shownin A. F: Daughter cells (arrowheads) resulting from transverse division of anepidermal cell. G: Vacuoles (v) form at both ends of each cell, and their adjoiningwalls appear to have thickened. H: Enlarged view of stoma visible in A (arrowhead inA). A pore or stoma has formed between the thickened walls of a pair ol guard cells(gc). Scale bars = 10 pm.J and K : Enlargement of two stomata in the abaxialface of the cotyledon shown inE. The guard cells have two zones of wallthickening (arrows). Also shown aresubsidiary cells (sub), epidermis (ep) with thick cuticle (c), tanniniferous hypodermalcell (tc) and mesophyll cells (me) with airspaces between, but no substomatalchambers. Scale bars = 10 um.

trffir.uli,ifl"-s[fri';i*i#=arT*i*;rF -i:l-- f*F $:Fig. 5.29

Figure 5.30 : Further tissue development during germination of theembryo/seedling ol Prumnopitys taxifolia.A to E : Part of an embryo/seedling exhumed during germination. The cotyledons,shoot apex and hypocotyl were sectioned in TS still inside the female gametophyte(fg) (A, B). The emergent root tip was cut off and sectioned separately in LS (C).A : The embryo in TS at the level of the shoot apex (sa), showing cotyledons (c1 &c2), first two leaves (lf1 & lf2), and several leaf primordia (arrowheads). Theepidermis (ep) and tanniniferous cells (tc) are readily visible. For details of thetissues see D and E. Scale bar = 100 pm.B : The hypocotyl of the embryo in TS just below the cotyledons, Tissues includeepidermis (ep), tanniniferous hypodermalcells (tc), cortex (cx) with cells resemblingmesophyll of cotyledons (in A and D), two central bands of fibre/phloem (f/p1), foursmaller fibre/phloem strands (f/p2) branching off to the cotyledons accompanied byfour secretory canals (sc), and additional secretory canals (arrowheads) which havebranched from central canals at a lower level. These will terminate beneath thegrowing plumule. Scale bar = 100 pm.C : The root tip of the germinated embryo/seedling, in a slightly oblique median LS,showing root cap (rt), quiescent centre at the root apex (ra), cortex (cx),undifferentiated vascular cylinder (vc) and the location of the differentiatingendodermis (en). Scale bar = 100 pm.D : Enlarged view of part of the cotyledon, from the same section as that in A.Tissues are: abaxial epidermis (ep1), tanniniferous hypodermal cells (tc), mesophyll(me) with starch grains (sg), secretory canal (sc), fibre cells (f), phloem cells (ph),xylem cells (x), adaxial epidermis (ep2). Part of a leaf primordium (lp) is also visible.Scale bar = 10 Fm.E : Enlarged view of a growing leaf primordium (lp) from the same section as thatshown in A. One mitotic cell (arrowhead) is visible in the epidermal layer. Alsovisible are the adaxial epidermis (ep2) and mesophyll (me) of the cotyledon.Scale bar = 10 pm.

""A.tf,JBIVCcxDcfaEit-t--r''''a?_, 1oi .t tr,r "j:'f.. rpl- tf t I tt.-Fig. 5.30

Figure 5.31: In the germinating embryo/seedling ol Prumnopitys taxitolia,elongation growth in the cotyledons pushes the shoot apex out ofthe gametophyte.A - E : Part of an embryo/seedling, in median LS. This seedling had a hairpinshapedhypocotyl at least 20 mm long, but was slightly younger than the specimenshown in Fig. 5.33 E. The sections shown in A, D and E form a non-adjacent seriesof the upper hypocotyl.A : The elongating cotyledons (cot) are about 3.75 mm long. Growth is concentratedin the basal tissues ({t) (see enlarged detail in C). The growing plumule (plu) hasmore than six leaves, and the shoot apex is almost outside the female gametophyte(fg). One of the first two leaves (lf 1) is visible overarching the plumule. Fibre/phloemstrands (f/p) and secretory canals (sc) branch into the cotyledons from the vascularcylinder in the hypocotyl (h). Another secretory canal (anow) passes into the base ofthe plumule. Also visible is the gynospore wall (gw). Scale bar = 500 pm.B : Tip of a cotyledon in LS. The xylem strand (x) is terminated by transfusiontissue (tt). The secretory canal (sc) ends at the level of the xylem. Other tissues areepidermis (ep) with a thick cuticle (c), and mesophyll (me). Scale bar = 50 pm.C : Tissues in the base of one cotyledon (cot), near a developing leaf (lf) on theplumule (at asterisk in A). In the mesophyll are cells at different stages of mitosis(arrowheads). Some dividing cells (arrows) are also present in the young leaf. Scalebar = 50 pm.D : Detail of the upper hypocotyl (h) in LS (section between A and E). The broadband of fibre/phloem (f/p) ends below the top of the hypocotyl. Two main strands(arrow) enter the cotyledons (c1 & c2), and smaller strands (arrowhead) continue intothe plumule (plu). Also visible are epidermis (ep), tanniniferous cells (tc), cortex (cx)and secretory canals (sc), and xylem strands (x) in the base of the plumule. Scalebar = 100 ptm.E : Another view of the hypocotyl (h) in LS (last in the series A, D & E). Xylemstrands (arrowheads) enter the cotyledons (c1 & c2) from the xylem tissue (x) at thecentre of the vascular cylinder in the hypocotyl. Also visible are epidermis (ep),tanniniferous cells (tc), cortex (cx) and fibre/phloem (f/p). Scale bar = 100 pm.

tf1t. ',l;-r_plutlpAFig. 5.31