Plant Ultrastructure in the Scanning Electron Microscope

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Scanning Electron Microscopy

Volume 1986 Number 1 Part I Article 30

3-8-1986

Susan H. Barnes British Museum

Stephen Blackmore British Museum

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Recommended Citation Barnes, Susan H. and Blackmore, Stephen (1986) "Plant Ultrastructure in the Scanning Electron Microscope," Scanning Electron Microscopy: Vol. 1986 : No. 1 , Article 30. Available at: https://digitalcommons.usu.edu/electron/vol1986/iss1/30

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PLANTULTRASTRUCTURE IN THE SCANNINGELECTRON MICROSCOPE

Susan H. Barnes * and Stephen Blackmore

British Museum (Na tura l History), Cromwell Road, London SW? SBD, England

(Received for publication December 30, 1985, and in revised form March 08, 1986)

Abstract Introduction

Preparative techniqu e s which have been us ed The combination of high resolution scanning to study internal details of plant cells in the ele ctron microscopy and new preparatory scanning elec tron microscope are reviewed . A techniques have provided fine structural number of me thods hav e previously been described information concerning the in t ernal features of which involve selective extraction of materials cells . He re we review the development of th ese from freeze-fractured s urfaces and can be techniques from a botanical perspective and referred to as freeze-fracture and cytoplasmic present new findings in the field of pollen maceration. One of these t echniques which ontogeny . involves an extended period of cytoplasmic The tough, secondary walls of some plant maceration with dilute osmium tetroxide has been ce l ls (e.g. wood, diatoms, pollen grains) lend applie d to th e study of Cichorium intybus themselves to observation when cytoplasmic (chicory) pollen ontogeny. The results obtained, contents are absent or have been removed. incl udin g changes in th e numbers of mitochondria However, if images of delicate membranous and the form of endoplasmic reticulum during th e components are needed mor e elaborate methods of co ur se of dev el opment demonstrate th e value of preparatlon must be employed. An early advance the approach in showing the three dimensional involved th e snapping of critical point dried arrangement of organelles and wall layers. The material to reveal the internal surfaces of cells findings emphasise that pollen grains with (Troughton and Donaldson, 1972; Kessel and Shih, similar mature morphologies may differ in th e 1974). details of their ontogeny. Epoxy r es ins have bee n used to give support and rigidity to cells of soft tissues. Wodzicki and Humphre ys (1973) introduced a method in which leaves were embedded in resin and fractured. In a study of developing peristome teeth of th e moss Funaria Jarvis (1975) dissolved the resin from blocks which had been sec tion ed for transmission electron microscopy. Chambers and Hamilton (1973) dissolved incompletely polymerised resin from cut surfaces of wheat nodes to reveal the internal details of transfer cell walls. Frozen specimens have ideal characteristics for fracturing. Low temperature scanning elec tr on microscopy has been used to examine frozen hydrated plant cells . Where necessary, moisture may be sublimed partially, to etch fractured surfaces or totally, to achieve permanent freeze-dried specimens (e.g. Read and Beckett, 1985). The absence of artefacts caused ~ words: Cichorium intybus, Compositae: by chemical treatm ent and critical point drying Lactuceae, echinolophate, exine, freeze-fracture is a significant advantage of this approach (see and cytoplasmic maceration, ontogeny, plant reviews by Echlin and Moreton, 1976 and Robards, ultrastructure, pollen, primexine, tapetum. 1984). To obtain a high er level of d e tail of *Address for correspondence: organelles and membrane systems more complex British Museum (Natural History), Cromwell Road, procedures involving chemical fixation are London SW? 5BD, England. needed. Haggis (1970) pioneered this approach Telephon e : 01 - 589 6323 ex t 348 and many developments followed. Lim (1971)

281 S.H. Barnes and S.Blackmore devised a method in which specimens too small to in aldehyde fixed root tips by maceration in handle were allowed to self-fracture when frozen dilute aldehyde followed by dilute osmium in 75% ethanol. Sybers and Ashraf (1973) tetroxide. As an alternative to fracturing, fractured specimens after dehydration to absolute Inoue and Osatake (1984) freeze - polished thin ethanol. This t echniq ue was adapted by Humphreys specimens to expose intracellular components et al. (1974) for a variety of plant and animal before maceration. This development has yet to be specimens. In their study of Lilium and Solanum eval uat ed with plant material but may well prove Whellan et al. (1974) freeze-fractured fixed, to be very valuable. rehydrated anthers in Freon 22. Although their Preparative techniques which involve micrographs show what may be disruption of some ex t rac tion of th e cytoplasmic matrix af t er cytoplasmic components, th e chromosomes appear freeze -fracturin g can be described und er th e well preserved. The work of Yamada e t al. general heading of "freeze-fracture and (1983a,b) and Osumi e t al. (1984) on th e fine cytoplasmic maceration." The ex tended maceration structure of isolated etioplas t prolamellar method (Barnes and Blackmore, 1984a) has recently bodies provides a recent demonstration of the been us ed to study pollen development in results obtainable by fracturing dehydrated Compositae (Blackmore and Barnes, 1985; Barnes material. and Blackmore, 1986) and Liliaceae (Dickinson and The method of Haggis et al. (1976) involved Sheldon, 1986). Her e we demonstrate th e cryoprotection and freeze-fracturing before applicabili t y of this approach by presenting new fixation. The use of a cryoprotectant enabled findings on pollen ontogeny in Cichorium fracturing to be carried out in an aqueous intyb us L. (Compositae : Lactuceae). medium. When the fragments were thaw ed in Many workers have used the transmiss ion fixative some mobile cytoplasmic components were electron microscope to st udy pollen wall ext racted from th e fractured surfaces, which was morphogenesis since the initial research of necessary to provide observable thr ee dimensional Heslop-Harrison (1962, 1963). Pollen grains have structure. This method was later named the diverse and elabora t e walls and th e processes by "freeze-fracture, thaw, fix" techniqu e (Haggis which th ey are deposited have now been documented and Phipps-Todd, 1977; Haggis and Bond, 1979). in a wide range of plants (see reviews by Heslop Maruyama (1983) studied chromosomes of Vicia faba Harrison, 1968; Knox, 1984; Blackmore and Crane, using a method involving thaw-fixat~after 1986). However, several fundamental questions freeze-fracturing of fresh unfixed root tips. including th e mechanism by which patterning is Excellent images of organelles and imparted to the developing wall have yet to be membranous systems of animal cells were obtained answered (Dickinson and Sheldon, 1986). The by Tanaka (1981) and Tanaka and Nagura (1981) study of sporogenesis in the scanning elec tron using their O-D-O method which introduced osmium microscope may provide a more comple t e tetroxide fixation in hypotonic buffer prior to understanding of th e processes involved by freeze-fracturing in dimethyl sulphoxide. revealing the spatial relationships between Extrac tion of mobile components was achieved by organelles and developing wall layers. treatment with a very dilute solution of osmium The echinolophate pollen of I:_ intybus has tetroxide in the same hypotonic buffer. When a characteris t ic pat t ern of spiny ridges. The applied to our studies of botanical materials we highly derived nature of this pattern and the found that this technique resulted in incomplete stratification of the wall are of taxonomic maceration of th e cytoplasmic matrix. However, significance (Tomb, 1975; Blackmore, 1981, 1982, successful results were achieved with onion root 1984). Our programme of research aims to tips by Nagura e t al. (1983). By greatly contrast the development of pollen morphology in extending the macerating period, from two days to closely related species. two weeks, we obtained improved extraction of the stroma in chloroplast membrane systems (Barnes Materials and Methods and Blackmore, 1984a). This adaptation of Tanaka's original method has proved s ucc essful Cichorium intyb us was cultivated at Chelsea when applied to a range of botanical specimens Physic Garden, London. Following th e procedure (Barnes and Blackmore, 1984b; Blackmore and described in our earlier work (Barnes and Barnes, 1986). Cytoskeletal components in Blackmore, 1984a,b ) truncat ed anthers at various mesophyll cells were revealed by another stages of maturity were fixed in 1% osmium adaptation of Tanaka's t echniq ue. Fresh, unfixed tetroxide in M/15 phosphate buffer pH 7.4 for 2- l eaves were freeze-fractured in liquid nitrogen 16 hours at room temperature. The fixed anthers without the use of a cryoprotectant, macerat ed were washed in buffer and treated with 15%, 30% for two days and then fixed in osmium tetroxide and 50% aqueous dime thyl sulphoxide (DMSO) for 30 (Blackmore et al., 1984; Barnes et al., 1985; minutes in each solution. They were then frozen Giordano et al., 1985). Other variations have on a liquid nitrogen cooled metal block and proved useful when applied to animal tissu es . freeze-fractured with a razor blade and hammer. Tanaka and Mitsushima ( 1984 ) studied mat erial Fragments were thawed in 50% DMSO, rinsed in that had been perf us ed with an aldehyde prior to buffer and transf erred to buffered 0 . 1% osmium osmication (A-O-D-0 method). In our experience tetro xide for 14 days at 4° C. The solution was aldehyde fixation of plant material r ende rs th e replenished regularly to avoid surface cytoplasmic matrix difficult to extract. contamination. The specimens were refixed in 1% However, Maruyama ( 1985) obtained adequate osmium tetroxid e for 1 hour, treat ed with 2% extraction to observe chromosome ultrastructure aqueous tannic acid for 16 hours and 1% buffered

282 Plant Ultrastructure in the SEM osmi um tetroxide for 1 hour to enhance electrical cond uc ti vi t y . They were dehydrated through a graded series of acetone solutions and critical point dried using carbon dio xide. Acetolysed pollen was prepared from herbarium ma t erial (Spain: Gardiner and Gardiner 1889, BM) following th e method described by Erd tman ( 1960 ). Whole pollen grains and sections cut us ing a freezing microtome were treated with 90% acetic anhydride and 10% concentra t ed s ulph ur ic acid for 5 minutes at 100° C. The pollen grains were washed, dehydrated in ace to ne and pipetted onto stubs. Specimens were sputter coated and examined in a Hit achi S800 field emission scanning elec tron microscope at an accelerating vol t age of 8 kV.

Results and Discussion

An acetolysed pollen grain of ~ intyb us (Fig. 1) shows the characteristic echinolophate morphology . In section (Fig. 2) the t wo main layers of the pollen wall are evident . The elaborate out er layer, the ectexine, develops from a polysaccharide primexine whereas the inner layer, the endexine, is deposited on a system of lamellations. After meiosis tetrads of micros pores surrounded by thick callose walls are formed. The callose wall isolates the microspores from the tapetal environment during the deposition of Fig. 1 . Acetolysed echinolophate pollen of C. primexine around the outside of the plasma intybus. bar 10 µm. membrane (Barnes and Blackmore, 1986). At the e nd of the tetrad period the callose is digested by enzymatic activity. At this stag e in C. intybus (Fig. 3) the microspore cytoplasm contains abundant endoplasmic reticulum in the form of flattened sheets and anastomosing tubes. Other organelles present include mitochondria, plastids, vesicles and dictyosomes. Bluntly conical spines can be seen on the surface of the primexine which is thickened and contains rounded dense bodies (Fig. 4). Lenticular struc tur es, termed onci, serve to interrupt endexine deposition forming the endoapertures (Barnes and Blackmore, 1986). Primexine is produced by the microspores which are the gametophyte generation; subsequent components of the exine are mainly produced by the sporophytic tapetum. For example, the spines become acute by addition of tapetally derived sporopollenin as Heslop Harrison (1968) described in Cosmos bipinnatus. As the callose recedes further the tapetal cells become highly vacuolate and engulf the microspores (Figs . 5,7). The branching, tubular endoplas mi c re ticulum of the microspore cytoplasm is of a very different nature to th e ban ks of rough endo plasmic reticulum present in the invasive tap e tum. Where th e t ape tum and developing wall are in close proximity an in t erac ti on in th e form of granular strands can be seen (Fig. 6). These strands may be sporopollenin precursors produced by the tapetum which will later polymerise within the primexine Fig . 2 . Freezing microtome sec ti on of ace t oly s ed t o form the mature wall. The specific sites a t exine showing t he endexine (en) an d th e ec texin e which sporopollenin precursors accu mul a t e are comprising tectum ( t e), col umellae (c) and foot difficult to detect unt il considerable layer (arrow). bar 1 µm.

283 S.H. Barnes and S. Blackmore

Fig. 3. Late tetrad stage microspore showing Fig. 5. Mic rospore surrounded by highly primexine (p), oncus (o) and degenerating callose vacuolate invasive tapetum. bar lOµm. wall (ca). bar 1 µm.

Fig. 4. Detail of Fig. 3. showing callose wall Fig. 6. Detail of Fig. 5. showing rough (ca), primexine (p) with dense bodies (db), endoplasmic reticulum (rer) and granular strands plasma membrane (arrow) and endoplasmic reticulum between primexine and tapetum (arrows). bar lµm. (er). bar lµm.

284 Plant Ultrastructure in th e SEM sporopollenin has polymeris ed . In th e TEM th e primexine becomes darker in areas where sporopollenin precursors are deposited, whilst in the SEM these areas are apparent as textural variations, the denser bodies corresponding to the darkly staining regions. These dense bodies are occasionally seen to be continuous with a thin homogeneous layer at the base of the primexine (Figs. 6,7). Whether this layer represents the onset of en dexine formation or the start of the ectexinous foot layer remains to be established. It is probably endexine although none of the characteristic lamellations are yet evident. Alternatively, th e deposition of the foot layer at this stage would be significantly different to the interpretation of Horner and Pearson (1978) of events in Helianthus annuus (Compositae: Heliantheae). At a slightly later stage (Figs. 8,10) th e tap e tum loses its cellular integrity forming a periplasmodium. The microspore cytoplasm contains many small vesicles near the central nucleus and an abundance of anastomosing endoplasmic r etic u l um. Towards the periphery of the cytoplasm the nature of the endoplasmic reticulum alters to a convoluted system of curved sheets. These sheets are often looped concentrically, t ermina ting at th e base of th e prim exi ne (Fig. 8). At th e onci th e en doplasmic reticulum is eq ually abundant, however, the looped arrangement is less evident (Figs. 9,10). Fig. 7. Detail of Fig. 5, showing continuity As the endexine is deposited and th e between dense bodi es in the primexine and a thin primexine differentiates into ec t exine the outer dense layer (arrows). The microspor e cytoplasm surface, th e tectum, displays an unusual feature contains dictyosomes (d), plastids (p), vesicles not present in matur e pollen. Each (ve) and tubular endoplasmic retic ulum (er). microperforation is encircled by a raised annulus bar lµm. (Fig. 11 ), this character has not previously been encoun t ered in oth e r Lactuceae we have studied. This emphasises that features with similar matur e morphologies may differ in the details of their ontogeny. Later, the micros pores pass through a distinctive vacuolate stage. At th e onset of this per iod (Figs. 12,13) vacuoles begin to form, the quantity of endoplasmic reticulum decreases whereas the number of mitochondria increases considerably. The thick endexine and spongy, columellate ec t exine now have their mature morphology and the t ectal surface displays th e microperforations t ypical of th e Lactuceae. The tapetum is highl y organised, still containing extensive sheets of rough endoplasmic reticulum. The main phase of exine deposition has now ended and the function of th e tapetum in producing sporopollenin precursors is almost complete. Subsequently, the tapetum begins to degenerate (Fig. 14) and will eventually break down fully to form pollenkitt, the oily surface coating of mature pollen. The vacuoles enlarge and coalesce occupying most of the volume of the cell and displacing the organelles to th e periphery of the cytoplasm (Fig. 15). Before th e anthers dehisce the nucleus will divide mitotically and a final pecto-cellulosic wall layer, the intine, will be formed. These initial findings reveal certain Fig . 8. The endoplasmic reticulum of the differences between Cichorium intybus pollen microspore becomes curved and sheet-like towards ontogeny and that of other Compositae. The the periphery (arrows). bar 1 µm.

285 S.H. Barnes and S. Blackmore

Fig. 9. Microspore showing gradation in Fig. 11. Detail of developing exine with thick endoplasmic reticulum from a tubular system near endexine; each tectal perforation is surrounded the nucleus (n) to folded sheets at the by an annulus (arrows) . bar 1 µm. periphery. bar l0µm.

Fig. 10. Detail of Fig. 9. Showing an oncus (o) Fig. 12. The start of the vacuolate microspore and associated convolutions of smooth endoplasmic stage. Tapetum (t), vacuoles (v), oncus (o). reticulum (arrows). bar 1 µm. bar 10 µm.

286 Plant Ultrastructure in the SEM

Fig. 13. Detail of Fig. 12, the exi ne is Fig. 15. Detail of Fig. 14 showing endexine matur e, and the tapetum contains banks of rough (en), foot layer (arrows), vacuole (v), and endoplasmic reticulum (rer). bar 1 µm. displaced cy toplasm containing numerous mitochondria (m). bar 1 µm.

ann ular ornamentation of microperforations and the number and activity of th e dense bodies within the primexine will require further el ucidation in the course of our continuing comparative investigations.

Conclusions

The ability to observe the dynamic and complex processes of pollen development serves to illustrate the value of this approach . Changes in cytoplasmic organelles, tapetal activity and differentiating primexine can be clearly seen facilitating interpretations of functional organisation. A combination of this me thod and conventional transmission electron microscopy, which has the additional advantage of differentially staining wall layers, will provide valuable information and therefore assist in the study of comparative pollen ontogeny.

References

Barnes SH, Blackmore S. (1984a). Scanning electron microscopy of chloroplas t Fig. 14. At the late vacuolate microspore stage ult rastructure . Micron and Microscopica Acta th e tapetum begins to degenerate. bar 10 µm. 15:187-194.

287 S.H. Barnes and S. Blackmore

Barnes SH, Blackmore S. (1984b). Freeze Haggis GH, Bond EF. (1979). Three dimensional fracture and cytoplasmic maceration in botanical view of the chromatin in freeze-fractured chicken scanning electron microscopy. J. Microsc. erythrocyte nuclei. J. Microsc. 115:225-234. 136:RP3-4. Heslop-Harrison J. (1962). Origin of exine. Barnes SH, Blackmore S, Claugher D. (1985). Nature 195:1069-1071. Scanning electron microscopy of plant Heslop-Harrison J. (1963). Ultrastructural ultrastructure. Hitachi Instrument News 17:4-7. aspects of differentiation in sporogenous Barnes SH, Blackmore S. (1986). Some tissues. Symp. Soc. exp. Biol. 25:277-300. functional features during pollen development. Heslop-Harrison J. (1968). The pollen grain In: S. Blackmore & I.K. Ferguson (eds), Pollen wall. Science 161:230-237. and Spores: Form and Function. Academic Press, Horner HT, Pearson CB. (1978). Pollen wall London. In press. and aperture development in Helianthus annuus Blackmore S. (1981). Palynology and (Compositae: Heliantheae). Amer. J. Bot. intergeneric relationships in subtribe 65:293-309. Hyoseridinae (Compositae: Lactuceae). Bot. J. Humphreys WJ, Spurlock BO, Johnson JS. Linn. Soc. 82:1-13. (1974). Critical point drying of ethanol Blackmore S. (1982). Palynology of subtribe infiltrated cryofractured biological specimens Scorzonerinae (Compositae: Lactuceae). Grana for scanning electron microscopy. Scanning 21:149-160. Electron Microsc. 1974: 275-282. Blackmore S. (1984). The Northwest European Inoue T, Osatake H. (1984). Freeze-polishing Pollen Flora, 32. Lactuceae. Rev. Palaeobot. method for observing intracellular structures by Palynol. 42:45-85. scanning electron microscopy. J. Electron Blackmore S, Barnes SH, Claugher D. (1984). Microsc. 33:356-362. Scanning electron microscopy of cytoskeletal Jarvis LR. (1975). A simple method for components in Aucuba japonica leaves. J. exposing plastic embedded structures for scanning Ultrastruct. Res. 86:215-219. electron microscopy. J. Microsc. 105:115-117. Blackmore S, Barnes SH. (1985). Cosmos pollen Kessel RG, Shih CY. (1974). Scanning Electron ontogeny: a scanning electron microscope study. Microscopy in Biology - A Students Atlas on Protoplasma 126:91-99. Biological Organisation. Springer-Verlag, Blackmore S, Barnes SH. (1986). Freeze Berlin. fracture and cytoplasmic maceration of pollen Knox RB. (1984). The pollen grain. In:B.M. grains. Grana, in press. Johri (ed.), Embryology of Angiosperms. Blackmore S, Crane PR. (1986). The Springer-Verlag, Berlin. 197-271. systematic implications of pollen and spore Lim DJ. (1971). Scanning electron microscopic ontogeny. In: C.J. Humphries (ed.), Ontogeny observations on non-mechanically cryofractured and Systematics. Columbia University Press, New biological tissue. Scanning Electron Microsc. York. In press. 1971: 259-264. Chambers TC, Hamilton CD. (1973). Scanning Maruyama K. (1983 ) . Stereoscopic scanning electron microscopy of transfer cells: a new electron microscopy of the chromosomes in Vicia method for preparation of plant tissues. J. faba (Broad beans). J. Ultrastruct. Res. 82:322- Microsc. 99:65-68. 326. Dickinson HG, Sheldon JM. (1986). The Maruyama K. (1985). Dilute aldehyde treatment generation of patterning at the plasma membrane on aldehyde-fixed cells for observing chromosomes of the young microspore in Lilium. In: S. in situ with the scanning electron microscope. Blackmore & I.K. Ferguson (e~Pollen and J. Microsc. 139:265-269. Spores: Form and Function. Academic Press, Naguro T, Murai S, Takeuchi A, Iino A. (1983). London. In press. Internal cell structures of onion root tip cells Echlin P, Moreton R. (1976). Low temperature (a scanning electron microscopic study using the techniques for scanning electron microscopy. Q-D-0 method). J. Electron Microsc. 32,227. Scanning Electron Microsc. 1976; 1:753-762. Osumi M, Yamada N, Nagano M, Murakami S, Baba Erdtman G. (1960). The acetolysis method, a N, Oho E, Kanaya K. (1984). Three dimensional revised description. Svensk Bot. Tidskr. 54:561- observations of the prolamellar bodies in 564. etioplasts of squash Cucurbita moscha ta. Giordano S, Basile A, Salerno P, Castaldo Scanning Electron Microsc. 1984; I:111-119. Cobianchi R. (1985). Cytoskeletal elements in Read ND, Beckett A. (1985). The anatomy of the the liverworts Lunnularia cruciata and mature perithecium in Sordaria humana and its Conocephalum conicum revealed by SEM. Giorn. significance for fungal ----iiiulticellular Bot. Ital. 119:33 (abstract). development. Can. J. Bot. 63:281-296. Haggis GH. (1970). Cryofracture of biological Robards AW, (1984). Fact or artefact - a cool material. Scanning Electron Microsc. 1970: 99- look at biological electron microscopy. Proc. 104. Roy. Microsc. Soc. 19:195-208. Haggis GH, Bond EF, Phipps B. (1976). Sybers HD, Ashraf M. (1973). 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288 Plant Ultrastructure in th e SEM

Tanaka K, Nagura T. (1981) . High r esol ution T. Ino ue: Now we perform the cytoplasmic scanning electron microscopy of cell organelles maceration using 0 . 1% osmium tetroxide at 20°c in by a new specimen preparation method. Biomed. our laboratory. This can shorten your specimen Res. 2:63-70 . preparation time . Tanaka K, Mitsushima A. (1984). A preparation Authors: We will try this interesting suggestion method for observing intracellular structures by with our specimens . scanning electron microscopy. J. Microsc . 133 :213 - 222. Tomb AS. (1975). Pollen morphology in tribe Lactuceae (Compositae). Grana 15:79-89. Troughton J, Donaldson LA. ( 1972 ) . Probing Plant Structure. Chapman and Hall, London. Whellan EDP, Haggis GH, Ford EJ , Dronzek B. (1974). Scanning electron microscopy of meiotic chromosomes of plants in situ. Can. J. Bot. 52,1438-1440. Wodzicki TJ, Humphreys WJ. (1973). Fracturing plastic embedded plant material for scanning electron microscopy. Micron 4:1-9. Yamada N, Nagano M, Murakami S, Ikeuchi M, Oho E, Baba N, Kanaya K, Osumi M. (1983a). Preparation for observation of fine structure of biological specimens by high resolution SEM. J. Electron Microsc. 32:321-330. Yamada N, Seki M, Osumi M, Baba N, Oho E, Kanaya K, Murakami S. (1983b). Analysis of three dimensional structure from scanning electron microscopy. J. Elec tr on Microsc. 32,279 (Abstract).

Discussion With Reviewers

J.R. Rowley: Do the "dense bodies" in the primexine form from evaginations of the plasma membrane as Fig . 4 suggests? T. Inoue: What do you think the granular substances on the fractured surfaces of the dense bodies (Fig. 4) and oncus (Fig . 10) are? Authors: The highly granular material of the oncus and early primexine is mostly polysaccharidic. We consider the less gran ular dense bodies form by the accumulation of sporopollenin precursors within the primexinous glycocalyx and not from plasma membrane evagina t ions .

J.R. Rowley: It has been reported that pollen grains become located in the vac uome of plasmodial tap etal systems. What have your results shown regarding the relationship between tap etal cells and microspores? Authors: Our results show that the microspores become intimately surrounded by tap e tal cells which often have a discontinuous plasma membrane.

T. Ino ue: What is the approximate thickness of the coated metal and what kind of metal and instrument did you use? Authors: The specimens were coated with gold to an approximate thickness of 10-15 nm using a Polaron Equipment Limited E5000 sputter coater for 90 sat 2 kV.

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