J. Cell Sci. ia, 741-763 (1973) 741

Printed in Great Britain

CAPSELLA EMBRYOGENESIS: THE CENTRAL

CELL

PATRICIA SCHULZ

Department of Biology, Rosary College, River Forest, Illinois 603^5, U.S.A.

AND W. A. JENSENDepartment of Botany, University of California, Berkeley, California 94720, U.S.A.

SUMMARY

The central cell is the binucleate cell of the angiosperm megagametophyte which contains thepolar nuclei and participates in double fertilization. The structure of the mature central cell,the fusion of the polar nuclei and the primary endosperm nucleus were studied with the electronmicroscope. The central cell cytoplasm appears very active and has an extensive ER, manymitochondria, dictyosomes, microbodies, polysomes, chloroplasts with well developed granaand starch and lipid reserves. A single, giant mitochondrion appears in the cytoplasm near thepolar nuclei at the time of fertilization, but its origin, fate and function are not known. Cyto-plasmic aggregates of dense, granular material are associated with the primary endospermnucleus and structurally resemble the nucleolus and similar aggregates in the nucleoplasm. ftis suggested that these cytoplasmic perinuclear bodies may represent extruded nucleolarmaterial. The central cell cytoplasm does not undergo any notable structural reorganization asa result of fertilization. The relationship of the central cell to the other cells of the maturemegagametophyte and its possible role in embryogenesis is discussed.

INTRODUCTION

The central cell is the largest cell in the angiosperm megagametophyte. It is thebinucleate cell which remains after the egg, synergids and antipodals have formedfrom the 8-nucleate embryo sac. Its 2 polar nuclei fuse with one of the male nucleiduring the unique event of double fertilization to form a triploid endosperm tissuewhich envelops the developing embryo. Light-microscope studies of the central cellhave been known for some time and have focused primarily on the polar nuclei(Maheshwari, 1950). Results of recent electron-microscope studies reveal that it is avery interesting and highly active cell (Cocucci & Jensen, 1969 a, b; Diboll & Larson,1966; Diboll, 1968; Eyme, 1965; Godineau, 1966; Jensen, 1965; Mikulsa& Rodkiewicz,1967a, b; Plisko, 1971; Van Went, 1970a, b; Vazart, 1969; Vazart & Vazart, 1965,1966) but its role in embryogenesis is still unclear. In a comprehensive investigationof Capsella embryogenesis (Schulz & Jensen, 19680-*:, 1969, 1971) the structure ofthe mature central cell and nuclear events at the time of fertilization were studied.The purpose of this report is to shed more light on the relationship of the central cellto the other cells of the megagametophyte and on its functional role in embryogenesis.The early development of the free nuclear endosperm and the cellularization and laterdevelopment of the endosperm in Capsella will be treated in subsequent papers.

742 P. Schulz and W. A. Jensen

MATERIALS AND METHODS

Plants of Capsella bursa-pastoris (L.) Medic, the shepherd's purse, were grown in the green-house from seeds collected at the Botanical Garden, University of California, Berkeley. Dis-sected whole ovules were fixed in 6% glutaraldehyde (GA) buffered with either 006 M phos-phate at pH 6-8 or o-i M cacodylate at pH 72 for 4 h at 4 °C. The tissues were washed for 1 hwith changes of buffer and postfixed with 2 % unbuffered OsO4 containing 4 % sucrose for1S h at 4 °C. Dehydration was achieved through a graded acetone series. Ovules remainedovernight in 70% acetone containing 1 % uranyl nitrate. The material was embedded in Epon812 (Luft, 1961) and sectioned with a diamond knife. Sections were stained on grids with leadcitrate (Reynolds, 1963) for 1 min and observed with a Zeiss EM 9A electron microscope.

Material prepared for light microscopy was fixed in glutaraldehyde, embedded in Epon,sectioned at 1 •$ fim and stained with the periodic acid/Schiff (PAS) reaction for the localizationof insoluble carbohydrate (Jensen, 1962). Observations were made with a Zeiss light microscope.

RESULTS

General considerations

Capsella has a monosporic 8-nucleate, 7-celled curved megagametophyte (145 x25 /<m) of the Polygonum type (Henry, 1958) (Fig. 1). The central cell, often termedthe embryo sac, lies between the egg apparatus and the antipodals and containsthe 2 polar nuclei (Fig. 2) which rest next to one another just above the egg. It issurrounded by a cell wall and a plasma membrane. The wall is continuous except atthe chalazal end of the egg and synergids where it is irregular and, in some areas,entirely absent (Fig. 9). Plasmodesmata occur only in the common walls separating thecentral cell from the other cells of the megagametophyte (Schulz & Jensen, 1968a, b,1971). At the chalazal end of the embryo sac, in a limited area surrounding theantipodals, there is a common wall between the central cell and the crushed adjacentchalazal nucellus (Schulz & Jensen, 1971). A thin electron-dense line, presumed acuticle, separates the central cell from the cells of the inner integument which borderit laterally (Fig. 12). The central wall may be thickened in localized areas, commonlyat the micropylar and chalazal ends. These areas represent incompletely resorbedaccumulations of wall material from the destroyed nucellus (Henry, 1958). In themature megagametophyte there are no central cell wall projections like those reportedin flax (Vazart & Vazart, 1966), sunflower (Newcomb & Steeves, 1971), Californiapoppy (Negi, 1972) and Lobelia (Torosian, 1972). However, both micropylar andchalazal wall projections are produced in the central cell of Capsella during earlyembryo development (Schulz & Jensen, 1969, 1971). The central cell has a largecentral vacuole surrounded on 3 sides by a thin peripheral cytoplasm that contains feworganelles. There is a dense accumulation of cytoplasm that contains the polar nucleiand many organelles at the micropylar end near the egg apparatus (Fig. 1).

In this report nuclear phenomena will be discussed first followed by a considerationof the central cell cytoplasm.

The polar nuclei and the fusion nucleusDuring megagametogenesis the polar nuclei come, one from each pole, and rest,

one slightly above the other, near the developing egg and synergids (Fig. 2). They

Central cell of Capsella 743

are large (~8xi2/ tm), elongated and flattened on their facing surfaces. There isalways a space of cytoplasm, containing organelles, between the sister nuclei. Eachpolar nucleus contains a large, dense nucleolus (~7/<m) and usually one micro-nucleolus (~ i /im) (Lafontaine, 1965, 1968) which may indent the surface of thenucleolus or be completely separated from it (Fig. 2). The peripheral portion of thenucleolus consists of dense granules and fibrillar elements suspended in an amorphousmatrix (Figs. 2, 3). The granules (15-20 nm) are slightly smaller than cytoplasmicribosomes. The core of the nucleolus is predominantly fibrillar in nature but maybe invaded in some areas by the particulate elements of the periphery. The nucleolusalso contains several vacuoles of varying size. Small single-membrane vesicles are some-times seen in the nucleoplasm close to the nuclear membrane (Figs. 3, 4). The nuclearmembrane, containing many pores, is continuous with the ER in a few places. Theseconnexions increase greatly in number during the early development of the endosperm(Schulz & Jensen, unpublished).

Nuclear fusion begins shortly after the polar nuclei come together. Fusion at firstis slow but accelerates as the egg approaches maturity. The polar nuclei are usuallycompletely fused at the time of fertilization (Figs. 1, 6) although in some cases they arepartially fused. At the initiation of fusion the nuclear envelopes of both nuclei evaginateand make contact at several points along their facing surfaces (Fig. 3). At these contactpoints the outer nuclear membranes fuse (Fig. 4). This is followed by the fusion ofthe 2 inner nuclear membranes (Fig. 5). Thus nuclear bridges are formed which allowcontinuity of nucleoplasm. Pockets of cytoplasm containing organelles are trappedbetween these bridges (Fig. 3). The bridges widen by the apparent resorption ofnuclear membrane and squeeze out the trapped cytoplasm until they merge and nuclearfusion is completed. This process of nuclear fusion is similar to that observed in cotton(Jensen, 1964; Jensen & Fisher, 1967). The formation of the fusion nucleus (Fig. 6)is accompanied by the complete fusion of the 2 nucleoli. The elongated fusion nucleusis cupped over the egg apparatus but remains separated from it by a layer of cytoplasm.Nucleoplasmic vesicles and ER attachments are often seen (Fig. 6).

The primary endosperm nucleus

The details of the path of the male gamete from the degenerated synergid (Schulz &Jensen, 1968 a) to the fusion nucleus have not been seen in Capsella nor documentedat the electron-microscope level for any species (see Jensen, 1972). These events occurrapidly and are difficult to catch in fixed material. Although the fusion of the egg andsperm nuclei begins before the fusion of the sperm and fusion nuclei, this latter fusionis completed first and the primary endosperm nucleus divides almost immediatelyafter it is formed (Guignard, 1902). For this reason the primary endosperm nucleusis also a difficult stage to find.

The primary endosperm nucleus is more rounded than the fusion nucleus but isslightly flattened on the side facing the zygote (Fig. 7). It has a large, dense, vacuolatenucleolus composed of particulate and fibrillar elements suspended in a dense matrixin an arrangement similar to that of the polar nucleoli. A unique feature of the primaryendosperm nucleus is the presence of aggregates of dense material in the perinuclear

744 P- Schulz andW.A. Jensen

cytoplasm (Figs. 7, 8). These aggregates consist of granules (15-20 nm) embeddedin a dense matrix and strongly resemble the material in the periphery of the nucleolusand similar aggregates in the nucleoplasm. At present no evidence has been seen forthe transfer of these bodies from the nucleus to the cytoplasm. They are only observedin close association with the primary endosperm nucleus and their fate is not known.

The central cell cytoplasm

A striking feature of the central cell cytoplasm is the presence of numerous large(~ 1-1-5 x 2-5-3-5/mi), w e u developed chloroplasts (Fig. 10). These plastids aresharply contrasted with those of the egg, synergids, antipodals and integuments, whichare smaller and have poor internal organization (Schulz & Jensen, 1968a, b, 1971).Plastids are primarily concentrated in the region immediately surrounding the polarnuclei and the primary endosperm nucleus, although some are found in the lateraland chalazal cytoplasm. The internal membranes are organized into stacks of 3-5fused thylakoids. They also contain single, vesiculate lamellae and small vesicles whichoriginate from the inner membrane of the plastid envelope (Fig. 10) and are presumedto contribute to the development of grana and stroma lamellae. The chloroplast matrixis filled with ribosomes (Kislev, Swift & Bogorad, 1965) and contains osmiophilicdroplets (Figs. 10-12). Clear areas in the matrix are traversed by fine fibrils presumedto contain DNA (Kislev et al. 1965). The presence and amount of starch in theseplastids is variable and is probably dependent on physiological conditions at the timeof fixation (Figs. 7, 10, 12). Most chloroplasts are elongated and ellipsoidal but someare cup-shaped (Fig. 11) (Newcomb, 1967; Schulz & Jensen, 1968a, b, 1969). Thecytoplasm which fills the pocket in cup-shaped plastids usually contains one or twoshort, vesiculate pieces of rough ER and is less dense than neighbouring cytoplasm.The altered appearance of this cytoplasm suggests a possible biochemical interactionwith the chloroplast. Plastids with central constrictions are frequently seen and areinterpreted as dividing organelles (Fig. 12).

Numerous spherical or oval mitochondria (~ 0-8-1-2 x 1-2-1-9 /tm) with shortvesiculate cristae are randomly distributed in the central cell cytoplasm (Figs. 9, n ,12). The mitochondrial matrix contains ribosomes, intramitochondrial granules andfibrils presumed to contain DNA (Bisalputra & Bisalputra, 1967). Mitochondria par-titioned by a single continuous crista (Fig. 11) are interpreted as dividing organelles(Tandler, Erlandson, Smith & Wynder, 1969).

A single, remarkably large (2-6 /tm diam.) mitochondrion is sometimes seen in thecentral cell cytoplasm close to the polar nuclei in both fertilized and unfertilized ovules(Figs. 13, 14). This giant mitochondrion has short vesiculate or tubular cristae, intra-mitochondrial granules and ribosomes, and its matrix is packed with fibrils apparentlycontaining DNA. This organelle appears only at the time of fertilization and is notseen after the first division of the primary endosperm nucleus.

Long, single profiles of rough ER are scattered throughout the central cell cytoplasm(Figs. 6, 10). The ER frequently parallels the surface contours of the nucleus (Fig. 6)and various organelles, especially plastids (Fig. 10). As mentioned previously, ERconnexions with the nuclear envelope (Fig. 6) are infrequent but increase greatly in

Central cell of Capsella 745

number during the early development of the endosperm. Short, vesiculate pieces ofER closely parallel the plasma membrane where the central cell borders the egg(Fig. 9), synergids and integuments (Fig. 12). Crystals, similar in size and appearance(0-04 x 0-5-1-2 fim) to those observed in the egg, synergids and zygote (Schulz &Jensen, 1968a, b), occur in the ER cisternae (Fig. 15). The chemical nature of thesecrystals has not been determined.

The central cell cytoplasm is filled with ribosomes. These appear for the most partas polyribosomes both free and attached to the ER (Figs. 3-5, 8). No notable changesin the pattern and distribution of ribosomes have been observed from the formationof the fusion nucleus to the division of the primary endosperm nucleus.

Active dictyosomes with 3-6 cisternae (Figs. 3, 16) are randomly distributed and donot appear to have any particular orientation with respect to the cell wall. Microbodies(glyoxysomes) (Frederick, Newcomb, Vigil & Wergin, 1968) of varying size and shapeoccur throughout the cytoplasm (Figs. 15, 16) and are frequently seen in close associa-tion with lipid droplets (spherosomes) (Fig. 16). Ribosomes immediately adjacent tolipid droplets often stain more intensely and are arranged in straight chains perpen-dicular to the surface of the lipid droplet (Fig. 16).

In addition to the large central vacuole (Fig. 1) numerous small vacuoles occur inthe cytoplasm (Figs. 2, 3). Both large and small vacuoles are devoid of structuralcontents. Multivesicular bodies are of rare occurrence in the central cell.

DISCUSSION

This study further emphasizes the unique character of the cytoplasms of thedifferent cells of the mature megagametophyte (Schulz & Jensen, 1968a, b, 1971).The central cell, with its extensive ER, numerous and well developed chloroplasts,many mitochondria, dictyosomes, microbodies and polysomes appears to be engagedin intense metabolic activity. It stands in sharp contrast to the egg (Schulz & Jensen,19686) and antipodals (Schulz & Jensen, 1971), which have fewer organelles, mono-somes, little ER and practically no dictyosomes. In cotton (Jensen, 1965) a similarcontrast in the apparent metabolic conditions of the egg and central cell is attributedto the rapid enlargement of the central cell and the fact that the primary endospermnucleus divides almost immediately after fertilization, whereas the cotton zygote doesnot divide for 3-5 days during which time many organizational changes take place inthe cytoplasm.

In its high degree of metabolic activity the central cell resembles the synergids whichare thought to function in the absorption, metabolism and transport of nutrients fromthe adjacent integuments to the egg (Jensen, 1965; Schulz & Jensen, 1968a). Thesynergids are modified for absorption by the massive wall ingrowths of the filiformapparatus (see Gunning & Pate, 1969). The presence of well developed central cellwall projections in sunflower (Newcomb & Steeves, 1971), Lobelia (Torosian, 1972),California poppy (Negi, 1972) and flax (Vazart & Vazart, 1966) suggests that in thesespecies the central cell may also be modified for the absorption of large quantities ofnutrients from surrounding tissues. The absence of wall projections in the mature

48

746 P. Schulz and W. A.Jensen

megagametophyte of Capsella supports the conclusion that in this species the centralcell is probably not absorbing large amounts of nutrients directly from the integuments.The presence of many large, well developed chloroplasts suggests instead that auto-trophy may be an important factor in its nutrition.

Interruptions in the chalazal wall of the egg (Fig. 9) allow a freer exchange betweenthe egg and central cell and suggest the possibility that the central cell may also beinvolved in the nutrition of the egg. Although similar wall-less areas between thesynergids and the central cell (Schulz & Jensen, 1968a) facilitate gamete transfer(Linskens, 1968; Jensen, 1972) they may also reflect metabolic interaction. However,speculations of this kind, based on static electron images, need to be substantiatedwith biochemical studies.

In many species (Diboll, 1968; Diboll & Larson, 1966; Jensen, 1965; Torosian,1972; Vazart & Vazart, 1965) the central cell contains starch and lipid reserves which,in Capsella, are quickly utilized during the early development of the endosperm(Schulz & Jensen, unpublished). Certain classes of microbodies (glyoxysomes) containthe enzymes for /^-oxidation of fatty acids and the glyoxylate cycle which convertsthe acetyl CoA units to succinate. Succinate is then fed into mitochondria andultimately converted to sucrose by reverse glycolysis (Cooper & Beevers, 1969a, b;Canvin & Beevers, 1961). The intimate association of microbodies with lipid droplets(spherosomes) (Fig. 16) in the central cell of Capsella indicates the possible initiationof the conversion of stored fats to sugar to supply energy for subsequent development.Microbodies are also seen in close association with endosperm spherosomes which arebeing utilized during castor bean germination (Vigil, 1970).

' Megamitochondria' have been experimentally induced in animal cells by shock,injury and specific nutritional deficiencies (Tandler, Erlandson & Wynder, 1968;Tandler et al. 1969). These oversized mitochondria are thought to arise by directenlargement and/or the fusion of smaller organelles. Reversal of riboflavin deficiencyin mice by injection causes megamitochondria to return to normal size by partitioningand subsequent division of the giant organelles.

The appearance of a single, giant mitochondrion in the same place at the same timein apparently healthy ovules of Capsella seems to discount injury or nutritional defi-ciency as a cause. The presence of large amounts of fibrillar material suggests that itmay serve as a reservoir of mitochondrial DNA and possibly give rise to many normal-sized mitochondria during the early development of the endosperm. At present wehave no evidence to indicate the possible origin, function or disposition of this unusualorganelle.

In several species changes have been observed in the fine structure of the centralcell immediately following fertilization. There is a notable increase in ER and polysomeformation in orchid (Cocucci & Jensen, 1969 a, ft) and Petunia (Van Went, 1970 ft) andan increase in dictyosome activity in Petunia (Van Went, 19706) and corn (Diboll,1968). These changes are thought to reflect increased metabolic activity and theorganization of protein-synthesizing machinery for the rapid differentiation of theprimary endosperm cell (Van Went, 1970ft). In Capsella the central cell in the maturemegagametophyte is already quite active and does not undergo any notable structural

Central cell of Capsella 747

reorganization as a result of fertilization. It is premature to speculate on the reasons forthese differences. A great deal more information is needed on this subject to elucidatethe precise biochemical events associated with double fertilization in angiosperms.

Figs. 7 and 8 give evidence for the possible transfer of large masses of nucleolarmaterial from the primary endosperm nucleus to the cytoplasm. It is well known thatthe nucleolus is the site of synthesis of ribosomal RNA and that ribosome subunitsare probably transferred from the nucleus to the cytoplasm where they are organizedinto functional ribosomes (Perry, 1965, 1967; Rodgers, 1968). The extrusion of largemasses of nucleolar material is commonly observed in animal cells, especially devel-oping oocytes, and has been studied in detail at both the light- and electron-microscopelevels (see Raven, 1961; Hay, 1968; Scharrer & Wurzelmann, 1969). Nucleolarextrusion has also been reported in nurse tissue (Bentfeld, 1971) and from the pro-nuclei of the rat within a precise time period (4-5-11 h) after fertilization (Szollosi,1965). Reports of nucleolar extrusion in plant cells are brief and unclear (Hadek &Swift, 1962).

Various mechanisms have been postulated for the transfer of nucleolar material tothe cytoplasm. Most commonly it appears that dispersed nucleolar material passesthrough nuclear pores and reaggregates in the perinuclear cytoplasm (Hay, 1968;Scharrer & Wurzelmann, 1969; Kessel, 1966). Less frequently the material is envel-oped by a portion of the nuclear membrane and eventually pinched off into the cyto-plasm (Szollosi, 1965). Staining procedures (Szollosi, 1965) and incorporation studies(Bentfeld, 1971) show that extrusion bodies are similar in composition to the nucleolus.Most investigators (Hay, 1968; Scharrer & Wurzelmann, 1969; Bentfeld, 1971;Szollosi, 1965) agree that nucleolar extrusions probably represent the mass transportof ribosomal precursors and possibly other kinds of RNA to the cytoplasm. The fateof extrusion bodies is not known (Szollosi, 1965; Kessel & Beams, 1963). They areonly observed close to the nuclear membrane and are presumed to be transformed ordisappear subsequently.

The perinuclear aggregates associated with the primary endosperm nucleus inCapsella strongly resemble the material in the periphery of the nucleolus and similaraggregates in the nucleoplasm. These structures have not been seen in the egg, zygoteor synergids nor has their composition or possible mechanism of transfer from thenucleus been determined. In view of the fact that it is well known that nucleolarsubstances are continually being transported to the cytoplasm, it seems plausible toconsider the possibility that nucleolar extrusion occurs in the primary endospermnucleus. Furthermore, as a result of the fusion of the 2 polar nuclei and the malenucleus there is an enormous amount of nucleolar material available as well as ademand by rapidly growing central cell and endosperm for ribosomes and other kindsof RNA involved in protein synthesis. Further studies are needed to determine theorigin, composition and fate of these perinuclear bodies.

This research was supported by NSF Grant GB 3-1101.

748 P. Schulz and W. A. Jensen

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VAZART, B. & VAZART, J. (1965). Infrastructure de l'ovule de Lin, Linum usitatissimum L.: Lescellules du sac embryonnaire. C. r. hebd. Acad. Sci., Paris 261, 3447-3450.

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VAZART, B. & VAZART, J. (1966). Infrastructure du sac embryonnaire du Lin (Linum usitatisswtumL.). Rev. Cytol. Biol. ve'g. 24, 251-266.

VAZART, J. (1969). Organisation et ultrastructure du sac embryonnaire du Lin (Limim usitotis-simum L.). Rev. Cytol. Biol. vig. 24, 251-266.

VIGIL, E. L. (1970). Cytochemical and developmental changes in microbodies (glyoxysomes)and related organelles of castor bean endosperm.,?. Cell Biol. 46, 435-454.

{Received 25 August 1972)

Fig. 1. Phase micrograph of mature megagametophyte showing the egg (e), onesynergid (sy), the central cell (cc) and the fusion nucleus (Jn). The fusion nucleus liesin a dense accumulation of starch-laden cytoplasm at the micropylar end of the centralcell near the egg apparatus. PAS. x 1100.Fig. 2. The polar nuclei (pn) each have one large nucleolus (nu) and a smaller micro-nucleolus (mri). v, vacuole. GA-OsO4. x 9625.

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Fig. 3. Fusing polar nuclei (pn) showing cytoplasmic pockets between nuclear bridges.The nucleoli (mi) are cut through the periphery which contains many dense granules.d, dictyosome; v, vacuole. GA-OsOj. x 17500.Fig. 4. Enlargement of a detail in Fig. 3. The outer nuclear membranes of the polarnuclei are fused. Small single-membrane vesicles (vs) sometimes appear in thenucleoplasm near the nuclear membrane. Note nuclear pore (short arrow). GA-OsOj.x 61 750.Fig. 5. Nuclear bridge between fusing polar nuclei (pn). The outer and inner mem-branes of the 2 nuclei are continuous. GA-OsO4. x 61750.

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Fig. 6. Fusion nucleus with huge nucleolus. Note the long strands of ER (er) parallelingthe nuclear envelope, e, egg. GA-OsO4. x 12000. Inset: continuity of the ER with theouter nuclear membrane of the fusion nucleus. Note also the small vesicle (arrow) inthe nucleoplasm. GA-OsO4. x 18240.Fig. 7. The primary endosperm nucleus. Dense bodies (arrows) occur in the cyto-plasm near the nuclear envelope, p, starch-containing plastid; z, zygote. GA-OsO4.x 12000.

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Fig. 8. A portion of the primary endosperm nucleus showing the similarity betweenperipheral nucleolar material (nu) and dense aggregates in the nucleoplasm (arrow)and perinuclear cytoplasm (double arrow). GA-OsO4. x 40250. Inset: high magnifica-tion of perinuclear aggregate. GA-OsO4. x 65 520.

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Fig. 9. The central cell (cc) and the chalazal end of the egg (c) in an area where thecell wall is absent and the plasma membranes are closely apposed. Gaps, containingclumps cf dense material (dm) also occur between the 2 cells. Short, vesiculate piecesof ER parallel the plasma membranes (arrows). Note the difference in size and appear-ance of the mitochondria (w) in the 2 cells. GA-OsO4. x 62700.Fig. 10. Large, well developed chloroplasts of the central cell containing fused thyla-koids and single vesiculate lamellae and small vesicles which originate from theimagination of the inner membrane of the plastid envelope (arrow). Plastids alsocontain ribosomes, osmiophilic droplets and clear areas traversed by fine fibrilspresumed to contain DNA. Long, single strands of rough ER (er) often run parallel toplastids. GA-OsO.,. x 35750.

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Fig. I I . Cup-shaped plastid (p) and a dividing mitochondrion (in). The cytoplasmin the pocket of the plastid contains short, vesiculate pieces of rough ER and is lessdense than surrounding cytoplasm. GA-OsOj. x 22750.Fig. 12. A dividing plastid (p) containing starch, ribosomes, and many osmiophilicdroplets. Short, vesiculate pieces of ER (arrows) parallel the plasma membrane ofthe central cell ice). An electron-dense line (cuticle) separates the wall of the centralcell from the wall of the inner integument (in), m, mitochondrion. GA-OsO4. X 18900.Fig. 13. Giant mitochondrion in unfertilized ovule packed with tiny fibrils and con-taining short, vesiculate cristae, and intramitochondrial granules. GA-OsO^. X 43 700.

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Fig. 14. Giant mitochondrion in fertilized ovule showing vesiculate and tubular cristae,intramitochondrial granules, ribosomes and fibrillar material. Compare with neigh-bouring mitochondrion (m). GA-OsOj. x 36340.Fig. 15. Elongated crystals (cr) in ER cisternae. Mitochondria (w) have tubular cristaeand a matrix containing fine fibrils, mb, microbody. GA-OsOj. x 50350.Fig. 16. A microbody (mb) in close association with a lipid droplet (/). Straight chains ofribosomes are oriented perpendicular to the surface of the lipid droplet (arrows).d, dictyosome. GA-OsO4. x 36400.

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