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 Title Page
 Copyright
 Acknowledgement
 Table of Contents
 List of Figures
 Introduction
 Materials and methods
 Observations
 Interpretation and discussion
 Summary
 Literature Cited
 Biographical sketch
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Title: electron microscope study of megaspore development in Dendrobium orchids.
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Title: electron microscope study of megaspore development in Dendrobium orchids.
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Table of Contents
    Title Page
        Page i
    Copyright
        Page ii
    Acknowledgement
        Page iii
        Page iv
    Table of Contents
        Page v
        Page vi
    List of Figures
        Page vii
        Page viii
        Page ix
        Page x
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
    Materials and methods
        Page 6
        Page 7
        Page 8
        Page 9
    Observations
        Page 10
        Page 11
        Page 12
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    Interpretation and discussion
        Page 130
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    Summary
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    Literature Cited
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    Biographical sketch
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    Copyright
        Copyright
Full Text
AN ELECTRON MICROSCOPE STUDY OF MEGASPORE DEVELOPMENT IN DENDROBIUM ORCHIDS
By
HERBERT WILLIAM ISRAEL
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA December, 1962


Copyright by Herbert William Israel 1962


ACKNOWLEDGMENTS
It is with sincere gratitude that the author acknowledges the patient support, encouragement, and review rendered by his advisory committee chairman, Dr. Yoneo Sagawa, without whose continued selfless assistance this study would have been quite improbable.
To the members of his advisory committee, Drs. R. H. Biggs, A. D. Conger, J. R. Edwardson, J. H. Gregg, and G. R. Noggle, the author extends his gratitude for the advice and criticism afforded.
The financial support provided by the United States Department of Health, Education, and Welfare in the form of a National Defense Education Act-Title IV Graduate Fellowship, and by the General Biological Supply House, Inc., Chicago, Illinois, in the form of the Turtox Scholarship for 1962-1963 is herewith gratefully acknowledged.
The writer wishes to thank Jones and Scully, Inc., Miami, Florida, and Dale Shaffer Orchids, Miami Beach, Florida, for the plant materials used in this study.
To the Department of Anatomy, School of Medicine, J. Hillis Miller Health Center, the University of Florida, the


author offers his gratitude for the use of their RCA EMU-3C electron microscope.
Finally, the writer extends warmest thanks to his wife and children whose tolerance, understanding, and devotion were required extensively throughout the duration of this work.


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS................... iii
LIST OF FIGURES................... vii
INTRODUCTION..................... 1
MATERIALS AND METHODS................ 6
Description of Plants............... 6
Description of Microscopy............. 7
OBSERVATIONS..................... 10
Background.................... 10
Quiescent Phase.................. 11
Proliferative Phase................ 13
Nucellar Phase .................. 15
Sporogenic Phase ................. 17
Prophase..................... 18
Metaphase.................... 22
INTERPRETATION AND DISCUSSION ............ 130
Cell Wall..................... 131
Cell Membrane................... 133
Ribosomes..................... 137
Endoplasmic Reticulum............... 139
Golgi-Apparatus.................. 140
Mitochondria ................... 14 2
Plastids..................... 145
Phragmosomes ................... 146
Fat Droplets................... 148
Vacuoles..................... 151
The Nucleus.................... 153
Nuclear Envelope ................. 154
Nucleolus..................... 161
Chromatin..................... 162
Overview of Development.............. 163


SUMMARY.......................171
LITERATURE CITED...................!75
BIOGRAPHICAL SKETCH ................. 189


LIST OF FIGURES
Figure Page
1. Flowering raceme of Dendrobium XJaquelyn
Thomas................... 25
2. External post-pollination ovary enlargement. 25
3. Internal post-pollination ovary enlargement. 25
4. Longitudinal section of an ovary in the late
proliferative stage............. 27
5. Late proliferative papillae.......... 27
6. Tip of a long, narrow quiescent placental
papilla................... 29
7. Tip of a proliferative placental papilla ... 29
8. Tip of a nucellar placental papilla...... 31
9. Tip of a sporogenic placental papilla..... 31
10. Tip of a prophase placental papilla...... 33
11. Tip of a metaphase placental papilla..... 33
12. Peripheral cell of a quiescent papilla .... 35
13. Portions of four cells in a quiescent
papilla................... 37
14. Portions of three quiescent papilla cells. . 37
15. Peripheral cell of a quiescent papilla .... 39
16. Internal cell of a quiescent papilla apex. 41
17. Wall between two cells of a quiescent papilla. 43
18. Oblique section of a dividing cell in a
proliferative papilla............ 45


19. Oblique section of a telophase mitotic figure
in a proliferative papilla......... 47
20. Developing cell plate between two newly
divided cells of a proliferative papilla 49
21. Portion of a cell in a proliferative papilla 51
22. Portion of a nucleus in a proliferative
papilla cell................ 53
23. Interphase nucleus in a cell of a prolifera-
tive papilla................ 55
24. Lipid droplets in a basal cell of a prolifera-
tive papilla................ 57
25. Lipid droplets in a basal cell of a prolifera-
tive papilla................ 57
26. Cell in the nucellar row of a nucellar
papilla................... 59
27. Cell in the nucellar row of a nucellar
papilla................... 61
28. Portion of the nucleus in a nucellar-row cell
of a nucellar papilla............ 63
29. Portions of three basal cells in a nucellar
papilla................... 65
30. "Secreting" plastids in a basal cell of a
nucellar papilla .............. 67
31. Volume relationships in developing Dendrobium
ovules................... 69
32. Diagram of modifications in bilaminar
systems................... 71
33. Apical cell in the nucellar row of an early
sporogenic papilla ............. 73


34. Portion of an enlarging apical cell in the
nucellar row of a sporogenic papilla .... 75
35. Enlarging apical cell in the nucellar row
of a sporogenic papilla........... 77
36. Tip of a sporogenic papilla.......... 79
37. Apical portion of an enlarging megaspore
mother cell in a late sporogenic papilla 81
38. End portions of two basal cells in a sporo-
genic papilla................ 83
39. Early-prophase megaspore mother cell ..... 85
40. Nucleolus of a prophase megaspore mother cell. 87
41. Perinuclear portion of a mid-prophase mega-
spore mother cell.............. 89
42. Perinuclear portion of a mid-prophase mega-
spore mother cell.............. 91
43. Perinuclear portion of a mid-prophase mega-
spore mother cell.............. 93
44. Mid-prophase papillar tip........... 95
45. Late-prophase papillar tip .......... 97
46. Portion of a late-prophase megaspore mother
cell.................... 99
47. Portion of a late-prophase megaspore mother
cell....................101
48. Apical portion of a late-prophase papillar tip 103
49. Late-prophase papillar tip .......... 105
50. Mitotic metaphase figure in a proliferating
primary integument ............. 107


51. Organelle formation at the cell membrane in
a proliferating primary integument .... 109
52. Basal (funicular) cell of a late-prophase
papilla.................. Ill
53. Late-prophase funicular cells........ 113
54. Primary integument cells of the late-
prophase papilla............. 115
55. Early-raetaphase papillar tip ........ 117
56. Early-raetaphase megaspore mother cell. ... 119
57. Early-metaphase megaspore mother cell. 121
58. Perinuclear portion of an early-metaphase
megaspore mother cell........... 123
59. Mid-metaphase megaspore mother cell..... 125
60. Peripheral cell of the secondary integument
in a metaphase papilla.......... 127
61. Portion of a peripheral cell in the second-
ary integument of a metaphase papilla. 129
62. Portion of the bilaminar envelope of a pro-
plastid in a secondary integument cell
of a metaphase papilla.......... 129


INTRODUCTION
Robert Brown (1831), in the concluding remarks of the major portion of his paper concerning the organs and mechanism of fertilization in the milkweed and orchid families, states, "I even venture to add, that in investigating the obscure subject of generation, additional light is perhaps more likely to be derived from a further minute and patient examination of the structure and action of sexual organs in Asclepiadeae and Orchideae, than from any other department either of the vegetable or animal kingdom." That this challenge by one of modern biology's earliest and most eminent scholars has been virtually buried in the subsequent one hundred and thirty years of scientific probing is one of many surprising facts in the history of science.
Of the many concise morphogenetic accounts presented in his paper, one bears particular consideration; namely, that Brown recognized and described the major difference between the two groups of orchids (Monandrae and Diandrae) with respect to the ontogenetic status of the ovarian placenta at anthesis. He precisely related that in some orchids the ovules were not nearly ready for fertilization even


after the pollen tubes penetrated the cavity of the ovary. He also noted that the undifferentiated ovule "consists of a minute papilla projecting from the pulpy surface of the placenta"; and that pollination initiated a developmental sequence in the ovules, which he accurately described.
The initiation of ovule development by pollination or the so-called "post-pollination phenomenon" is unique to the more highly modified and commercially desirable members of the family Orchidaceae. Except for occasional studies (Guignard, 1886; Massart, 1902; Heusser, 1914; Afzelius, 1916; Schnarf, 1929; Laibach, 1932; Sesha-giriah, 1941; Duncan and Curtis, 1942a,b; Hagerup, 1944; Swamy, 1949a,b; Maheshwari, 1950; Hsiang, 1951; Magli, 1958; Poddubnaya-Arnoldi, 1960a,b; Jones, 1961; Niimoto and Sagawa, 1961,1962) treating the gross aspects of both the initiation mechanism and the timing of the morphogenetic sequence, which varies between four days and ten months among the various orchids, little work approximating a "minute and patient examination" of post-pollination mega-sporogenesis in orchids has been forthcoming.
The complex processes centered around development may be approached at many levels and from many avenues. The


analysis of raegasporogenesis in orchids has been attempted solely by way of the techniques of light microscopy in their most basic forms. Owing to the small size of the cells (2 5p) little beyond the nuclear events and some rather striking modifications in cell size, shape, and position have been observed. Data are being presented which substantiate the view that cytoplasm plays a major role in cytodifferentiation (Briggs and King, 1957; Kaufmann and Gay, 1958; Rudnick, 1958; Van Fleet, 1959; Fischberg and Blackler, 1961; Stone, 1962).
The electron microscope with its inherent hundredfold increase in resolution provides a fresh approach to the subject. The hazardous interpretations encountered a decade ago are rapidly dissipating owing to the intimate and relevant correlations between the findings of electron microscopy and those of other approaches to the field of cellular ultra-structure (Epstein, 1959; Mercer, 1960; Schmidt, 1960). A review of the accumulating literature in the field reveals that comparatively little has been produced in the area of developmental biology. Several reasons for the dearth may be proposed: (1) Complete familiarity with the light microscopic aspects of the developmental sequence is necessary. (2) The sequence must occur in sufficient quantity; prefer-


ably in a synchronized system. (3) The sequence must be readily adaptable to the rigors of current electron microscopic techniques; that is, radically isolated in time and space, amenable to severe preparatory manipulations, and convincingly reproducible. The growth and differentiation of the female ovum meets many of the above criteria; and it is, consequently, providing fertile ground for recent developmental studies (Kessel and Kemp, 1962).
A review of the leading journals reveals that in the botanical fields the number of electron microscopic studies has been very low. Research on megasporogenesis and oogenesis in plants is virtually nonexistent. In a series of papers, Camefort (1959a,b; 1960; 1961a,b) has reported on the morphogenesis of the oosphere in the pine, Pinus lari-cio. Jensen (1962) has recently described the mature cotton megagametophyte with little reference to its development. On the family Orchidaceae, only a brief paper (Chardard, 1959) dealing with chromosome structure has been found.
The foregoing is particularly disconcerting in that, despite Brown's remarks in 1831, the special advantages of developing orchid ovules as a source of material for an intensive fine-structural analysis of development have not been utilized. The tissue layers are markedly thin, thus


permitting rapid light microscopic analysis by the smear technique. The sequence of development can be initiated by pollination, and is rigidly paced (Duncan, 1959). The number of developing ovules within a given ovary may approach one million (Correll, 1950), while individual developmental sequences among the ovules remain highly synchronized (Duncan, 1959). The tissue is easily prepared for electron microscopy. The ovules are small several cells in diameter and protrude conveniently into the ovarian cavity. The developmental modifications are readily observable and sequential in practically all immediately adjacent tissues, thus easing the burdens of interpretation. In order to restrict the time dimension, it was found expedient to limit the current study to the early phases of megasporo-genesis; commencing with the undifferentiated ovule prior to pollination, and concluding with the events of meiotic metaphase I.
On the basis of the foregoing remarks, and with a desire to contribute both to the understanding of plant cytoarchitecture and to a broad program at the University of Florida examining cellular proliferation, growth, and differentiation, the present investigation was undertaken.


MATERIALS AND METHODS
Description of Plants Plants of the genus Dendrobium were utilized in this study because of their ready availability; their continuous, copious production of blossoms; and because the basic investigations pertaining to timing, cellular modifications, and nuclear events had previously been pursued (Niimoto and Sagawa, 1961). Being self-incompatible, five of the plants (D. XJaquelyn Thomas, UF #1171; D. XJaquelyn Thomas dark, UF #1172; D. XAlice Spaulding X D. strati-otes, UF #1177, UF #1178, UF #1179) were induced to differentiate ovules by intercrossing with pollen of D. XEthel Kawamoto X D. gouldii blue, UF #1181. The sixth plant D. XJaquelyn Thomas dark Shaffer, UF #1397) was activated by selfing. Flowers from the plants were utilized one to two weeks after they came into bloom during the period November, 1960, through November, 1962. Insofar as chronologically and morphologically the cellular events are very similar in all six plants, a common description will be adhered to throughout this presentation.


Description of Microscopy For purposes of introduction and orientation, gross structural changes in intact and freshly excised tissue were photographically recorded on Kodak Panatomic X sheet film with a 4 x 5 inch camera used in conjunction with a 6-3/8 inch Graflex optar lens (Figs. 1,2) or the optical system of a Wild-Heerbrugg M20 stereo-dissecting microscope (Figs. 3,4,5) .
Material for light and electron microscopy was selected on the basis of previously determined developmental schedules (Swamy, 1949a; Niimoto and Sagawa, 1961). Early work indicated that for most phases of the sequence a sampling interval of two days was most efficient. Freshly excised ovaries were rapidly opened and placental ridges were removed under the dissecting microscope using new surgical blades. Three samples of the excised tissue corresponding to the major divisions of the parietal placenta of Dendrobium orchids (see "OBSERVATIONS Background") were fixed as follows: (1) For subsequent smear analysis, one portion for three hours at 60 C. in Carnoy's solution; (2) for electron microscopy, one portion for one hour at 20 C. in an unbuffered 2.5 per cent aqueous solution of potassium permanganate (Mollenhauer, 1959), and one portion for eighteen hours at 20 C. in a buffered 1 per cent aque-


ous solution of osmium tetroxide, after the methods of Siegesmund et al. (1962). To circumvent injury phenomena (Mollenhauer et al., 1960), individual ovules free from placental attachment were never used. To avoid possible developmental differentials along the long axis of the ovary (Sagawa, 1962), material for the simultaneous fixations was always selected from the same transverse plane. Following fixation, material for electron microscopy was dehydrated at twenty-minute intervals at room temperature in a gradient ethanol series and embedded in Araldite epoxy resin (Epoxy Resins). Thin sections obtained with glass knives on a Servall Porter-Blum microtome were picked up on clean, uncoated copper grids and examined with an RCA EMU-3C electron microscope. Three and one-quarter by 4 inch Kodak medium contrast lantern slide plates were suitably enlarged on Kodabromide F-3, F-4, and F-5 paper.
This study utilized many ovaries from which numerous ovules were excised and observed. Ovules embedded in transparent plastic are well adapted to thin-sectioning techniques because the worker can not only ascertain and adjust their spatial orientation, but also determine their relative degree of development when viewing them in a dissecting microscope.
Whereas time is an important dimension in the


observations described herein, an effort to categorize the developmental events based on major modifications of the placental tissue will be adhered to. The sequential stages have been labeled thusly: (1) The Quiescent Phase. (2) The Proliferative Phase. (3) The Nucellar Phase. (4) The Sporogenic Phase. (5) The Prophase. (6) The Metaphase.


OBSERVATIONS
Background
In Dendrobium orchids as many as twenty blossoms may be formed in an acropetal sequence (Fig. 1) on each of a dozen or more racemes during a given interval within the reproductive life of a plant. Each blossom is comparatively small, having at anthesis a very slender ovary which expands noticeably (Fig. 2) after application of pollen to the stigma. Within the enlarging ovary, the paired tripartite parietal placenta, initially undifferentiated, proliferates rapidly and extends into the ovarian cavity which enlarges concomitantly (Fig. 3). Each of the three pairs of placental ridges, coursing along the periphery of the ovary (Fig. 4) the entire length of its long axis, is covered with numerous papilla-like protrusions (Fig. 5).
During the Quiescent Phase prior to pollination the numerically few undifferentiated papillae are seen to range in shape from short, blunt structures three cells long and eight cells broad to long narrow forms (Fig. 6) twelve cells long and three cells broad. Three to four days following pollination the Proliferative Phase commences during which


the papillae become mitotically active (Fig. 7). The cellular proliferation, frequently involving dichotomous branching of the broad papillae, continues at a diminishing rate until approximately thirty days after pollination by which time the number of papillae has increased markedly. By thirty-three days the Nucellar Phase is characterized by a central column of three to seven flattened, cylindrical cells covered by a uniseriate nucellar epidermis (Fig. 8). Thereafter the subapical cell (apical in the column) now called the megaspore mother cell enlarges rapidly thus initiating the Sporogenic Phase (Fig. 9). By approximately day fifty the primary integument has begun enveloping the greatly enlarged megaspore mother cell, the nucleus of which is found to be in the meiotic Prophase (Fig. 10). The secondary integument has grown around the above system with its archesporial nucleus now in the meiotic Metaphase I by the fifty-fifth day following pollination (Fig. 11).
Quiescent Phase The Quiescent Phase (Fig. 6) is marked by a conspicuous absence of division figures. As in all developmental stages, the papilla is enveloped by an intact lipid-bearing cuticle. The cells near the distal portion of the papilla are observed to possess high nucleo-cytoplasmic ratios


indicative of real or potential meristematic activity which are lower in the cells located immediately proximal to them. The more proximal cells are also marked by large vacuoles. All the cells maintain numerous intercellular connections or plasraodesmata which are seen associated with the nuclei either directly through contact with the paired-membrane component of the envelope (Pig. 12) or indirectly via envelope continuity with the endoplasmic reticulum (Fig. 13). In this phase of development the nuclear envelope is always seen to be unusually "active" as evidenced by the bleblike vesicles (Fig. 12), discontinuities (Fig. 14), and numerous extensions (Figs. 12,13) sometimes associated with organelles (Fig. 14). At times the nuclear envelope appears to be composed of three membranes (Fig. 16). Pores ca. 400a in diameter are frequently encountered in the nuclear envelope (Figs. 12,15). The cytoplasm contains comparatively few inclusions (Figs. 6,12-16). The golgi-apparati (Figs. 15,16) with their low number of stacked lamellae terminating in enlarged vesicles are typical of those in young plant tissue (Whaley et al., 1959). Mitochondria are equally typical in their small sizes and shallow cisternae (Figs. 12,13,15,16). Paired, branched, and/or cup-shaped mitochondrial complexes (Manton, 1961a) are rarely observed. Proplastids (Figs. 12,14,15) exhibiting rudimentary internal


structure are few in number and are distinguishable by their lamellar spacing and differential affinity to potassium permanganate (Whaley et al., 1960a). The extensive cellular membrane system (endoplasmic reticulum) is quite sparse (Figs. 12-16). The vacuoles of the dense apical cells appear small and branched whereas in the more basal cells they are observed to be considerably larger and less branched (Figs. 6,12,15,16). Lipid droplets are seen as electron opaque bodies either solid or hollow; single, paired, or aggregated (Figs. 6,12,13); and/or electron transparent areas (Fig. 16). The limiting cell membrane is generally smooth, adhering closely to the cell wall. Its double leaflet structure (Robertson, 1962) is often visible at points of distortion and/or displacement (Fig. 17).
Proliferative Phase Duplication and growth in all their manifestations appear to be the theme of the Proliferative Phase. Conspicuous mitotic figures are observed in the dermal and sub-dermal layers of the extending papillae (Figs. 7,18,19). The division figures exhibit all of the "typical" plant cell organelles (Figs. 18,19). Although the Proliferative Phase is evidenced by a marked increase in organelles (Fig. 21),


there are generally fewer seen in the cells actually undergoing division (Figs. 18,19). The endoplasmic reticulum, so extensive in the micrographs of apical meristematic regions (see Whaley et al. 1960a? Porter and Machado, 1960), is generally reduced to a few peripheral fragments which maintain intercellular "communication" with contiguous cells throughout the mitotic maneuver (Figs. 18,19). The paired-membrane element in the micrographs is, primarily, displaced nuclear envelope (Figs. 18,19). "Phragmosomes," reported by Porter and Caulfield (1960), Porter and Machado (1960), and Manton (1961b), were not seen in or near the developing phragmoplast (Figs. 19,20). Evidence for equational organelle partition across the equatorial zone as observed in some animals (Tahmisian et al., 1956) is totally lacking. Organelle duplication appears to be a process of binary fission and/or branching (Figs. 19,21). Occasional lipid droplets are seen in all figures. The chromosomal complement, "sticky" in light microscopic handling, is observed to be decidedly cohesive (Figs. 18,19) thus negating an analysis of the individual components. The nucleoli (and other RNP-rich areas such as ribosomes) which are lightly stained with potassium permanganate (Fig. 8) (see Mollen-hauer, 1959) are observed to be remarkably distinct, displaying internal modifications when fixed with osmium


tetroxide (Figs. 22,23). Never are delimiting structures found associated with the nucleoli. In the interphase nucleus, two nucleoli are seen closely associated with the organizer chromatids and the nuclear envelope. Also frequently seen associated with the nucleoli are dense hetero-chromatic regions often called "false nucleoli" or chromo-centers (De Robertis et al.. 1960; Peveling, 1961). Internal differentiation is seen to consist of vacuole-like regions (Fig. 22) and electron dense granules (Fig. 23) which are seen well in later phases (Fig. 40). Prior to mitotic breakdown, the nucleoli are observed in close association with each other (Fig. 23).
In the more basal, highly vacuolated cells of the proliferative protrusion, large aggregates of lipid droplets are found (Figs. 24,25). The possible origin and function of these droplets will be discussed in the following sections .
Nucellar Phase Cessation of mitotic activity in the papilla is followed closely by an alignment and enlargement of a central column of cells commonly referred to as the nucellar row (Fig. 8). The maneuver renders the protrusion three cells in diameter. In the nucellar column, the nucleo-cytoplasmic


ratio remains high (Fig. 26) while vacuolar volume is low. The cells maintain intimate continuity with their peripheral environs by way of numerous plasraodesmata. The cell membrane remains smooth and "inactive." The membrane-organelle fraction of the cytoplasm is diffuse and "unspecialized" (Fig. 26). Occasionally lipid droplets are observed in the cells. The nuclei maintain many pores (Figs. 8,26,27). Rarely is more than one nucleolus per cell observed. The nucleolar contents are heterogeneous; often three and four regions of differential fixation are noted (Fig. 27). Granules ca. 120A in diameter appear to be the elementary unit of the nucleolus (Fig. 28). The epidermal layer of cells enveloping the nucellar row is modified to accommodate the enlarging central component (Fig. 8). This layer, referred to as the nucellar epidermis or nucellar integument, is composed of cells similar to the nucellar row except for their smaller size and frequent possession of two nucleoli (Fig. 9).
In the more basal cells of the protrusion, lipid droplets are seen aggregated (Fig. 29) as in the previous phase. Plastids in this region are sometimes observed "secreting" membrane bound units into the cytoplasm (Fig. 30).


Sporogenic Phase
Morphologically there is little save its position and high nucleo-cytoplasmic ratio (Pigs. 31,33) that predisposes the apical cell of the nucellar row to its future role as a megaspore mother cell. During sporogeny, the most obvious event occurring in the apical region of a papilla is the enlargement of the mother cell. It is also observed to occupy a proportionally greater volume of the papillar tip (Fig. 31). The cytoplasm is heavily endowed with ribosomes (Fig. 34) which appear both "free" and "attached" to the membrane system. The proplastids, mitochondria, golgi, "phragmosomes," and endoplasmic reticulum appear to increase in number (compare Fig. 33 with Figs. 35,36). Ofttimes, binary division of organelles can be seen (Figs. 35,36,37). Many of the proplastids continue to show internal regions considerably less electron opaque than their immediate surroundings (Fig. 35). These clear areas are believed to be rich in starch (see Bal and De, 1961). An occasional lipid droplet is observed in the cytoplasm.
A striking change becomes evident during the Sporogenic Phase; namely, the loss of plasmodesmata previously existing between the megaspore mother cell and its immediate cellular environment (compare all figures numbered below 34 with all those above 34). As far as can be ascertained,


rhopheocytosis (see Policard and Bessis, 1962) or micro-pinocytosis (Fig. 37) is initiated simultaneously with the disappearance of the plasmodesmata. Rhopheocytotic vesicles are observed throughout the papillar tip, with the highest incidence being observed in the enlarging megaspore mother cell and its immediate cellular environment. Primarily, the vesicles are internally homogeneous; however, at times internal modifications are observed (Fig. 49). Never are vesicular structures observed within the walls. The nuclear envelope has many pores (Fig. 36). The fused nucleoli show no increase in volume.
Prophase
In terms of observable morphological changes, the prophase papillar apex presents the greatest amount of activity. Whereas the relative volume of the archesporial nucleus decreases during the rapid growth of the Sporogenic Phase, it again increases during the early Prophase (Fig. 31). The early-prophase megaspore mother cell (Fig. 39) exhibits a large central nucleus which lacks pores in its envelope. The single, large nucleolus retains its status quo volume and internal heteromorphology (Fig. 40). The peripheral cytoplasm displays proplastids, mitochondria, "phragmosomes,* lipid droplets, rudimentary golgi, and an


extensive elaboration of the membrane network including numerous rhopheocytotic vesicles (Fig. 39). The earliest observable deviation from the above-described status is a marked increase in the cytoplasmic volume (Fig. 31) accompanied by a striking multiplication of the organelle population (Fig. 41) via binary fission (Fig. 42) and cup-proliferation (Figs. 42,43). Rhopheocytosis continues at an accelerated pace with extensive vesicular and cisternal elements penetrating the ground cytoplasm (Figs. 41,44). Despite the rapid build-up of cytoplasmic structures, the ribosomal fraction has become very dilute and appears to be primarily attached to membranes (Figs. 43,46). With the synthesis of cytoplasm, the nucleus becomes rather ameboid, and again exhibits pores (Fig. 44). The nucleolus remains spheroid, heteromorphic, and of unaltered volume. The enveloping cytoplasm contains all forms of membrane expression and lipid droplets. Rhopheocytosis continues.
The ameboid appearance of the nucleus is less noticeable as the nuclear envelope is seen forming numerous pores by a marked "break-up" of envelope continuity (Figs. 45,47,49). The pores are round in tangential view (Fig. 46) and cylindrical in oblique sections (Fig. 47). In face
iew a central granule is sometimes seen in a pore (Fig.
>) Often, annulate lamellae coursing parallel to the


nuclear envelope, and looking very much like it, are observed (Pig. 47). The cytoplasm retains the numerous components described above. The ribosomal fraction remains diffuse and attached. In the immediately adjacent milieu of the ribosome, small distinct granules (15A 20A) are observed (Fig. 46). Rhopheocytotic activity is still observed (Fig. 48). As the megaspore mother cell approaches metaphase and the nuclear envelope continues to dissipate, many of the organelles are seen to possess a single limiting membrane in places (Fig. 49).
As the archesporial cell passes through prophase, two grossly obvious events occurs (1) The entire apical half of the papillar structure recurves 180 on its central axis thus rendering the apex of the unit in close opposition to the base of the protrusion. (2) The primary integument grows up around the papillar tip enveloping it in the integumentary cup. The region of integument profileration is highly mitotic, exhibiting division figures (Fig. 50) very similar to those described in the Proliferative Phase. In the rapidly growing integumentary cells, a striking modification of the cell membrane is observed. At isolated locations along the membrane, small invaginated "loops" appear (Fig. 51a). These are seen in various sizes (Fig. 51b). Others are observed to be connected to the cell membrane


through a narrow isthmus whose limiting membrane is actually continuous with the cell membrane (Pigs. 51c,51d). Still others are seen totally divorced from the limiting cell membrane; and as distinct differentiating organelles, both proplastids and mitochondria.
The basal protrusion cells, which may now be said to compose the funiculus, are observed to be highly vacuolated with few intercellular plasmodesmata (Pig. 52). In the region distal to the vacuolated cells, and near the cross-sectional plane at which the integument originates, are seen some cells with internally homogeneous lightly stained vacuoles and others with darkly stained vacuoles showing some heterogeneity (Figs. 53, 54). Never are the two types of vacuoles observed in the same cell. The structures often appear stellate and much branched. Some are highly heterogeneous. Not only are dark, stellate vacuoles seen in the protrusion proper, but also in the integuments (Pig. 54), and in the "disorganizing" nucellar epidermis (Pigs. 45,48). This is the only period in the entire developmental sequence during which these structures are observed.


Metaphase
In the transition from the Prophase to the Metaphase, the primary integument appears to envelop totally the papillar tip. The cells of the primary integument exhibit a minimum of vacuolation (Figs. 11,55). Together with the remaining cells of the nucellar row, the nucellar epidermis is continuously resorbed by the meiotic megaspore mother cell. The abundant, dark stellate vacuoles of the late Prophase are virtually gone (Fig. 55). The secondary integument (Fig. 11) which will not totally envelop the megasporial cell until after fertilization is seen at the base of the complex (Fig. 55). The papillar tip is isolated from the surrounding integuments by an intact lipid-containing cuticle (Figs. 11,55,59). With the progressive disintegration of the nuclear envelope the organelle population of the mother cell is observed to be more diffuse (Fig. 56). The process continues with an obvious reduction in the number of "classical" organelle shapes (Fig. 57). Many of the yet identifiable organelles display external "breakdown symptoms" (Figs. 57,58). Rhopheocytosis is observed less frequently (Figs. 57,59). Little remains of the nuclear envelope and/or the abundant prophase organelle contingent as the development is seen in the Metaphase (Fig. 59). Distinct "phragmosome bodies" are seen in


the cytoplasm (Pigs. 56,58,59). A considerable portion of the cells of the nucellar epidermis is composed of nuclei.
At the same time, the primary integument is observed to be dense; the cells appear filled with cytoplasm and nuclei (Fig. 11). The secondary integument is highly vacuolated (Figs. 11,60,61). The vacuoles are lightly stained, very large, and often invested with organelle-like inclusions (Figs. 60,61). The cells maintain plasmodesmata with each other (Fig. 60). The organelles appear "immature." In fact, never in the whole sequence are photosynthetic-type chloroplasts observed. Each of the paired elements of the proplastid and/or mitochondrial envelope is resolved to be composed of two 20A unit-membranes (Fig. 62).


Fig. 1. Flowering raceme of Dendrobium XJaquelyn Thomas. Approximately XI/3.
Fig. 2. External post-pollination ovary enlargement. Lefts Pre-pollination stage. Center: Early proliferative stage. Right: Late proliferative stage. Approximately X2/3.
Fig. 3. Internal post-pollination ovary enlargement. Left: Cross-section of a freshly excised ovary in the pre-pollination stage. Approximately XI5. Center: Cross-section of a freshly excised ovary in the early proliferative stage. Approximately X15. Right: Cross-section of a freshly excised ovary in the late proliferative stage. Approximately X15.




Fig. 4. Longitudinal section of an ovary in the late proliferative stage. Note the numerous papillae on the ridges running horizontally in the illustration. Approximately X60.
Fig. 5. Late proliferative papillae. Left: Portion of freshly excised placental ridge. Approximately XI20. Center: Same as left. Approximately X250. Right: Same as left. Approximately X500.




Key to symbols used on all electron micrographs:
c ground cytoplasm ne nuclear envelope
ch chromatin nin nucellar integument
cm cell membrane np nuclear pore
cu cuticle nu nucleolus
cw cell wall pd plasmodesma
dv dark vacuole ph phragmosome
er endoplasmic reticulum pin primary integument
g golgi apparatus PP proplastid
1 lipid droplet rp ribosomal particle
m mitochondrion rv rhopheocytotic vesicle
mc mitochondrial crista sin secondary integument
mm megaspore mother t tonoplast
n nucleus V vacuole
Fig. 6. Tip of a long, narrow quiescent placental papilla. Note the dense apical cells, and the large vacuoles (v) in the more basal cells. KMn04 fixation. Approximately X4,500.
Fig. 7. Tip of a proliferative placental papilla. Note the cuticle (cu) which is partially disrupted by the preparatory techniques. Note also the prominent mitotic figure (arrow). KMn04 fixation. Approximately X7,000.




Fig. 8. Tip of a nucellar placental papilla. Note the division of the papilla into a prominent, central row three cells long; and the uniseriate nucellar integument (nin) enveloping them. KMnO^ fixation. Approximately X10,000.
Fig. 9. Tip of a sporogenic placental papilla. Note the enlarging apical cell, now referred to as the megaspore mother cell (mm), of the nucellar row. KMn04 fixation. Approximately X8,000.




Fig. 10. Tip of a prophase placental papilla. Note the greatly enlarged megaspore mother cell, the flattened cells of the nucellar integument (nin), and the developing primary integument (pin). KMnO^ fixation. Approximately X4,200.
Fig. 11. Tip of a metaphase placental papilla. Note the megaspore mother cell (mm) now enveloped by the dissociating nucellar integument (nin), the primary integument (pin), and the secondary integument (sin). KMn04 fixation. Approximately X2,700.




Fig. 12. Peripheral cell of a quiescent papilla. The nuclear envelope (ne) shows considerable "blebbing" (arrows), extensions (hollow arrows), and pores (np). Organelles are few in number (m,pp). Small vacuoles (v) are seen in the cytoplasm. Lipid droplets are seen as very dark inclusions (1). The endoplasmic reticulum (er) is very sparse. Plasmodesmata (pd) are maintained between the cells by crossing through the cell wall (cw). KMnO^ fixation. Approximately X24,000.




Fig. 13. Portions of four cells in a quiescent papilla. Note the continuity of the nuclear envelope (ne) with the plasmodesmata (pd) via the endoplasmic reticulum (er). Nuclear extensions (hollow arrows) are observed. Note the large vacuole (v) with its thin tonoplast (t) in the lower left cell. Note also the "phragmosome" (ph) in the right-hand cell. KMnO^ fixation. Approximately X26,000.
Fig. 14. Portions of three quiescent papilla cells. The marked nuclear discontinuity (arrow) is seen associated with several organelles. It is also seen in contact with several plasmodesmata. KMnO^ fixation. Approximately X21,000.




Fig. 15. Peripheral cell of a quiescent papilla. The large central nucleus has very discrete pores. The organelles are very few in number, and comparatively "un-specialized." Several small vacuoles are seen in the cytoplasm. Small tubules (arrows) permeate the ground cytoplasm (c). They are believed to be elements of the endoplasmic reticulum. KMn04 fixation. Approximately X22,000.




Fig. 16. Internal cell of a quiescent papilla apex. The limiting cell wall is traversed by many plasmo desraata. The cytoplasm is invested with few organelles, and small vacuoles. Transparent cytoplasmic regions (1) mark locations of lipid droplets which are either poorly fixed or have lost the outer dark sheath of lipoprotein in subsequent maneuvers of preparatory technique. The bilaminar nuclear envelope appears distinctly trilaminar at the arrow. KMnO^ fixation. Approximately X18,500.




Fig. 17. Wall between two cells of a quiescent papilla. Note the cell membrane (cm) which seldom is seen adhering to the cell wall, and often at points of distortion (arrows) is observed to be composed of two elements or unit-membranes. Inasmuch as the cells were seen to be highly vacuolated, the scattered ribosomes (rp) are typical of those found in differentiating cells. OsO^ fixation. Approximately X170,000.




Pig. 18. Oblique section of a dividing cell in a proliferative papilla. The scattered endoplasmic reticulum is observed to be continuous with the many plasmodesmata interconnecting the cell with its cellular environment. The organelles are few in number. The displaced nuclear envelope persists as large plates which occasionally show pores (np). The bilaminar membranes often appear to have supernumerary elements (arrows). KMn04 fixation. Approximately X16,000.


r ^ i
PP
ch
pd
np


Fig. 19. Oblique section of a telophase mitotic figure in a proliferative papilla. The newly formed daughter cells maintain intercellular connections via plasmodesmata. The displaced nuclear envelope exhibits distinct pores seen in tangential section. An ameboid proplastid is believed to be in the process of replication by division. The newly forming cell plate is observed in the lower center of the cell(between arrows). It is composed of cytoplasmic membrane elements. KMnO^ fixation. Approximately X24,000.


\
y
ch


Fig. 20. Developing cell plate between two newly divided cells of a proliferative papilla. Note the complete absence of "phragmosomes" in or near the cell plate (cp). Note also the supernumerary aspect of the bilaminar systems (arrows). KMn04 fixation. Approximately X21,500.




Fig. 21. Portion of a cell in a proliferative papilla. The cytoplasm is marked by a large fraction of organelles; some seen as dividing forms. The endoplasmic reticulum is observed to be extensive. A tangential section of a nucleus reveals its bilaminar envelope together with a number of nuclear pores. KMnO^ fixation. Approximately X32,000.




Fig. 22. Portion of a nucleus in a proliferative papilla cell. The granular nature of the nucleolus is somewhat evident. The "penetration" of the nucleolus by the organizer chromosome (ch) is clearly seen together with the interposed "vacuole" (arrow), and the appended chromocenter (cc). The complete structure is closely appressed to the nuclear envelope. OSO4 fixation. Approximately X90,000.




Fig. 23. Interphase nucleus in a cell of a proliferative papilla. The closely appressed nucleoli are clearly visible in the center of the nucleus. Each nucleolus has an appended chromocenter (cc). The organizer chromatids (ch) are very close to each other. The nuclear envelope is observed to be very uniform; displaying a number of nuclear pores in places. Heavy deposits of chromatin can be seen closely adhered to the nuclear envelope (upper left region of nucleus). OSO4 fixation. Approximately X54,000.




Fig. 24. Lipid droplets in a basal cell of a proliferative papilla. Note particularly the hollow nature of the potassium permanganate fixation of the droplets. The droplets were frequently observed to be aggregated in similar fashion in many vacuolated cells of the papilla. KMnO fixation. Approximately X16,500.
Fig. 25. Lipid droplets in a basal cell of a proliferative papilla. This is a portion very similar to the region of the cell in the previous figure. Note especially the "chatter effect" produced frequently in the thin sectioning of lipid droplets fixed in this manner. OSO4 fixation. Approximately XI5,000.




Fig. 26. Cell in the nucellar row of a nucellar papilla. Note the distinct nuclear pores. Note also the manifestations of the lipid droplets. KMnO^ fixation. Approximately X17,500.


m
o


Pig. 27. Cell in the nucellar row of a nucellar papilla. Note the differential staining of the nucleolus; believed to be produced by the pre-meiotic fusion of the two nucleoli. Note also the "penetration" of the nucleolus by the two organizer chromatids (ch). The nuclear envelope is invested with many pores. 0s04 fixation. Approximately X24,000.


/
np -
ch ch
.'$>
nil


Fig. 28. Portion of the nucleus in a nucellar-row cell of a nucellar papilla. Note the dense granules which comprise the major portion of the nucleolus. The bilaminar nuclear envelope is observed to be considerably distorted (arrows). The "collars" of the nuclear pores assume "hour-glass" shapes under the distorting stress. Cytoplasmic ribosomes (rp) are very similar in size and staining capacity to the nucleolar granules. OsO^ fixation. Approximately X96,000.




Fig. 29. Portions of three basal cells in a nucellar papilla. Note particularly the wrinkled lipid droplets believed to be produced by osmotic stresses during fixation. Note also the modifications of the bilaminar systems (arrows). KMn04 fixation. Approximately X30.000.




Fig. 30. "Secreting" plastids in a basal cell of a nucellar papilla. The membrane-bound entities appear to be formed on the lamellae of the plastids (hollow arrows) from whence they move into the space between the bilaminar elements of the plastid envelope (arrows), and then into the cytoplasm (1). KMnO^ fixation. Approximately X65,000.




Fig. 31. Volume relationships in developing Den-drobium ovules. Measurements taken from "typical" electron micrographs of sagittal sections of papillae in the various developmental phases.


NUCELLAR SPOROGENIC PROPHASE METAPHASE
DEVELOPMENTAL PHASE


Pig. 32. Diagram of modifications in bilaminar systems. Row A: Phenomena as seen in electron micrographs, that is, from the top. Row B: Horizontal displacement within the thickness of a section; believed to produce conditions observed in Row A. 1. No horizontal displacement. 2. Equal shift to the right. 3. Unequal shift to the right. 4. Equal shift to the right accompanied by distortion of the unit-membrane component. 5. Unequal shift to the right accompanied by distortion of the unit-membrane component. 6. Right-left arc.


HM\\II
ill ill iii iii ill ii
ill iii iii iii iii
ii iii iii iii iii
iii iii iii iii iii i i
9
V
CM rO


Fig. 33, Apical cell in the nucellar row of an early sporogenic papilla. Note the large nucleus, the sparse organelle fraction, and the intercellular plasmodesmata. A proplastid is observed in binary division in the upper portion of the cell. KMnC>4 fixation. Approximately X22,000.




Fig. 34. Portion of an enlarging apical cell in the nucellar row of a sporogenic papilla. The ribosomes appear to be both "free" (in clumps) and "attached" (in definite patterns on membranes? arrows). 0s04 fixation. Approximately X92,000.




Fig. 35. Enlarging apical cell in the nucellar row of a sporogenic papilla. The cytoplasm in this stage contains organelles of all types. In the upper left-hand corner of the cell a proplastid is seen dividing. The proplastids also show starch granules (arrows). The nuclear envelope has many distinct pores. KMn04 fixation. Approximately Xll,500.


k
PP
er
np
m


Fig. 36. Tip of a sporogenic papilla. Note the lack of intercellular plasmodesmata, the numerous cytoplasmic organelles in the enlarging apical cell, and the comparatively reduced nuclear volume of the apical cell. The nucellar integument appears somewhat flattened. KMn04 fixation. Approximately X5,000.


/A0'
n
n


Fig. 37. Apical portion of an enlarging megaspore mother cell in a late sporogenic papilla. Note the abundant cytoplasmic components. Proplastids are observed in division. A distinct "phragmosome" is seen. The cytoplasm appears heavily invested with rhopheocytotic vesicles (rv) which can be seen forming at the cell membrane (arrow). KMnO^ fixation. Approximately Xll,500.




Fig. 38. End portions of two basal cells in a sporogenic papilla. The obliquely sectioned vacuolar tono-plast (t) is observed to be composed of two unit-membranes (arrow). One intercellular plasmodesmata is seen to be tubular (hollow arrow). KMn04 fixation. Approximately X70,000.




Fig. 39. Early-prophase megaspore mother cell. The large, central nucleus with its envelope which lacks pores is observed to contain an ellipsoidal nucleolus and prophase chromosomes. The cytoplasm contains extensive ramifications of the endoplasmic reticulum. Organelles are numerous. Golgi-bodies all appear quite rudimentary in form. Numerous rhopheocytotic vesicles are seen in the cytoplasm, and being formed at the cell membrane. KMnO^ fixation. Approximately X8,000.




Fig. 40. Nucleolus of a prophase megaspore mother cell. Note the internal heterogeneity in the fixation-staining of the basic granules. OsO fixation. Approximately X92,000.




Fig. 41. Perinuclear portion of a mid-prophase megaspore mother cell. Note the numerous mitochondria, the immature golgi-apparati, and the distinct "phragmosomes." KM11O4 fixation. Approximately XI7,000.




Pig. 42. Perinuclear portion of a mid-prophase megaspore mother cell. Note the organelle duplication by binary fission (arrow) and cup-proliferation (hollow arrow). Note also the immature golgi-apparatus, and the extensive fenestrations of the cytoplasmic membrane system (f). KMn04 fixation. Approximately X35,000.


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