J. Cell Sci. 8, 289-301 (1971) 289

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FINE STRUCTURE STUDY OF POLLEN

DEVELOPMENT IN HAEMANTHUS

KATHERINAE BAKER

I. FORMATION OF VEGETATIVE AND GENERATIVECELLS

JEAN M. SANGER AND W.T.JACKSONDepartment of Biological Sciences, Dartmouth College, Hanover, New HampshireO3755, U.S.A.

SUMMARY

When microspores of the African blood lily divide, they form pollen grains which consist of2 cells of unequal size. This is accomplished when the microspore nucleus is displaced from thecentre of the grain prior to division. The displacement is always towards the side of the grainopposite the furrow, and large vacuoles form in the cytoplasm between the furrow and thenucleus. During cell division the cell plate curves around one daughter nucleus and fuses withthe pollen wall to enclose the generative cell. The cell-plate attachment always occurs with thewall that is opposite the furrow of the grain. Most of the microspore's organelles become incor-porated in the larger vegetative cell, whereas the generative cell has few, if any, plastids andonly a small number of other organelles. The wall around the generative cell is composed offinely fibrillar material enclosed within 2 unit membranes. The generative cell eventuallybecomes detached from the pollen wall, becomes spheroidal, and moves to a position near thecentre of the pollen grain. At the same time, the large vacuoles disappear from the vegetativecell and the number of organelles increases substantially.

INTRODUCTION

Cell division in microspores of flowering plants is noteworthy for several reasons.The division is a polar one, resulting in daughter cells of unequal size. Furthermore,the smaller of the 2 cells initially attaches to the pollen wall and later lies entirelywithin the cytoplasm of the larger cell. Finally, these 2 cells differ greatly in their sub-sequent development and functions.

Most of the published ultrastructural studies of microspore division and earlypollen development were undertaken without the benefit of glutaraldehyde-osmiumfixation (Bopp-Hassenkamp, i960; Diers, 1963; Larson, 1965; Sassen, 1964; Maru-yama, Gay & Kaufmann, 1965). Two exceptions are the recent studies of Heslop-Harrison (1968) and Angold (1968). The present study using glutaraldehyde fixationwas undertaken to determine how the microspore nucleus is displaced before the polardivision, what ultrastructural differences exist between the 2 cells, how the generativecell is attached to the pollen wall and, finally, in what manner it becomes detached.

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290 J. M. Sanger and W. T. Jackson

MATERIALS AND METHODS

The cells examined in this study were obtained from plants of the African blood lily, Haeman-thus katherinae Baker, grown in the Murdough Experimental Greenhouse at DartmouthCollege. The colour and length of the anthers give an approximate indication of their develop-mental stage. Thus, anthers change from white to yellow to orange as they mature to a finallength of about 5 mm. To determine the exact stage of development, however, the contents ofthe anther were stained with acetocarmine and examined with the light microscope.

Anthers which were to be examined in the electron microscope were first cut into pieces1-2 mm long One piece was stained with acetocarmine to determine the developmental stageand the other pieces were fixed for 1 h at room temperature in 15 % glutaraldehyde buffered atpH 70 in 005 M phosphate buffer. The pieces were washed in at least 4 changes of phosphatebuffer over a 1-2 h period before being post-fixed in 1 % OsO4 for 1 h. Dehydration was accom-plished in 5-min steps in a graded series of ethanol followed by two 5-min changes in propy-lene oxide. Two different plastic mixtures were used for embedding: (a) 1:1 mixture of Epon812 and nadic methyl anhydride (Luft, 1961) and (b) Epon 812, Araldite 506, and dodecyl-succinic anhydride (Mollenhauer, 1964). Sections were cut with a diamond knife on an LKBUltrotome III and picked up on uncoated 200-mesh grids. They were stained for 2 h in asaturated uranyl acetate-water solution then post-stained in Reynolds's lead citrate (Reynolds,1963) before being examined with the Zeiss EM 9 A electron microscope.

OBSERVATIONS

A microspore of H. katlierinae about to undergo mitosis is characterized by asculptured wall and a large nucleus which has been displaced from the cell's centretowards one side of the grain (Fig. 1). The nucleus is always displaced in a particulardirection; that is, to the side of the grain opposite the furrow. Vacuoles form betweenthe nucleus and the furrow. Present also at this stage is a scattering of lipid droplets,0-2-2-0 /<m in diameter, as well as other typical cell organelles. Plastids have fewlamellae and contain large starch inclusions.

By the time chromosome condensation has begun, the organelles exhibit a polarityin their cytoplasmic distribution (Fig. 2). Most of the plastids, mitochondria, and lipidbodies are displaced toward the furrow side of the cell. Around the nucleus is a narrowzone devoid of the larger organelles and containing short, unaligned microtubules(Fig. 3). The plastids lack starch at this stage but sometimes contain small osmiophilicbodies (Fig. 2) and often appear to be dividing (Fig. 4).

As mitosis continues, the majority of organelles remain associated with the chromatinmass that becomes incorporated into the vegetative cell. In Fig. 5 there are no plastidson the generative cell's side of the cell plate. In fact, a plastid was seen in only 2 of allthe sections of generative cells examined. Two-celled pollen grains fixed shortly aftercompletion of mitosis contain spherical nuclei (Fig. 6). Large vacuoles are present inboth cells, and the wall of the generative cell is attached to the pollen wall at pointsopposite the furrow.

The boundary between generative and vegetative cytoplasms consists of 2 cellmembranes separated by a gap which varies in width from 45 to 120 nm. The 2 cellmembranes are the same width (10 nm) as the cell membrane which borders the intine.The outer leaflets of each cell membrane (i.e. those bordering the gap, as opposed to thecytoplasm) appear more dense. This is also true for the cell membrane leaflet which

Fine structure of developing pollen 291

borders the intine of the pollen grain. The generative wall consists of a finely fibrillarcomponent sandwiched between the 2 unit membranes (Figs. 7, 8).

At the stage when the large vacuoles have disappeared from both cells of the pollengrain (Fig. 9), the generative cell detaches from the pollen wall and becomes sur-rounded by vegetative cytoplasm. Before detachment occurs, membrane fragments canbe seen between the intine and the plasma membrane (Fig. 8). These fragments areconcentrated along the junction between the generative cell and the pollen wall. Theyare not seen in other areas of the wall at this stage, nor are they seen during any otherstages of pollen development. Microtubules are positioned along both sides of thegenerative cell at its junctions with the pollen wall.

The areas of contact of generative cell wall and pollen wall move closer together(Figs. 6, 7) until the generative cell finally is pinched off. At this time it is sphericaland moves to the centre of the pollen grain, where it is surrounded by lipid droplets(Fig. 9). The vegetative cell at this stage has lipid droplets scattered throughout thecytoplasm in addition to those which surround the generative cell. The cytoplasmcontains large numbers of mitochondria and plastids which were not evident beforegenerative cell detachment.

DISCUSSION

Cell division of a microspore produces a 2-celled pollen grain which early in itsdevelopment consists of a cell within a cell. These 2 cells are destined to play quitedifferent roles: the generative cell, embedded in the cytoplasm of the vegetative cell,produces 2 sperm by a subsequent division; and the vegetative cell produces the pollentube in which the generative cell divides and through which the sperm gain access tothe egg. That 2 such cells should differ markedly from the time of their origin is notsurprising. This is readily documented by light and electron microscopy. The distri-bution of organelles during division of the microspore produces a vegetative cell con-taining most of the microspore's cytoplasm and a generative cell containing very littlecytoplasm.

There is no evidence from this study or other published reports (Angold, 1968;Heslop-Harrison, 1968) that a particular ultrastructural event can be causally relatedto the displacement of the nucleus prior to division. No peripheral microtubules wereever seen demarcating the boundary of the future cell plate as has been reported forthe polar cell division giving rise to wheat stomatal cells (Pickett-Heaps & Northcote,1966). The cell plate curving around the generative nucleus is hemispherical. The cellwall, which forms subsequently, consists of unidentified finely fibrillar materialbetween 2 unit membranes. In some micrographs the material of the generative cellwall has the same appearance as the intine with which it is initially contiguous andwhich has been reported to be composed of cellulose and polyuronides (Bailey, i960).The generative cell wall in H. katherinae does not have the same electron density as isseen in the callose walls of the generative cells of orchid pollen (Heslop-Harrison,1968).

The detachment or pinching off of the generative cell follows the same general pattern19-2

292 J. M. Sanger and W. T. Jackson

as reported for orchid (Heslop-Harrison, 1968) and bluebell pollen (Angold, 1968). Inaddition, microtubules are present in H. katherinae at the junction of the generativecell wall and pollen wall and appear to run parallel to these walls. Since the micro-tubules are present both within and outside the generative cell at the junction it isdifficult to assign a role to them in the detachment of the cell.

The origin of the small vesicles found within the junction between the generative celland pollen wall could not be determined. There was no indication that they derivedfrom dictyosomes. Since the vesicles are present only prior to generative cell detach-ment, they may be contributing cell wall material to the generative cell. Heslop-Harrison (1968) has suggested that detachment of the orchid generative cell is causedby the growth of the generative cell wall over the surface of the intine, followedby the separation of the 2 walls. It is possible that such a process also occurs in H.katherinae, although the mechanism by which the generative wall changes from ahemispherical to a spherical state before detachment still remains to be explained.

Plastids are excluded almost entirely from the generative cell. The localization ofthese organelles can be seen clearly in the microspore by telophase of the mitoticdivision. Some of the other types of cell organelles are included in the cytoplasm of thefuture generative cell during telophase and it is not clear how the plastids are excluded.They are much larger than any of the other cytoplasmic organelles and this may be afactor in their exclusion. The absence of plastids from generative cells has been re-ported in several other species of pollen (Sassen, 1964; Larson, 1965; Heslop-Harrison,1968).

During and for a time after microspore mitosis, plastids increase in number,apparently by division, and decrease in size. Starch is not present in the plastids of thenewly formed pollen grain. However, it is found in the pre-mitotic microspore and inthe mature pollen grain (Sanger, 1968).

Osmiophilic bodies are also present in the cytoplasm of the microspore and thepollen grain. Judging from their appearance in osmium-fixed material, they probablycontain lipids (Sanger, 1968) and are termed here lipid bodies. Angold (1968) reportsthe appearance of similar lipid droplets in the vegetative cytoplasm of bluebell pollenafter the generative cell has been formed. In H. katherinae, as in bluebell pollen, thesedroplets ensheath the generative cell. In H. katherinae pollen, however, they are alsopresent in the cytoplasm of the generative cell and are scattered throughout thevegetative cell as well.

We thank Dr Peter K. Hepler for his suggestions and Dr Joseph W. Sanger for his assistancein various stages of this study.

This investigation was supported by PHS Microbiology Training Grant 3 TO 1 00961-0551to the first author and by a grant to the second author from the Agriculture Research Service,U.S. Department of Agriculture, grant no. 12-14-100-7981 (34) administered by Crops ResearchDivision, Beltsville, Maryland.

REFERENCES

ANGOLD, R. E. (1968). The formation of the generative cell in the pollen grain of Endynrionnon-scriptus (L). J. Cell Sci. 3, 573-578.

BAILEY, I. W. (i960). Some useful techniques in the study and interpretation of pollen mor-phology. J. Arnold Arbor. 41, 141-151.

Fine structure of developing pollen 293

BOPP-HASSENKAMP, G. (i960). ElektronenmikroskopischeUntersuchungen an Pollenschlauchenzweier Lilaceen. Z. Naturf. B 15, 91-94.

DIERS, L. (1963). Electronenmikroskopische Beobachtungen an der generativen Zelle vonOenotliera hookeri Torr. et Gray. Z. Naturf. B 18, 562-566.

HESLOP-HARRISON, J. (1968). Synchronous pollen mitosis and the formation of the generativecell in massulate orchids. J. Cell Sci. 3, 457-466.

LARSON, D. A. (1965). Fine-structural changes in the cytoplasm of germinating pollen. Am. J.Bot. 52, 139-154.

LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. biophys. biocfievi.Cytol. 9, 409-414.

MARUYAMA, K., GAY, H. & KAUFMANN, B. P. (1965). The nature of the wall between genera-tive and vegetative nuclei in the pollen grain of Tradescantia paludosa. Am. J. Bot. 52,605-610.

MOLLENHAUER, H. H. (1964). Plastic embedding mixtures for use in electron microscopy.Stain Technol. 39, 111-114.

PICKETT-HEAPS, J. D. & NORTHCOTE, D. H. (1966). Cell division in the formation of thestomatal complex of young leaves of wheat. J. Cell Sci. 1, 121-128.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208-212.

SANGER, J. M. (1968). An Ultrastructural Analysis of Pollen Development in Haemanthiskatlierinae Baker. Ph.D. thesis, Dartmouth College.

SASSEN, M. M. A. (1964). Fine structure of petunia pollen grain and pollen tube. Acta bot.veerl. 13, 175-181.

{Received 29 June 1970)

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294 J- M. Sanger and W. T. Jackson

Fig. i. Mature microspore prior to cell division. Vacuoles (v) are positioned betweenthe nucleus and the furrow (/). Plastids (p) usually contain starch (arrows), and lipidbodies (/) are distributed throughout the cytoplasm, x 3600.Fig. 2. Microspore during prophase of mitosis. Most of the organelles lie betweenthe furrow and nucleus. The plastids sometimes contain osmiophilic droplets (arrows).The area in brackets is shown at higher magnification in Fig. 3. x 3700.

Fine structure of developing pollen

296 J. M. Sanger and W. T. Jackson

Fig. 3. Higher magnification of the area in brackets in Fig. 2. Randomly arrayedmicrotubules (arrows) are present adjacent to the nucleus (;»). x 22000.

Fig. 4. A plastid which seems to be dividing (dp) is present in the cytoplasm of amicrospore undergoing mitosis, x 28000.

Fig. 5. Telophase of microspore mitosis. Most of the organelles are between thefurrow (/) and the cell plate (cp). A plastid which seems to be dividing (dp) is adjacentto the chromatin (vcfi) of the future vegetative cell. Mitochondria (in), but no plastids,are present between the cell plate and the chromatin (gch) of the future generativecell, x 4500.

Fine structure of developing pollen 297

298 J. M. Sanger and W. T. Jackson

Fig. 6. Newly formed pollen grain. The generative cell wall (gw) is attached to thepollen wall opposite the furrow of the grain. Both nuclei are approximately sphericaland of equal size, although in this figure they appear unequal in size due to plane of thesection, x 4700.

Fig. 7. Fusion of the generative (gw) and the pollen cell walls (pw). The generativewall is formed of finely fibrillar material (arrows) located between 2 unit membranes.Membrane fragments (inf) are located in the area of fusion of the generative wall withthe intine (t) of the pollen wall, x 27000.

Fine structure of developing pollen 299

3<x> J. M. Sanger and W. T. Jackson

Fig. 8. A high-magnification view of membrane fragments (mf) found in the areaof fusion of the 2 walls, and the 2 unit membranes (arrows) adjacent to the genera-tive wall. Microtubules (mf) are found in both the generative (gc) and vegetative(vc) cells in the area of fusion, x 75 coo.Fig. 9. Pollen grain soon after detachment of the generative cell. Lipid bodies (I)surround the generative cell and are found scattered in the cytoplasm of both cells.The vegetative nucleus (vn) is no longer spherical. There has been a great increase inthe number of organelles within the cytoplasm of the vegetative cell, x 4500.

Fine structure of developing pollen