Chapter 1

MALE GAMETOGENESIS
Development and Structure ofSperm

DARLENE SOUTHWORTH 1 and SCOTT RUSSELL 2

1Department of Biology, Southern Oregon University, 1250 Siskiyou Blvd., Ashland, OR

97520 and 2Department of Botany and Microbiology, University of Oklahoma, Norman, OK

73019, U.S.A.

1. Introduction

The full range of gene expression leading to male gamete formation in

flowering plants begins with determination of the stamen whorl in flower
development and ends with release of mature sperm into the embryo sac near
the egg and central cell. In this review we focus on events within the anther
from meiosis to anthesis and follow the development of sperm to their
discharge into the embryo sac. Chapter 3 in this volume covers pollen tube
growth. Here we review the principles of microsporogenesis gleaned from
ultrastructural work several decades ago (Heslop-Harrison, 1968, 1971) and
then describe recent advances and new approaches to male gametogenesis.

1.1. OVERVIEW OF MALE GAMETOGENESIS

In anthers of very young flower buds, a column of microsporocytes

(colloquially called pollen mother cells) is formed in the center of the future
anther sac. With the completion of meiosis, pollen mother cells produce
haploid microspores (typically four per microsporocyte) that differentiate
into pollen (Bedinger, 1992). Microspores develop a complex cell wall, take
up nutrients, and differentiate. Each haploid cell divides asymmetrically into
a larger vegetative cell and a smaller generative cell, both enclosed within
the pollen grain wall. The vegetative cell will not divide again, but will
develop a long extension that is the pollen tube. The generative cell divides

S.S. Bhojwani and W.Y. Soh (eds.), Current Trends in the Embryology ofAngiosperms, 1-16.
© 2001 Kluwer Academic Publishers.
2 Chapter 1

either in the pollen grain or in the pollen tube to form two sperm cells.
Development of pollen is supported by the tapetum; ablation of tapetal cells
interrupts pollen maturation (Mariani et al., 1990). Recent reviews have
emphasized diverse aspects of male gametogenesis: an overview of
gametogenesis and fertilization (Southworth, 1996), the cytoskeleton of
sperm and generative cells (Palevitz and Tiezzi, 1992), association of two
sperm cells with the vegetative nucleus in the male germ unit (Mogensen,
1992), and gene expression in the developing male gametophyte
(Mascarenhas, 1989; Twell, 1994).

2. Microsporogenesis

2.1. MEIOSIS

Microsporocytes are encased in a special callose wall so that after meiosis,

the four haploid microspores are held together (Fig. IA). At the completion
of meiosis, microtubules radiate from telophase nuclei (Brown and Lemmon
1991, 1992a,b; Pickett-Heaps et al., 1999). This system of microtubules
creates nuclear-cytoplasmic domains that were initially interpreted as
spindles (Heslop-Harrison, 1971). However, their formation between non-
sister nuclei makes this interpretation incorrect. The intersection of radiating
microtubules, including those from non-sister nuclei, determines the location
of phragmoplasts creating the cell walls that delineate individual pollen
grams.
Haploid gene expression in the microspore is extensive (Twell, 1994).
One gene expressed in tapetum and in microspores, Bcpl, is essential for
pollen development (Xu et al., 1995). Linker histones have been shown in
transgenic tobacco to be essential for normal meiosis and for pollen
development (Prymakowska-Bosak et al., 1999).

2.2. EXINE AND INTINE DEVELOPMENT

The exine is patterned on microspore plasma membrane surfaces within the

callose wall (Fig. lA-G) (for reviews see Heslop-Harrison, 1968, 1971). A
primexine matrix is deposited between the microspore plasma membrane
and the encasing callose wall (Fig. 1A, E, F). Within this matrix, sites of
condensation of unknown substances create depressions ("crypts") in the
plasma membrane (Fitzgerald and Knox, 1995). Subsequently, on plasma
membrane peaks, pro bacula appear as the first evidence of exine components
(Fig. 1 F, G). After the callose wall is hydrolyzed, sporopollenin is deposited
from the tapetum onto the thickening exine, but by this time, the pattern is
already established (Fig. 1H). Following exine formation, the intine, a
glycan cell wall, forms from within the microspore. In spite of detailed
Male Gametogenesis 3

studies of exine development in diverse families over the intervening

decades (e.g., Dickinson and Sheldon, 1986; Rowley and Dahl, 1977;
Takahashi and Skvarla, 1991 ), no prevailing theory of exine patterning has
been developed.

Figure I. Vigna unguiculata pollen development. A. Unpatterned microspore. B. Unpatterned

microspore released from tetrad by pressure. Apertures bulge, but surface of microspore is
smooth. C. Transitional stage of unpatterned microspore with surface depressions. D.
Microtubules radiate from nuclear surface and curve under the cell surface (confocal). E.
Microspore with regular depressions in the surface that coincide with future lumina of
reticulum. F. Microspore with patterned primexine reticulum (still within callose wall). G.
Muri of reticulum slightly thicker. H. Mature pollen grain with reticulum. A-G, X 1500. H, X
600
4 Chapter 1

One hypothesis for the ongm of exine pattern involves rigid

microtubules, according to the cellular tensegrity model (Ingber, 1993;
Southworth and Jernstedt, 1995), and pressure within the cavities in the
callosic tetrad. In Vigna unguiculata, with a thick, coarse reticulate exine
pattern on mature pollen, microtubules radiate from the nucleus after
cytokinesis (Fig. lD) (Southworth and Jernstedt, 1995). As the microspore
secretes a primexine matrix into the space between the plasma membrane
and the restricting callose wall, pressure is created by osmotic pressure and
radiating microtubules within the microspore and by hydration of the
primexine surrounding them (Fig. lA-E). Microtubules move to minimize
the pressure and create protruding regions of the plasma membrane on which
primexine elements form, thus laying down the exine pattern. This
hypothesis is based on changes in the location of microtubule ends at the
plasma membrane during exine patterning. The callose wall is an essential
feature of pollen development because mutants with premature dissolution of
callose do not survive (Worrall et al., 1992)
Another approach to exine patterning involves mutants of Arabidopsis
thaliana. In one mutant, dexl, the reticulate pattern is lost (Paxson-Sowders
et al., 1997). In the tetrad, no pattern of plasma membrane protuberances
forms to generate the arrangement of the probacula bases. Sporopollenin is
deposited from the tapetum, but the exine pattern fails to form. In two other
mutants of Arabidopsis thaliana, the four haploid cells do not separate
completely, remaining together as a tetrad (Preuss et al., 1994).
During late maturation of pollen, after the exine has developed, additional
materials are deposited on the surface and in the interstices of the exine.
These materials are essential for subsequent steps in pollen germination
(Preuss et al., 1993).

3. Development of the Male Gametophyte

3.1. FIRST DIVISION OF MICROSPORE

The asymmetric division of the microspore apparently activates a divergence

of cellular programmes to be initiated in the generative and vegetative cells
(Twell, 1994; Eady et al., 1995; Park et al., 1998). An apparently unique
cellular event among higher plants is the migration of the generative cell
from its position against the intine (Fig. 2) to the interior of the vegetative
cell where it remains immersed for the rest of its life (Russell et al., 1996).
As a result, the metabolic needs of the generative cells and later the sperm
may be met exclusively by the vegetative cell, except for those functions
crucial to the fate of the sperm. Thus, the generative cells and sperm require
less expression of housekeeping genes. Functions unique to male germ cells
Male Gametogenesis 5

include cell recognition, fusion mechanisms and post-fusion triggers for the
delivery of hereditary information to the egg and central cell.

Figure 2. Generative cell in immature pollen of Plumbago zeylanica. The generative cell
contains a full complement of organelles. Adjacent to the generative cell is the vegetative
nucleus. ER, Endoplasmic reticulum; M, Mitochondria; N, Nucleus; P, Plastids; V, Vacuoles;
VN, Vegetative nucleus. X 7000

Contact between the vegetative cell and the generative cell is close,
forming a type of cell-cell junction in which membranes closely parallel
each other (Southworth, 1992). No membrane bridges link vegetative and
generative cells. Although the separation distance between the cell
membranes of the vegetative and generative cells is uneven in aldehyde-
fixed thin sections, in freeze-fractured or freeze-substituted pollen, the
separation distance is uniform, and plasma membranes are parallel (Cresti et
al., 1987; Southworth et al., 1989a). In freeze-fractured pollen, the surface of
the generative cell is indented. Distinctive fracture patterns of parallel ridges
occur on the inner vegetative cell membrane at the indentations. The ridged
6 Chapter 1

pattern suggests a junction, either for maintaining cell contact or for passage
of materials but does not precisely resemble tight or gap junctions.
Undoubtedly, the vegetative cell is responsible for nourishment of the
generative cell and for controlling its cell cycle; however, there is no direct
evidence for transfer of molecules between them. The vegetative cell stores
food during its development within the anther and later takes up nutrients
from the style during pollen tube growth.

3.2. DEVELOPMENT OF THE GENERATIVE CELL

The generative cell differentiates and develops structural properties that are
later inherited by the sperm. After the first pollen mitosis, the wall around
the generative cell decreases and in many species disappears completely.
The generative cell becomes more spherical and lies in a cavity or pocket
within the vegetative cell. A generative cell is surrounded by two
membranes: its own cell membrane and the inner cell membrane of the
vegetative cell pocket. Generative cells are closely associated with the
vegetative nucleus, often with a cellular extension entwined through the
vegetative nucleus but always separated from it by the two cell membranes
(Mogensen, 1992; Yu and Russell, 1993).
Chromatin in the generative nucleus condenses to become denser than that
in the vegetative nucleus (Tanaka, 1997). This correlates with changes in
histones that apparently increase the packing density of gametic DNA. In
lily, generative cells and sperm synthesize male-specific histones that are
variants of histones H2B and H3 (Ueda and Tanaka, 1995a,b). Genes for
H2A and H3 substitution histones were subsequently isolated from
generative cell eDNA libraries of lily and are also specific to male germ cells
(Xu et al., 1999b). These altered histones are not found in vegetative cell
nuclei. No specific function has been demonstrated yet, although they
correlate with chromatin condensation in generative cells and in sperm and
suggest a diminished capacity for transcription.
Generative cells and their descendent sperm cells have distinctive
physiological and biochemical characteristics (Chaboud and Perez, 1992)
including the ability to transcribe their own mRNA (Blomstedt et al., 1996),
translate polypeptides (Zhang et al., 1993), and express a genetic programme
distinct from that of the pollen vegetative cell (Xu et al., 1999a). The male
germ cells under express routine "housekeeping" genes, and instead appear
to import pollen compounds required to meet the nutritional and
physiological needs of the gametes. Genes expressed in the male gamete
lineage appear to include an enriched number of control genes, including
cyclins, protein kinases and a distinct form of ubiquitin that has a unique
pattern of activity that differs from that of the pollen (Xu et al., 2000). A
DNA repair gene similar to human ERCCJ has been found expressed in the
male germ cell lineage but not elsewhere, suggesting that special protection
Male Gametogenesis 7

of the DNA occurs in the male gametes but not in the somatic cells (Xu et
a/., 1998).
The cytoskeleton of generative cells is distinct from that of somatic cells.
Immature isolated generative cells are spheroidal, with a meshwork of
microtubules (Zhou and Yang, 1991). As microtubules elongate, the
generative cell becomes ellipsoidal and finally spindle-shaped with
cytoplasmic extensions at one or both ends of the cell. Parallel microtubule
bundles form a slightly spiraled cage or basket of microtubule bundles in the
thin cytoplasmic layer around the nucleus (Del Casino et a/. 1992;
Bohdanowicz et a/., 1995). The extensions vary in precise shape and
location, but consistently include ends of microtubule bundles. Microtubule
bundles are not regularly bridged to the plasma membrane or to the nuclear
envelope (Cresti et al., 1990).
Evidence for actin microfilaments in generative cells is conflicting
although microfilaments have been observed in vegetative cells (Taylor et al.
1989; Lancelle et al., 1987; Palevitz and Liu, 1992; Knox et al., 1993). In
Brassica, actin filaments encircle the generative cell, but it was not clear
whether the actin was in the generative cell or in the vegetative cell
surrounding it (Hause et al., 1992). Knox et al. (1993) showed short actin
microfilaments in generative cells of lily in isolated vegetative cell
protoplasts in culture. In contrast to the rapid movement of organelles in the
vegetative cell, cytoplasmic streaming is minimal in generative cells
(Pierson et al., 1990). This is consistent with an absence or impoverishment
of actin microfilaments which are typically involved in cytoplasmic
streaming (Palevitz and Liu, 1992).

3.3. DIVISION OF GENERATIVE CELL TO FORM SPERM

The generative cell divides to form two sperm. When pollen grains are
released from the anther, they are either bicellular, with one vegetative cell
and one generative cell, or tricellular, with one vegetative cell and two sperm
cells, depending on the timing of generative cell division.
Timing of cell division is hereditary, but regulative mechanisms are
unknown. Generative cell division within the pollen grain apparently
involves a typical mitosis with the formation of a cell plate. Division within
the pollen tube, however, is unusual in that spatial constraints in the
generative cell confine the cell plate to a highly oblique or longitudinal
orientation within the pollen tube (Terasaka and Niitsu, 1989). A skewed
spindle is formed, with chromosomes on the metaphase plate nearly parallel
to the length of the pollen tube. The cell plate is reduced.
The cytoskeleton of the generative cell gives rise to the cytoskeleton of
the sperm cell. In tobacco, generative cell microtubule bundles disassemble,
then reorganize as a spindle apparatus, followed by formation of new
microtubule bundles in sperm (Palevitz, 1993; Yu and Russell, 1993). In
8 Chapter 1

Tradescantia, generative cell microtubule bundles participate directly in

spindle formation by kinetochore-fibre capture (Palevitz and Cresti, 1989;
Palevitz, 1990; Liu and Palevitz, 1991, 1992; Palevitz and Tiezzi, 1992).
Interphase microtubules do not disassemble. Microtubule bundles are present
during prophase and become part of the spindle apparatus. After cytokinesis,
the original microtubule bundles of generative cells form the microtubule
bundles of sperm. In sperm, microtubule bundles generally consist of fewer
microtubules than in generative cells but not precisely half as many (Cresti
et al., 1990; Yu and Russell, 1993).
Generative cell division forming the two sperm cells apparently triggers a
morphogenetically controlled programmatic change correlated with a major
transition in proteins (Bedinger and Edgerton, 1990). Notably, there are no
reports of generative cells acquiring the ability to function as gametes
without the occurrence of an ensuing cell division (Battaglia, 1980, 1989).

4. Gamete Structure

4.1. SPERM

Sperm cells in flowering plants are structurally reduced compared to other

plant cells. Each sperm cell contains a nucleus and a slender cytoplasm that
usually contains heritable cytoplasmic organelles: mitochondria and, in some
species, plastids (Russell, 1991). Specialized features of sperm include an
absence of organized cell walls, a small cytoplasmic volume, condensed
chromatin, linear microtubule bundles and an elongated, spindle shape, with
long extensions (Fig. 3). Sperm cells are not directionally motile and have
limited ability to change their shape.
As with the generative cell, the sperm in pollen grains and tubes are
surrounded by two membranes: the sperm cell membrane plus the inner cell
membrane of the vegetative cell. Sperm cell shape ranges from spheroidal to
highly elongate, with one or more thin extensions 30 J.lm or longer. They are
unusually small, as little as 3 J.lm or smaller in diameter, with few organelles,
little cytoplasm, and condensed chromatin. The cytoskeleton is similar to
that in generative cells, consisting of a basket or cage of microtubule bundles
arranged around the nucleus.
The small size of sperm cells is derived from the small size of the
generative cell, from their lack of growth, and from cytoplasmic diminution
observed in serial reconstructions (Yu and Russell, 1992). Vesicles or
cytoplasmic bodies containing membranous organelles and rarely
microtubu1es pinch off from sperm cells (Mogensen and Rusche, 1985; Yu
et al., 1992). Vesicles remain in pockets in the vegetative cell. Further loss
of cytoplasm may occur at fertilization when a cytoplasmic body is left
Male Gametogenesis 9

outside the egg (Mogensen, 1982, 1988), although male cytoplasmic

reduction may be far less in other species (Russell et al., 1990). Typically,
sperm cells lack plastids, with the consequence that exclusively maternal
plastids are inherited in the resulting embryo (Hagemann and Schroder,
1989; Corriveau and Coleman, 1988). The extent of biparental plastid
inheritance in plants with sperm plastids is largely determined by maternal
permissiveness, however, and not just the number of sperm plastids (Zhu et
al., 1993).
Sperm nuclei are oval to elongate with most of the nuclear volume
occupied by condensed chromatin in a tightly packed mass that fluoresces
brightly with DNA-fluorochromes. In thin sections, condensed chromatin is
unevenly stained. Nucleoli are difficult to identify at some stages.
Occasional nuclear "vacuoles" or spheroidal zones free of chromatin, are
observed (Southworth and Knox, 1989). Mitochondria have also been
observed in nuclei of sperm cells in plants where division of generative cells
occurs in the pollen tube and are apparently trapped there during
reconstitution of the nuclear envelope under close conditions in the pollen
tube (Yu and Russell, 1994c). The nuclear envelope lies in contact with
condensed chromatin, and nuclear pores are sparse to absent (Southworth et
al. 1989b; Southworth, 1990). Sperm organelles include mitochondria,
plastids (in some species), ribosomes, endoplasmic reticulum, dictyosomes
and vesicles. No structure comparable to an acrosome has been detected.
As in generative cells, bundles of microtubules, with a predominantly
axial orientation, branch and rejoin to form a slightly twisted basket or cage
of microtubule bundles surrounding the sperm nucleus (Cresti et al., 1992,
Palevitz and Tiezzi, 1992; Pierson and Cresti, 1992; Knox et al., 1993).
Microtubule bundles show no particular association with the plasma
membrane or nuclear envelope, although the small volume of cytoplasm and
prominent nucleus restricts to a cortical position, close to both nuclear and
cell plasma membranes. Similar patterns of microtubule bundles are found in
sperm cells located in tricellular pollen at anthesis and in sperm cells formed
in pollen tubes of bicellular pollen. Microtubule bundles terminate in the
cellular extensions. Because microtubule bundles join and diverge, the
number of bundles and the microtubules per bundle changes along the length
of the sperm so that a single cross-section provides little quantitative
information.
Sperm of some species are dimorphic with respect to size and number of
organelles (Russell, 1991; Zhu et al., 1992; Yu and Russell, 1994b). Other
species show slight differences in size and shape, but no distinct dimorphism
(Mogensen and Rusche, 1985). In Plumbago, with the most dimorphic sperm
described so far, the sperm closest to the vegetative nucleus had typically no
plastids and over 200 mitochondria while the other sperm contained fewer
mitochondria and over 20 plastids (Russell, 1984). Isolation of two
cytologically distinct populations of sperm cells in Plumbago zeylanica
10 Chapter 1

(Russell, 1984, 1991 ), which differ in function during fertilization (Russell,

1985, 1992), may shed light on whether preferential fertilization depends on
different programmes in the two types of sperm cells present in the pollen
and provide differentially expressed eDNA libraries (Zhang et al., 1998).

Figure 3. Diagrammatic reconstruction of the male germ unit of Nicotiana tabacum,

illustrating the leading sperm (Svn) associated with the vegetative nucleus (VN), the trailing
sperm (Sua), and the pattern of cytoplasmic reduction. Sperm produce enucleated cytoplasmic
bodies (ECB) and vesicle-containing bodies (unlabelled arrows and arrowheads), one of
which (asterisk) is visible in the trailing cytoplasm of the vegetative cell (VC). G, Golgi body;
L, lipid body; M, mitochondrion; NU, nucleolus; PTW, pollen tube wall ; V, vacuole; W, sperm
crosswall. X 4000
Male Gametogenesis 11

Changing conditions of the sperm cell surface are indicated during

maturation. Just after sperm cell formation, the two cells can fuse
spontaneously if placed in apposition, in vitro, whereas at later stages, sperm
cells must be exposed to enzymes including cellulase and pectinase and
incubated in dilute Ca2+ to trigger fusion (Tian and Russell, 1998). This
suggests that the surface becomes less susceptible to spontaneous fusion and
presumably more specific in its recognition of the female target cells. A
transitory aniline blue-fluorescent wall material remains between the two
adjacent sperm cells (Russell et al., 1996; Yu and Russell, 1993), assuring
that the cells remain separated during passage in the pollen tube.

4.2. MALE GERM UNIT

Sperm in pollen tubes remain in close association with each other and with
the vegetative nucleus. In many species, one sperm is attached, via a long
extension, to the vegetative cell membrane appressed to the vegetative
nucleus. The second sperm is attached to the opposite end of the first either
by membrane contact or by extracellular matrix. This tripartite structure
(Fig. 3 ), two sperm plus vegetative nucleus, is called the male germ unit
(Dumas et al., 1984; Mogensen, 1992; Yu et al., 1992). Contacts between
vegetative cell and generative cell are transient, separating during division of
the generative cell and reforming as a contact between one sperm and the
vegetative cell (Palevitz, 1993; Yu and Russell, 1994a). The two sperm cells
and vegetative nucleus remain together during pollen tube growth and
passage of sperm through the pollen tube, but separate quickly when the
pollen tube is ruptured. This association serves to deliver two sperm cells
simultaneously to the embryo sac for double fertilization. Although the male
germ unit travels proximally to the calcium-rich pollen tube tip and high
concentrations of calcium appear to compromise sperm viability (Zhang et
a!., 1995), calcium must be present in the anther at sufficient concentration
during pollen development to assure fertilization success (Tian et al., 1998).
The male germ unit is not independently motile, but transported through
associations between microfilament bundles in the pollen, and myosin on the
outer surface ofthe vegetative nucleus and germ cells (Heslop-Harrison and
Heslop-Harrison, 1989; Tang et al., 1988). Myosin II is involved in the male
germ unit; myosins I and V are found in association with nearby pollen tube
organelles (Miller et al., 1995). This association serves to deliver two sperm
cells simultaneously to the embryo sac for double fertilization. When
released from the pollen tube, the male germ unit loses its outer vegetative
cell membrane (Zhang et al., 1999). Myosin of the pollen cytoplasm
apparently becomes charge-bound to the sperm plasma membrane
facilitating further movement in the embryo sac (Russell, 1996; Zhang and
Russell, 1999). Ultimately, the sperm cells separate with gametic fusion
(Russell, 1992).
12 Chapter 1

5. Conclusions

The major trend in the study of male gametogenesis is a more molecular

approach. Use of mutants, particularly of Arabidopsis, creation of eDNA
libraries from microspores, generative cells and sperm, development of
transgenic plants using pollen-specific promoters, and characterization of
male gamete-specific gene expression have created opportunities for further
investigation. A second trend in the descriptive approach is use of new
methods for preparation of specimens: rapid-freezing and freeze substitution
for fixing immature stages, antibodies to cytoskeletal components, in-situ
hybridizations, and confocal microscopy.

6. Acknowledgements

This work was funded m part by NSF-RUI grant IBN-9816945 to D.

Southworth.

7. References

Battaglia, E. (1980) Embryological questions. 1. On the occurrence ofthe last step of the male
gametophyte evolution in Spiranthes (Orchidaceae), Annali Bot. 39, 1-7.
Battaglia, E. (1989) Embryological questions. 15. Does the case spore-nucleus=egg-nucleus
occur in angiosperm female gametophytes? Annali Bot. 48, 7-48.
Bedinger, P.A. (1992) The remarkable biology of pollen, Plant Cel/4, 879-887.
Bedinger, P.A. and Edgerton, M.D. (1990) Developmental staging of maize microspores
reveals a transition in developing microspore proteins, Plant Physiol. 92, 474-479.
Blomstedt, C.K., Knox, R.B. and Singh, M.B. (1996) Generative cells of Lilium longiflorum
possess translatable mRNA and functional protein synthesis machinery, Plant Mol. Bioi.
31, 1083-1086.
Bohdanowicz, J., Ciampolini, F. and Cresti, M. (1995) Striped projections of the outer
membrane of the generative cell of Convallaria majalis pollen, Sex. Plant Reprod. 8, 223-
227.
Brown, R.C. and Lemmon, B.E. (1991) The cytokinetic apparatus in meiosis: Control of
division plane in the absence of a preprophase band ofmicrotubules, in C. Lloyd (ed.), The
Cytoskeletal Basis of Plant Growth and Form, Academic Press, London, pp. 259-273.
Brown, R.C. and Lemmon, B. E. (1992a) Control of division plane in normal and griseofulvin-
treated microsporocytes of Magnolia, J Cell Sci. 103, 1031-1038.
Brown, R.C. and Lemmon, B.E. (1992b) Cytoplasmic domain: A model for spatial control of
cytokinesis in reproductive cell of plants, EMSA Bulletin 22, 48-53.
Chaboud, A. and Perez, R. (1992) Generative cells and male gametes: Isolation, physiology,
and biochemistry, Inti. Rev. Cytol. 140, 205-232.
Corriveau, J.L. and Coleman, A.W. (1988) Rapid screening method to detect potential
biparental inheritance of plastid DNA and results for over 200 angiosperm species, A mer.
J Bot. 75, 1443-1458.
Cresti, M., Lancelle, S.A. and Hepler, P.K. (1987) Structure of the generative cell wall
complex after freeze substitution in pollen tubes of Nicotiana and Impatiens, J Cell Sci.
88, 373-388.
Male Gametogenesis 13

Cresti, M., Murgia, M. and Theunis, C.H. (1990) Microtubule organization in sperm cells in
the pollen tubes of Brassica oleracea L., Protoplasma 154, 151-156.
Cresti, M., Blackmore, S. and Van Went, J.L. (1992) Atlas of Sexual Reproduction in
Flowering Plants, Springer-Verlag, New York.
Del Casino, C., Tiezzi, A., Wagner, V.T. and Cresti, M. (1992) The organization of the
cytoskeleton in the generative cell and sperms of Hyacinthus orienta/is, Protoplasma 168,
41-50.
Dickinson, H.G. and Sheldon, J. (1986) The generation of patterning at the plasma membrane
of the young microspore of Lilium, in S. Blackmore and I. Ferguson (eds), Pollen and
Spores: Form and Function, Academic Press, London, pp. 1-17.
Dumas, C., Knox, R.B., McConchie, C.A. and Russell, S.D. (1984) Emerging physiological
concepts in fertilization, What's New Plant Physiol. 15, 17-20.
Eady, C., Lindsey, K. and Twell, D. (1995) The significance of microspore division and
division symmetry for vegetative cell-specific transcription and generative cell
differentiation, Plant Cel/7, 65-74.
Fitzgerald, M.A. and Knox, R.B. (1995) Initiation of primexine in freeze-substituted
microspores of Brassica campestris, Sex. Plant Reprod. 8, 99-104.
Hagemann, R. and Schroder, M.B. ( 1989) The cytological basis of the plastid inheritance in
angiosperms, Protoplasma 153, 57-64.
Hause, G., Hause, B. and Van Lammeren, A.A.M. (1992) Microtubular and actin filament
configurations during microspore and pollen development in Brassica napus cv. Topas,
Can. J Bot. 70, 1369-1376.
Heslop-Harrison, J (1968) Pollen wall development, Science 161, 230-237.
Heslop-Harrison, J. (1971) Wall pattern formation in angiosperm microsporogenesis, Symp.
Soc. Exp. Bioi. 25, 277-300.
Heslop-Harrison, J. and Heslop-Harrison, Y. (1989) Myosin associated with the surfaces of
organelles, vegetative nuclei and generative cells in angiosperm pollen grains and tubes, J.
Cell Sci. 94, 319-325.
Ingber, D. (1993) Cellular tensegrity: Defining new rules of biological design that govern the
cytoskeleton, J Cell Sci. 104, 613-627.
Knox, R.B., Zee, S.Y., Blomstedt, C. and Singh, M.B. (1993) Male gametes and fertilization
in angiosperms, New Phytol. 125, 679-694.
Lancelle, S.A., Cresti, M. and Hepler, P.K. (1987) Ultrastructure of the cytoskeleton in
freeze-substituted pollen tubes of Nicotiana alata, Protoplasma 140, 141-150.
Liu, B. and Palevitz, B.A. (1991) Kinetochore fiber formation in dividing generative cells of
Tradescantia. Kinetochore reorientation associated with the transition between lateral
microtubule interactions and end-on kinetochore fibers, J Cell Sci. 98, 475-482.
Liu, B. and Palevitz, B.A. (1992) Anaphase chromosome separation in dividing generative
cells of Tradescantia. Changes in microtubule organization and kinetochore distribution
visualized by antitubulin and CREST immunocytochemistry, Protoplasma 166, 122-133.
Mariani, C., De Beuckeller, M., Truettner, J., Leemans, J. and Goldberg, R. B. (1990)
Induction of male sterility in plants by a chimaeric ribonuclease gene, Nature 347, 737-
741.
Mascarenhas, J.P. (1989) The male gametophyte of flowering plants, Plant Cell1, 657-664.
Miller, D.D., Scordilis, S.K. and Hepler, P.K. (1995) Identification and localization of three
classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata C., Cell Sci.
108, 2549-2563.
Mogensen, H.L. (1982) Double fertilization in barley and the cytological explanation for
haploid embryo formation, embryoless caryopses, and ovule abortion, Carlsberg Res.
Commun. 47,313-354.
Mogensen, H.L ( 1988). Exclusion of male mitochondria and plastids during syngamy in
barley as a basis for maternal inheritance, Proc. Nat/. Acad. Sci. USA 85, 2594-2597.
14 Chapter 1

Mogensen, H.L. (1992) The male germ unit: Concept, composition, and significance, Intl.
Rev. Cytol. 140, 129-147.
Mogensen, H.L. and Rusche, M.L. ( 1985) Quantitative analysis of barley sperm: Occurrence
and mechanism of cytoplasm and organelle reduction and the question of sperm
dimorphism, Protoplasma 128, 1-13.
Palevitz, B.A. (1990) Kinetochore behavior during generative cell division in Tradescantia
virginiana, Protoplasma 157, 120-127.
Palevitz, B.A. (1993) Organization of the mitotic apparatus during generative cell division in
Nicotiana tabacum, Protoplasma 174,25-35.
Palevitz, B.A. and Cresti, M. ( 1989) Cytoskeletal changes during generative cell division and
sperm formation in Tradescantia virginiana, Protoplasma 150, 54-71.
Palevitz, B.A. and Liu, B. (1992) Microfilaments (F-actin) in generative cells and sperm: An
evaluation, Sex. Plant Reprod. 5, 89-100.
Palevitz, B.A. and Tiezzi, A. (1992) The organization, composition and function of the
generative cell and sperm cytoskeleton, Intl. Rev. Cytol.140, 149-185.
Park, S.K., Howden, R. and Twell, D. (1998) The Arabidopsis thaliana gametophytic
mutation gemini pollen! disrupts microspore polarity, division asymmetry and pollen cell
fate, Development 125, 3789-3799.
Paxson-Sowders, D.M., Owen, H.A. and Makaroff, C.A. (1997) A comparative ultrastructural
analysis of exine pattern development in wild-type Arabidopsis and a mutant defective in
pattern formation, Protoplasma 198, 53-65.
Pickett-Heaps, J.D., Gunning, B.E.S., Brown, R.C., Lemmon, B.E. and Cleary, A.L. (1999)
The cytoplast concept in dividing plant cells: Cytoplasmic domains and the evolution of
spatially organized cell division, A mer. J Bot. 86, 153-172.
Pierson, E. S. and Cresti, M, ( 1992) Cytoskeleton and cytoplasmic organization of pollen and
pollen tubes, Inti. Rev. Cytol. 140, 73-125.
Pierson, E.S., Lichtscheidl, I.K. and Derkson, J. (1990) Structure and behaviour of organelles
in living pollen tubes of Lilium longiflorum, J. Exp. Bot. 41, 1461-1468.
Preuss, D., Lemieux, B., Yes, G. and Davis, R.W. (1993) A conditional sterile mutation
eliminates surface components from Arabidopsis pollen and disrupts cell signaling during
fertilization, Gene Dev. 7, 974-985.
Preus, D., Rhee, S.Y. and Davis, R.W. (1994) Tetrad analysis possible in Arabidopsis with
mutation of the QUARTET (QRT) Genes, Science 264, 1458-1460.
Prymakowska-Bosak, M., Przewloka, M.R., Slusarczyk, J., Kuras, M., Lichota, J.,
Killianczyk, B. and Jerzmanowski, A. (1999) Linker histones play a role in male meiosis
and the development of pollen grains in tobacco, Plant Cellll, 2317-2329.
Rowley, J.R. and Dahl, A.O. (1977) Pollen development in Artemisia vulgaris with special
reference to glycocalyx material, Pollen Spores 19, 169-184.
Russell, S.D. (1984) Ultrastructure of the sperm of Plumbago zeylanica II. Quantitative
cytology and three-dimensional organization, Planta 162, 385-391.
Russell, S.D. ( 1985) Preferential fertilization in Plumbago zeylanica: Ultrastructural evidence
for gamete recognition in an angiosperm, Proc. Nat!. Acad. Sci. USA 82,6129-6134.
Russell, S.D. ( 1991) Isolation and characterization of sperm cells in flowering plants, Annu.
Rev. Plant. Physiol. Plant Mol. Bioi. 42, 189-204.
Russell, S.D. (1992) Double fertilization, Inti. Rev. Cytol. 140,357-388.
Russell, S.D. (1996) Attraction and transport of male gametes for fertilization, Sex. Plant
Reprod. 9, 337-342.
Russell, S.D., Rougier, M. and Dumas, C. (1990) Organization of the early post-fertilization
megagametophyte of Populus deltoides. Ultrastructure and implications for male
cytoplasmic transmission, Protoplasma 155, 153-165.
Russell, S.D., Strout, G.W., Stramski, A.K., Mis1an, T.W., Thompson, R.A., Schoemann,
L.M. (1996) Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). I.
Descriptive cytology and three-dimensional organization, Amer. J Bot. 83: 1435-1453.
Male Gametogenesis 15

Southworth, D. (1990) Membranes of sperm and vegetative cells of Gerbera jamesonii, J.

Struct. Bioi. 103, 97-103.
Southworth, D. (1992) Freeze fracture of male reproductive cells, Intl. Rev. Cytol. 140, 187-
204.
Southworth, D. (1996) Gametes and fertilization in flowering plants, Curr. Top. Devel. Bioi.
34, 259-279.
Southworth, D. and Jernstedt, J.A. (1995) Pollen exine development precedes microtubule
rearrangement in Vigna unguiculata (Fabaceae): A model for pollen wall patterning,
Protoplasma 187, 79-87.
Southworth, D. and Knox, R.B. (1989) Isolation of sperm cells from Gerbera jamesonii
pollen, Plant Sci. 60, 273-277.
Southworth, D., Platt-Aloia, K.A., DeMason, D.A. and Thomson, W.W. (1989a) Freeze-
fracture of the generative cell of Phoenix dactylifera (Arecaceae), Se:x. Plant Reprod. 2,
270-276.
Southworth, D., Platt-Aloia, K.A. and Thomson, W. W. (1989b) Freeze-fracture of sperm and
vegetative cells in Zea mays pollen, J. Ultrastruct. Mol. Struct. Res. 101, 165-172.
Tang, X., Hepler, P.K. and Scordilis, S.P. (1989) Immunochemical and immunocytochemical
identification of a myosin heavy chain polypeptide in Nicotiana pollen tubes, J. Cell Sci.
92, 569-574.
Takahashi, M. and Skvarla, J.J. (1991) Exine pattern formation by plasma membrane in
Bougainvillea spectabilis Willd. (Nyctaginaceae), A mer. J. Bot. 78, 1063-1069.
Tanaka, I. ( 1997) Differentiation of generative and vegetative cells in angiosperm pollen, Se:x.
Plant Reprod. 10, 1-7.
Taylor, P., Kenrick, J., Li, Y., Kaul, V., Gunning, B.E.S. and Knox, R.B. (1989) The male
germ unit of Rhododendron: Quantitative cytology, three-dimensional reconstruction,
isolation and detection using fluorescent probes, Se:x. Plant Reprod. 2, 254-264.
Terasaka, 0. and Niitsu, T. (1989) Peculiar spindle configuration in the pollen tube revealed
by the anti-tubulin immunofluorescence method, Bot. Mag. (Tokyo) 102, 143-147.
Tian, H.Q. and Russell, S.D. (1998) The fusion of sperm cells and the function of male germ
unit (MGU) oftobacco (Nicotiana tabacum L.), Se:x. Plant Reprod. 11, 171-176.
Tian, H.Q., Zhang, Z. and Russell, S.D. (1998) Isolation of the male germ unit (MGU):
Organization and function in tobacco (Nicotiana tabacum L.), Plant Cell Rep. 18, 143-
147.
Twell, D. (1994) The diversity and regulation of gene expression in the pathway of male
gametophyte development, in R.J. Scott and A.D. Stead (eds), Molecular and Cellular
Aspects of Plant Reproduction, Cambridge University Press, Cambridge, pp. 83-135.
Ueda, K. and Tanaka, I. ( 1995a) The appearance of male gamete-specific histones gH2B and
gH3 during pollen development in Lilium longiflorum, Dev. Bioi. 169, 210-217.
Ueda, K. and Tanaka, I. (1995b) Male-specific H2B and H3 histones, designated gH2B and
gH3 in Lilium longiflorum, Planta 197, 289-295.
Worrall, D., Hird, D.L., Hodge, R., Paul, W., Draper, J. and Scott, R. (1992) Premature
dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco,
Plant Cell 4, 759-771.
Xu, H., Knox, R.B., Taylor, P.E. and Singh, M.B. (1995) Bcpl, a gene required for male
fertility in Arabidopsis, Proc. Nat!. Acad. Sci. USA 92, 2106-2110.
Xu, H., Swoboda, I., Bhalla, P.L. and Singh, M.B. (1999a) Male gametic cell-specific gene
expression in flowering plants, Proc. Nat!. Acad. Sci. USA 96, 2554-2558.
Xu, H., Swoboda, 1., Bhalla, P.L. and Singh, M.B. (1999b) Male gametic cell-specific
expression ofH2A and H3 histone genes, Plant Mol. Bioi. 39, 607-614.
Xu, H., Swoboda, I., Bhalla, P.L., Sijbers, A.M., Zhao, C., Ong, E.K., Hoeijmakers, J.H. and
Singh, M.B. (1998) Plant homologue of human excision repair gene ERCC1 points to
conservation of DNA repair mechanisms, Plant J. 13, 823-829.
16 Chapter 1

Xu, H., Swoboda, I., Zhang, Z., Liu,. K, Russell, S.D., Bhalla, P.L. and, Singh, M.B. (2000)
Developmental expression of polyubiquitin genes and distribution of ubiquitinated
proteins in generative and sperm cells, Planta 197 (in press).
Yu, H.S. and Russell, S.D. (1992) Male cytoplasmic diminution and male germ unit in young
and mature pollen of Cymbidium goeringii: A 3-dimensional and quantitative study, Sex.
Plant Reprod. 5, 169-181.
Yu, H.S. and Russell, S.D. (1993) Three-dimensional ultrastructure of generative cell mitosis
in the pollen tube of Nicotiana tabacum, Eur. J Cell Bioi. 61, 338-348.
Yu, H.S. and Russell, S.D (1994a) Male reproductive cell development in Nicotiana tabacum:
Male germ unit associations and quantitative cytology during sperm maturation, Sex. Plant
Reprod. 7, 324-332.
Yu, H.S. and Russell, S.D. (1994b) Populations of plastids and mitochondria during male
reproductive development in Nicotiana tabacum L.: A cytological basis for occasional
biparental inheritance, Planta 193, 115-122.
Yu, H.S. and Russell, S.D. (1994c) Occurrence of mitochondria in the nuclei of tobacco
sperm cells, Plant Cell6, 1477-1484.
Yu, H.S., Hu, S.Y. and Russell, S.D. (1992) Sperm cells in pollen tubes of Nicotiana tabacum
L.: three-dimensional reconstruction, cytoplasmic diminution, and quantitative cytology,
Protoplasma 168, 172-183.
Zhang, G., Gifford, D.J. and Cass, D.D. (1993) RNA and protein synthesis in sperm cells
isolated from Zea mays L. pollen, Sex. Plant Reprod. 6, 239-243.
Zhang, G., Williams, C.M., Campenot, M.K., McGann, L.E., Cutler, A.D. and Cass, D.D.
(1995) Effects of calcium, magnesium, potassium and boron on sperm cells isolated from
pollen of Zea mays L., Sex. Plant Reprod. 8, 113-122.
Zhang, Z., Xu, H., Singh, M.B. and Russell, S.D. (1998) Isolation and collection of two
populations of viable sperm cells from the pollen of Plumbago zeylanica, Zygote 6, 295-
298.
Zhang, Z. and Russell, S.D. (1999) Sperm cell surface characteristics of Plumbago zeylanica
in relation to transport in the embryo sac, Planta 208, 539-544.
Zhou, C. and Yang, H.Y. (1991) Microtubule changes during the development of generative
cells in Hippeastrum vittatum pollen, Sex. Plant Reprod. 4, 293-297.
Zhu, T., Mogensen, H.L. and Smith, S.E. (1992) Heritable paternal cytoplasmic organelles in
alfalfa sperm cells: Ultrastructural reconstruction and quantitative cytology, Eur. J Cell.
Bioi. 59, 211-218.
Zhu, T., Mogensen, H.L. and Smith, S.E. (1993) Quantitative, three-dimensional analysis of
alfalfa egg cells in two genotypes: Implications for biparental plastid inheritance, Planta
190, 143-150.