Pollen Development

Pollen development and maturation
Paper: Reproductive Biology of Angiosperms

Lesson: Pollen development and maturation
Lesson Developer: Dr Bharti Chaudhry and Dr
Anjana Rustagi
College/ Deparment: Department of Botany, Ramjas
College and Department of Botany, Gargi College,
University of Delhi
Institute of Lifelong Learning, University of Delhi

Table of contents
Chapter: Pollen development and maturation
• Introduction
• Microsporogenesis
o Significance of callose during microsporogenesis
o Cytokinesis
o Microspore tetrads
• Microgametogenesis
o Vegetative cell (VC)
o Generative cell (GC)
o Sperm Cells
o Male Germ Unit (MGU)
 Significance of MGU
• Maturation of Pollen
• Rare features
o Pollen development in Cyperaceae
o Pollen grain embryo sacs
• Summary
• Practice Questions
• Glossary

POLLEN DEVELOPMENT AND MATURATION
Introduction
Pollen, the yellow dust that carries the male germ line of flowering plants is vital
for sexual reproduction and seed formation. Pollen grains develop within the
anther and are dispersed by dehiscence of the anther. The pollen grains
germinate on the stigma, the pollen tubes grow through the tissues of the stigma
and style, enter the embryo sac and discharge the male gametes for fertilization
and subsequent seed and fruit development. Pollen development involves the
development of microspores within the microsporangium (microsporogenesis)
followed by the development of the male gametophyte/pollen grain containing the
male gametes (microgametogenesis).
Microsporogenesis
The series of events that lead to the development of haploid microspores within
the microsporangium is known as microsporogenesis. It involves the division of
the microspore mother cells by meiosis followed by cytokinesis. The sporogenous
cells may directly function as microspore mother cells/ microsporocytes/
meiocytes/ pollen mother cells or they may undergo a few mitosis to add upto
their number before entering meiosis. Each microspore mother cell ( MMC/PMC)
by a meiotic division gives rise to a group of four haploid microspores. An
aggregate of four microspores is referred to as a microspore tetrad.
Meiosis
Meiosis is one of the most critical events during microsporogenesis and thus in the
development of pollen. Meiosis is an important component of the life cycle of every
reproducing organism because the doubling of chromosome number that takes
place at fertilization is balanced by the halving of the chromosomes at this stage.
Meiosis is always preceded by chromosome duplication in a diploid cell followed by
two successive nuclear divisions, meiosis I and meiosis II leading to the formation
of four haploid cells. Meiosis I is a reduction division that reduces the chromosome
number from diploid to haploid and meiosis II is more or less a normal mitosis.
During meiosis I, homologous chromosomes pair (synapse), exchange genetic
material (crossing-over) and separate (disjoin) from each other. During meiosis II,
the chromatids disjoin from each other. Therefore, apart from reduction in the
number of chromosomes by half, meiosis facilitates recombination of genetic
material, a significant feature of sexual reproduction.
Microsporogenesis includes two important structural features of meiosis:

1. Formation of a syncytium of microspore mother cells during the prophase
stage of meiosis. The syncytium ensures an efficient system for the
distribution of nutrients between the MMC’s of each sporangium and
accounts for the synchrony of meiosis within the microsporangium.
2. Isolation and insulation of individual microspore mother cells by a callose
wall (β-1,3-glucan polymer).
In the young anther, prior to initiation of meiosis, all anther wall layers, including
the tapetum and the sporogenous tissue show plasmodesmatal connections
between the cells of the same layer, as well as with those of adjacent layers.
After initiation of meiosis, the plasmodesmatal connections between the tapetum
and sporogenous tissue are severed, but are retained between cells of the same
layer. The onset of meiosis is also characterized by initiation of callose deposition
around individual microspore mother cells. Callose is deposited between the
plasma membrane and the wall of each MMC. The primary cell wall is finally
degraded in many species. Initially the callose wall is incomplete, with many gaps
in the sites of some plasmodesmata. These gaps enlarge later to form massive
cytoplasmic channels that connect adjacent microspore mother cells. The
cytoplasm and organelles can easily pass through these channels from one
meiocyte to the other. Therefore, all microspore mother cells of a sporangium
form a single multinucleated cytoplasmic entity, the syncytium. Eventually, the
callose wall around the microspore mother cells becomes continuous severing the
cytoplasmic channels. Completion of meiotic division results in the formation of
tetrads of microspores. The callose extends to the walls separating individual
microspores within the tetrads resulting in the isolation of individual microspores.

Callose
Callose is a polysaccharide in the form of β-1,3-glucan with some β-1,6-branches

and it exists in the cell wall of a wide variety of higher plants. Callose plays an
important role during a variety of processes in plant development and/or in
response to multiple biotic and abiotic stresses. Callose has two distinctive
properties
(1) High impermeability
(2) Rapid synthesis and easy degradation.
Callose is produced by callose synthases and it is degraded by β-1,3-glucanases.
Callose is deposited between the plasma membrane and the cellulosic cell wall
and it appears electron-lucent in a transmission electron micrograph. Callose can
be identified with aniline blue (as fluorochrome) by fluorescence microscope. It is
a common wall constituent in the sieve elements of phloem tissue around the
sieve pores. It plays a significant role in the reproductive biology of
angiosperms. It appears temporarily around the microsporocytes and
megasporocytes during microsporogenesis and megasporogenesis respectively.
It is present in the normal course of development in pollen tubes as callose
plugs. Callose accumulates in the walls of incompatible pollen grains and tubes
and in the papillae of stigma following rejection reaction during pollen-pistil
interaction. Callose deposition can also be induced by wounding, infection by
pathogens, aluminium, abscisic acid and other physiological stresses.
Significance of callose during microsporogenesis
• Isolation of meiocytes by a callose wall is essential for the normal

development of pollen. Lack of callose deposition or its early breakdown
results in pollen sterility or formation of non-viable pollen.
• Isolation by callose is necessary for the meiocytes to undergo transition
from the sporophytic phase to the gametophytic phase and for the
expression of gametophytic genome without interference either from other
spores or from the parent sporophytic tissue. According to Heslop-
Harrison, the callose functions as a molecular filter for the isolation of the
meiocytes by selectively preventing entry of some macromolecules like

thymidine and fluorescein diacetate and provides genetic autonomy to the

developing meiocytes.
• Isolation of young microspores by a callose wall is essential for an orderly
deposition of exine (Waterkeyn and Bienfait 1970). The callose wall
provides a template for the formation of species-specific exine sculpturing
pattern seen in mature pollen grains. In members of Cyperaceae, where
three of the microspores of a tetrad degenerate, a callose wall develops
around the microspore mother cell but does not develop around individual
microspores. Consequently, the exine is laid down on outer surface of the
microspores but not along the walls separating individual microspores.
Also, the mutants of Arabidopsis that produce no callose wall do not show
any ornamented exine.
• Callose protects the meiocytes from dehydration and also serves as a
source of nutrition to the developing microspores by providing soluble
carbohydrates following its breakdown. (Barskaya and Balina 1971).
• Callose wall also isolates the products of meiosis to prevent cell
coherence/ fusion and facilitates release of free cells after its dissolution.

b a b
ca
c d
e f
Figure 1. Microsporogenesis in Prosopis juliflora anthers (light micrographs)

(ca, callose; en, endothecium; ep, epidermis; ml, middle layer; mmc, microspore
mother cell; px, primexine; sp, sporogenous cell; t, tetrad; tp, tapetum)
a. T.S. floral bud (part) with tetrasporangiate anthers at sporogenous cell
stage. The anther wall is differentiated into epidermis, an endothecium,
two middle layers and secretory tapetum.
b. T.S. of an anther showing microspore mother cells enclosed in the callose
wall.
c. Anther locule enlarged from ‘b’ showing the thick callose wall investing
individual meiocytes on the inner side of cellulosic wall. Each microspore
mother cell contains a single nucleus with a prominent nucleolus.

d. Anther locule showing microspore mother cells connected by cytoplasmic

channels (arrow).
e. Portion of an anther locule showing initiation of simultaneous centripetal
constriction furrows indicated by arrows (cytokinesis stage).
f. Tetrads invested in callose wall. Each microspore of the tetrad shows the
initials of the exine (primexine) that stains with Periodic acid Schiff’s (PAS)
reagent.
Source: Author
!" #"
Figure 2. Microsporogenesis in Prosopis juliflora (Fluorescence microscopy)

a. Anther locule viewed by fluorescence microscopy showing each microspore
mother cell (black spot) enveloped by callose wall (white). The callose
deposition occurs initially along the corners of the meiocytes, the wall later
extends laterally and forms a continuum.
b. Microspore tetrads with tetrahedral symmetry embedded in thick callose
walls.
Source: Author

!" #"
$" %"
Figure 3. (a-d) Transmission electron micrographs of cross-sections through

anthers of Arabidopsis thaliana.
a. Premeiosis. Microsporocytes are surrounded by the tapetum, middle layer,

endothecium and epidermis.
b. Premeiosis. Higher magnification view of microsporocytes and tapetum.
Plasmodesmata connect microsporocyte to microsporocyte (arrowheads),

tapetal cell to tapetal cell (large arrow) and microsporocytes to tapetal

cells (small arrows). Each microsporocyte contains a single nucleus with a
prominent nucleolus.
c. Premeiosis II. Microsporocytes have secreted callose inside primary cell
walls. Membranes and vesicles can be seen embedded in the callose.
Original plasmodesmatal connections from microsporocyte to
microsporocyte are severed due to deposition of callose wall material
(arrowheads). Cells of the tapetum remain connected to each other by
plasmodesmata (small arrows).
d. Meiosis. Microsporocytes are isolated from each other by thick callose
walls secreted within their primary cell walls, positions of which are
indicated by arrowheads. Condensing chromosomes are present in the
nucleus of one microsporocyte. Tapetal cell protoplasts are located at a
distance from their inner tangential and radial walls with loss of
plasmodesmatal connections between adjacent cells (large arrow).
(C, Callose; Chr, chromosome; En, endothecial cells; EP, epidermis; M,
microsporocyte; ML, middle layer; N, nucleus; Nu, nucleolus; T, tapetal cells;
V, vacuole.)
Source: Owen and Makaroff (1995) ‘Ultrastructure of microsporogenesis and
microgametogenesis in Arabidopsis thaliana (L.) Heynh. ecotype
Wassilewskija (Brassicaceae)’ Protoplasma 185:7-21
Cytokinesis
The process that cleaves the MMC into separate microspore cells is called as
cytokinesis. Wall formation after the meiotic division in PMCs is of two types:
1. Successive cytokinesis is usually common in monocots. The first meiotic
division in the MMC is followed by a wall separating the two nuclei
resulting in the formation of a dyad cell. The wall formation is centrifugal
(cell plate extends from the center laterally). Callose is deposited on either
side of the plate. The two cells of the dyad undergo the second meiotic
division resulting in a tetrad. Tetrads resulting from successive division are
usually isobilateral (also called as tetragonal) or T-shaped or linear or
decussate.
2. Simultaneous cytokinesis is usually common in dicots. The first meiotic
division is not followed by wall formation. Therefore, a binucleate cell is
formed after meiosis I; there is no dyad stage. The two haploid nuclei
synchronously undergo the second meiotic division. Callose walls are
formed after the second meiotic division giving rise to a tetrad. The wall

formation in this type is centripetal (from the periphery towards the

center) that meet in the center of the cell to divide it into four parts
(furrowing). The tetrads formed by simultaneous cytokinesis are usually
tetrahedral or decussate.
Microspore tetrads
Each microsporocyte produces four microspores arranged in various patterns
resulting in differently shaped tetrads. The common arrangement of microspores
in a tetrad is tetrahedral or isobilateral. However, decussate, linear and T-shaped
tetrads are also seen in many species. In Aristolochia elegans all the five type of
tetrads are reported. All the four spores within a tetrad are completely isolated
from one another and from the spores in other tetrads of the locule by the callose
wall. The pollen wall begins to form while the spores are still enclosed in the
callose wall. The pollen wall is made up of an outer sculptured layer called as
exine and an inner smooth layer called as intine.
The initials of the exine (primexine) are laid down at the tetrad stage around
each microspore while enclosed in the callose wall. The primexine is a fibrillar
polysaccharide material. The presumptive apertures are also demarcated during
the formation of the primexine. Probaculum and Protectum are formed within the
primexine after accumulation of sporopollenin at specific sites within the
primexine. The sporopollenin is contributed by the microspore cytoplasm during
the tetrad stage. The microspores are subsequently released in the anther locule
by the breakdown of the callose wall by an enzyme callase secreted by the
tapetum. After the release of the microspores from the callose walls,
sporopollenin, secreted by the haploid microspore as well as the surrounding
tapetum, is incorporated into the developing wall.

!" #"
$" %"
Figure 4. Transmission electron micrographs of cross-section through anthers of

Arabidopsis thaliana
a. Cytokinesis stage. Haploid nuclei are located at the edge of the
microsporocyte. Wall ingrowths (indicated by arrowheads) are present at
the edges of the microsporocytes.
b. Tetrads. Three of the four microspores that make up each tetrad are
visible in the plane of section, surrounded by a common callose wall.
c. Tetrads. Within the callose, each microspore has secreted a wall, the
primexine (arrowheads). Deposition of material at specific sites within the

primexine results in formation of probaculae, some of which are indicated

by small arrows. Tapetal walls have completely degenerated.
d. Released microspores. Each microspore contains a single nucleus with a
prominent nucleolus. Residue of primexine is present at bases of bacula.
(C, Callose; Ms, microspore; N, nucleus; Nu, nucleolus)
microgametogenesis in Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija
(Brassicaceae)’ Protoplasma 185:7-21
! " #
Figure 5. Anthurium andraeanum. TEM Tetrads in an anther locule surrounded by

thick callose (TEM).
a. Tetragonal.
b. Decussate.
c. T-shaped.
(C, callose and PT, plasmodial tapetum)
Source: Furness and Rudall (1999) ‘Microsporogenesis in Monocotyledons’, Annals

of Botany 84, 475–499
Microgametogenesis
Microspores represent the beginning of the male gametophytic generation. The

series of events leading to the development of the male gametes within the
microgametophyte/pollen grains is called as microgametogenesis.
The microspores released from the callose walls are richly cytoplasmic with a
prominent centrally placed nucleus. The microspores/ pollen grains soon undergo

rapid expansion by the uptake of locular fluid and develop a characteristic exine
by the deposition of sporopollenin precursors on the exine initials laid down
during the tetrad stage. The cytoplasm of the pollen grain becomes highly
vacuolated (vacuolate pollen grain stage) and the nucleus is displaced from the
center to one side of the cell (Ring- vacuolate microspores). The displacement is
always in a particular direction and marks the position of the generative cell. A
major re-organisation takes place in the cytoplasm; most of the plastids and
mitochondria move away from the region of the nucleus, resulting in polarity of
organelle distribution. The pollen nucleus undergoes an asymmetric mitotic
division to form a larger vegetative cell (VC) and a smaller generative cell (GC).
The vegetative cell receives most of the plastids, mitochondria and lipid bodies of
the microspore and the generative cell receives a few organelles. The cytoplasm
of the VC and that of the GC is initially separated by two plasma membranes. The
wall of the GC is formed in between the two plasma membranes and adjoins the
intine on either side of the generative cell. The GC is then pinched off from the
wall and moves to the center of the VC. The wall material in many taxa has been
identified as callose that soon disappears. The VC and GC remain separated by
the membranes of the respective cells.
Vegetative cell (VC)

The VC completely engulfs the small GC and is a metabolically active cell with
high levels of RNA and protein synthesis. The VC is rich in plastids, mitochondria
and the number of organelles continue to increase until pollen maturation. The VC
of the mature pollen usually shows a distorted nucleus with dispersed chromatin
and often lacks a nucleolus. The nucleus does not synthesise DNA (remains at 1C
level). Reserve food material in the form of lipids or starch grains accumulates in
the VC.
Generative cell (GC)

GC is completely enclosed within the cytoplasm of the VC. The GC is spheroidal
initially but soon elongates and becomes spindle shaped/ lenticular. The elongate
shape of the GC is maintained by the microtubules oriented just below the
membrane parallel to the long axis of the cell. When the microtubules are
destroyed by exposure to colchicine, the generative cell reverts to the spheroidal
shape. The lenticular shape of the GC facilitates its movement into the pollen
tube. A characteristic feature of GC is the sparsity of organelles, especially of
plastids. This characteristic is inherited by the sperm cells, which thus lack
plastids in their cytoplasm in majority of the angiosperms. The maternal

inheritance of plastids is the norm in many flowering plants. However the GC

contains plastids in some species eg. Oenothera biennis, Medicago sativa,
Pelargonium zonale, Rhododendron indicane, Plumbago zeylanica etc., which
therefore, show biparental inheritance of plastids. The nucleus of the GC shows
condensed chromatin and undergoes DNA synthesis (increases to 2C level). There
is no appreciable synthesis of RNA and protein in the GC. The GC undergoes
second pollen mitosis to produce the sperms (the male gametes).
Sperm Cells
The sperms are formed by a mitotic division in the generative cell. The GC may
divide either inside the pollen grain (tricellular pollen) or in the pollen tube after
germination (bicellular pollen). The pollen grains of about 70% species are
bicellular (eg. Solanaceae, Rosaceae, Betulaceae) and in the remaining 30%
species tricellular ( eg. Asteraceae, Poaceae, Brassicaceae, Caryophyllaceae). The
sperm cells posses their own plasma membrane and a thin polysaccharide
extracellular matrix but lack a rigid structured true cell wall (Mc. Cue et al, 2011).
The sperm cells have a nucleus and a small amount of cytoplasm. The sperm cells
in many species are physically connected to each other and to the vegetative
nucleus forming a male germ unit.
Isolation of live sperm cells of both monocot and dicot flowering plants and using
them for in-vitro fertilization is now possible. In plants with 3-celled pollen, sperm
isolation involves osmotic bursting of freshly collected pollen. In plants with 2-
celled pollen it is possible only after pollen germination.

!" #"
$"
%" &"
Figure 6. Microgametogenesis in Prosopis juliflora anthers (light micrographs).

(gnu, generative cell nucleolus; en, endothecium; ep, epidermis; e, exine; gc,
generative cell; i, intine; ml, middle layer; p, polysaccharide grains; s1 and s2,
sperm cells; tp, tapetum; vc, vegetative cell; vn, vegetative cell nucleus; vnu,
vegetative cell nucleolus)
a. Portion of an anther locule to show the released microspores gorged with
perinuclear polysaccharide grains.
b. Portion of anther locule showing vacuolated pollen grains with vegetative
cell and newly formed generative cell appressed to the wall. The exine of

the pollen grains is well developed. The thickenings in the endothecium

are noteworthy.
c. Anther locule to show bicellular pollen grains with small generative cell
embedded in the vegetative cell cytoplasm.
d. Mature pollen grains showing spindle shaped generative cell. The
vegetative cell nucleus is irregularly lobed.
e. Portion of anther locule showing tricellular pollen with two lenticular
sperm cells present within the vegetative cell cytoplasm. The vegetative
nucleus and the pair of sperm cells are in close association.
Source: Author

!!"
% #
#"
$"
$
Figure 7. Transmission electron micrographs of cross-sections through anthers

of Arabidopsis thaliana
a. Released microspores. Microspores contain numerous vacuoles.
b. Ring-vacuolate microspores. Nucleus is displaced to side of microspore by a
single large vacuole.
c. Bicellular pollen grains. Generative cell has moved away from edge of
pollen grain. Osmiophilic bodies accumulate in cytoplasm of vegetative cell
at generative cell surface.

d. Mature pollen. Cytoplasm of vegetative cell is highly vacuolated and

contains many small osmiophilic storage bodies with electron-transparent
inclusions. Two sperm cells each contain a single nucleus. The exine is
sculptured and the intine is electron transparent.
(N nucleus, V vacuole, GC Generative cell, VC vegetative cell, In intine, SC sperm
cell, VN vegetative nucleus)
microgametogenesis in Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija
(Brassicaceae)’ Protoplasma 185:7-21
Male Germ Unit (MGU)

Male germ unit refers to a physical connection between two sperm cells and the
vegetative cell nucleus. In the bicellular pollen the association is formed in the
pollen tube and in the tricellular pollen, it is formed in the pollen grain. The term
“Male Germ Unit” was coined by Dumas et al. in 1985. MGU forms an effective
unit in which all the DNA, both nuclear and cytoplasmic, of male heredity is linked
together and travels as a single unit through the pollen tube. The sperm cell that
contacts the VN is usually termed SC1. The two sperms may be dimorphic in size,
shape and organelle content.
The first detailed description of physically connected dimorphic sperm cells were
from Plumbago zeylanica (Plumbaginaceae) by Russell and Cass 1981, Russell
1984. In Plumbago zeylanica, the pollen grain is tricelled. The two sperm cells are
directly linked together by a common extracellular matrix with potential
intercellular connections. One of the sperms (SC1/SVn) is consistently associated
with the vegetative nucleus through a cytoplasmic projection that is wrapped
around the vegetative nucleus. The surface of the VN is highly lobed possessing
many indentations and numerous nuclear pores on the VN surface. The
cytoplasmic projection of the SC attaches at the sites of these indentations,
referred to as clasping grooves (Russell, 1984). The two sperms differ in size,
shape and the number of organelles. The smaller sperm( SC2/Sua) is not
associated with the vegetative nucleus and contains more plastids (24) and fewer
mitochondria (40) as compared to the larger sperm. The larger sperm (SC1/Svn)
is closely associated with the vegetative nucleus and is rich in mitochondria (256)
but contains only a few plastids. The cytoplasmic projection may play a role in
dictating the ordered arrival of each SC to the embryo sac, thereby determining
which SC fertilizes the egg (Mc Cue et al. 2011). In Plumbago, SC1 preferentially
fuses with the polar nuclei, generating the triploid endosperm and SC2
preferentially fertilizes the egg cell (Russell 1985).

In Brassica campestris (tri-celled pollen), the sperm cells are not wrapped around
the vegetative nucleus but one sperm is connected to it by a cytoplasmic
projection about 10 µm long that is supported by the cytoskeleton of
microtubules (Mc Conchie and Knox 1985). The two interconnected sperm cells
exhibit sperm dimorphism with respect to size, shape and the number of
mitochondria but lack plastids in the sperm cells. The SC associated with the VN
has more number of mitochondria. These associations established within mature
pollen grains are maintained throughout pollen tube growth.
The occurrence of MGU has been observed in several species with 2-celled pollen
grains, where sperms are not formed until pollen germination such as Petunia
hybrida, Nicotiana tabacum, Rhododendron species, Aloe ciliaris, Sambucus nigra
etc. In Nicotiana tabacum, the two sperm cells present inside the pollen tube are
arranged in tandem and are closely associated with the vegetative nucleus, which
always takes the lead (Yu, Hu and Zhu 1989). The leading sperm cell (SC1) has a
long and narrow cytoplasmic projection, which lies within the enclaves of the
much lobed vegetative nucleus, thus forming a physical association. The trailing
sperm cell (SC2) and the SC1 are not only joined by a common transverse cell
wall but also are surrounded by a periplasm bounded by the plasma membrane of
the sperm cells and that of the vegetative cell, thus forming a structural
connection. The sperm cells are elongated with cytoplasmic projections at the
anterior end of the SC1 and at both ends of the SC2. The cytoplasm of both
sperm cells includes mitochondria, endoplasmic reticulum, dictyosomes,
ribosomes, small vacuoles and axially oriented microtubules but no plastids.
In most angiosperm species, the VN precedes the SCs in the growing pollen tube.
The cytoplasmic projection becomes reduced near the end of the pollen tube
growth and is severed following release of the SCs. The cytoplasmic projection is
known to posses a microtubule based cytoskeletal framework that may provide
structural support to the cytoplasmic projection. The most obvious potential role
of the cytoplasmic projection is the physical connection and stabilization of the
MGU in pollen tube.
In grasses, which are monocots with 3-celled pollen, neither the sperms are in
close association with the vegetative nucleus nor are they dimorphic (Triticum,
Hordeum, Zea mays, Triticale, Secale). However, a close association is formed
after pollen hydration and activation in Zea mays and after in vitro pollination in
Hordeum.
The MGU is not motile on its own. It is transported in the pollen tube through
association between the actin microfilament bundles in the pollen cytoplasm and
myosin on the surface of the sperm cells and the vegetative nucleus.

Figure 8.
a. Reconstruction of the two sperm cells of Plumbago zeylanica without the
associated vegetative nucleus. The larger of the sperm cells is associated with the
vegetative nucleus and is designated Svn. The sperm cell not associated with the
vegetative nucleus is labeled Sua. The cellular projection of the Svn is indicated
by arrowheads.
b. Reconstruction of the two sperm cells of Plumbago zeylanica and the
associated vegetative nucleus with superimposed profiles of mitochondria and
plastids. The Svn contains a majority of the mitochondria and two plastids near
the sperm crosswall (arrowheads). The Sua contains most (and usually all) of the
plastids and significantly fewer mitochondria. Note the gradation between the
main body of the Svn and its cellular projection
Source: Russel SD (1984) Ultrastructure of the sperm of Plumbago zeylanica II.
Quantitative cytology and three-dimensional organization Planta162:385-391

Figure 9. Schematic diagram of the male germ unit from a single pollen grain of
Brassica campestris showing placement of vegetative nucleus (VN), sperm cells
(SC1 and SC2) with nuclei (N), mitochondria (M) and arrays of microtubules
(MT).
Source: Mc Conchie et al (1985), Computer-assisted reconstruction of the male
germ unit in pollen of Brassica campestris. Protoplasma 127:57-63

30 Pollen development and maturation
Hong-Shi Yu et al.: Ultrastructure of sperm cells and the male germ unit in pollen tub
30 Hong-Shi Yu et al.: Ultrastructure of sperm cells and the male germ unit in pollen tubes of Nicotiana tabacum
!" #"
Figs. 1-4
Figure 10. Male Germ Unit in Nicotiana tabacum
a. Fluorescence micrograph showing the vegetative nucleus and the sperm

nuclei in a pollen tube of Nicotiana tabacum growing in a style.
b. A longisection of a pollen tube in the style, showing a MGU. Note the
cytoplasmic projections at both the anterior end of the SC 1 and the
posterior end of the SC2.
(CP, cytoplasmic projection; Nu nucleolus; S1 the leading sperm cell; S2 the

trailing sperm cell; SN sperm nucleus; TW transverse wall; V vacuoles; VC
vegetative cytoplasm; VN vegetative nucleus)
Source: Yu HS, Hu SY, Zhu C (1989) Ultrastructure of sperm cells and the male
germ unit in pollen tubes of Nicotiana tabacum. Protoplasma 152: 29–36
Figs. 1-4

Significance of MGU
• The occurrence of MGU, whether formed before or after pollen

germination, is probably a universal feature among the flowering plants.
• MGU linkage is necessary to direct the movement of sperms in the
growing pollen tube. During pollen tube growth destabilizing the MGU by
exposure to heat can result in dissociation of the SCs, the loss of SC
movement or the failure of the SCs to enter the growing pollen tube (Ge
et al. 2011).
• MGU may dictate the ordered arrival of each SC to the embryo sac and
ensure preferential fusion with the egg and the polar nuclei.
• Sperm to sperm linkage may have a role in reducing the incidence of
heterofertilization (the sperms involved in syngamy and triple fusion are
from different pollen tubes) that usually occurs in maize and barley.
• The association serves to deliver the two sperm cells simultaneously to the
embryo sac and ensures a co-ordinated development of embryo and
endosperm.
• The cytoplasmic projection and the male germ unit may facilitate close
communication and transfer of information between the somatic
vegetative cell nucleus and the germinal sperm cells via RNA and/or
protein transport (Mc. Cue et al. 2011).
• It may facilitate crop improvement by genetic engineering of the sperm
cell involved in syngamy and utilizing it for in-vitro fertilization.
Maturation of Pollen
The pollen grains undergo maturation prior to anther dehiscence to prepare
themselves for independent survival, interaction (cross-talk) with the female
sex organ, germination and pollen tube growth (Shi and Yang, 2010).
Maturation of pollen involves three major aspects:
• Dehydration/ dessication of pollen.

Pollen dehydration maintains the pollen in a metabolically inactive or
quiescent state. It usually causes a decrease in volume accompanied
by a change in pollen shape. Mature dehydrated pollen can survive in
dry state for extended periods of time and accommodate changes in
relative humidity of the environment to which they are exposed.

• Elaboration of pollen wall

The pollen wall is the most complex wall than any other plant cell wall
that enables the pollen to withstand physical abrasion, dessication and
UV-B radiation. The wall of the pollen grain comprises of two distinct
layers: the inner intine and the outer exine. The exine layer is highly
sculptured and ornamented. The different sculpturing patterns of the
exine have long been used for taxonomic classification and for forensic
identification. The exine is made up of an elaborate ektexine
comprising tectum, columella and foot layer and a less elaborate
endexine. The endexine is usually absent in monocotyledons. The
exine is made up of a complex and stable biopolymer, sporopollenin
that is resistant to non-oxidative, physical, biological and chemical
degradation processes (Blackmore, 2007). The exine is interrupted by
one or more circular or elongate sites called the apertures, through
which the pollen tube emerges. The exine is covered by a pollen coat
containing lipids, protein, pigments and aromatic compounds that fill
up the sculptured cavities of the pollen exine. The pollen coat is
derived from the tapetum and is called as the pollenkitt and tryphine.
The intine is a uniform layer that envelops the pollen protoplast and is
pecto-cellulosic in nature, comparable to the primary wall of the plant
cells.
• Accumulation of storage materials

The reserves accumulated in mature pollen are mainly carbohydrates
such as starch, sucrose, glucose and fructose or lipids. In most
Poaceae members, pollen grains contain a large amount of starch with
only a small quantity of soluble sugars. Large number of oil bodies are
formed in the pollen grains of Brassica napus, Gossypium hirsutum and
Arabidopsis thaliana (Shi and Yang, 2010). Starchy pollen grains tend
to be larger than starchless pollen (in which starch is hydrolyzed to
sugar). Starch rich pollen is usually associated with self pollination and
pollination by wind, birds and lepidopteron (moths and butterflies).
Lipid- rich pollen is usually associated instead with bee and fly
pollination (Baker and Baker, 1979).

Figure 11. Structure of pollen wall

Source: http://www.geo.arizona.edu/palynology/geos581/polwal.gif

Figure 12. SEM image of pollen grains of various species showing characteristic
exine sculpture.
Source:
https://en.wikipedia.org/wiki/Pollen#/media/File:Misc_pollen_colorized.jpg
Rare features
Pollen development in Cyperaceae

The members of Cyperaceae family eg. Cyperus, Kyllinga, Scirpus, Carex exhibit
a special mode of pollen development resulting in the formation of a unique
pollen type known as a pseudomonad or a cryptotetrad. In angiosperms as a rule,
each microspore mother cell gives rise to four functional microspores. In
Cyperaceae only one member of the tetrad is functional and the other three abort
and, thus, only one functional pollen grain is formed by each microspore mother
cell. Following meiosis in the microspore mother cell, three microspore nuclei are
cut off on one side and are separated from the functional nucleus by a wall. The
functional microspore nucleus divides to form a vegetative cell and a generative
cell. The newly formed generative cell undergoes another mitosis to form two
sperm cells. The non-functional nuclei may begin to divide but the divisions are
abortive and they eventually degenerate. The pseudomonads of Cyperaceae are
wedge shaped and highly polarized in cytological organization. The viable
microspore in the pseudomonad is always adjacent to the tapetum (abaxial pole)
and the three degenerated microspores are located in pointed adaxial portion of
the wedge shaped pollen grain. Unlike grasses, which undergo successive
cytokinesis, members of Cyperaceae undergo simultaneous cytokinesis after
completion of meiosis (Brown and Lemmon, 2000)

Figure 13. Development of Pollen grains in Cyperaceae

Source:
http://www.expertsmind.com/CMSImages/1231_Development%20of%20Pollen%
20in%20Cyperaceae.png

Figure 14. Cross section of an anther showing peripheral arrangement of

pseudomonads in the four locules. Functional uninucleate microspores are
appressed to the tapetum (abaxial) and the three abortive microspores are in the
center of the locule (adaxial).
Source: Brown RC and Betty EL (2000) The cytoskeleton and polarization during
pollen development in Carex blanda (Cyperaceae) American Journal of Botany
vol. 87 no. 1 1-11
Pollen grain embryo sacs
As a rule in angiosperms, the mature pollen grain is either two celled or three
celled. In some members of Liliaceae family, an increase in the number of nuclei
in the male gametophyte may lead to the formation of female gametophyte
(embryo sac) like structures. Nemec (1898) was the first to note embryo sac-like
pollen grains in the petaloid anthers of Hyacinthus orientalis and the phenomenon
since then is called as the Nemec phenomenon. Such abnormal embryo sacs are
also reported in Ornithogalum nutans, Leptomeria billardierii, Heuchera micrantha
and may arise from the divisions of microspore nucleus or vegetative nucleus or

generative nucleus or directly from the microspore mother cells. In Hyacinthus,

some of the microspores enlarge, escape from the spore wall and form sac-like
structures. In some of these embryo sacs the nucleus undergoes three mitotic
divisions like the nucleus of the functional megaspore to form eight nuclei, which
organize like an 8-nucleate embryo sac. The Nemec phenomenon is probably an
expression of the dominance of the female potency over the male in microspores.
Summary
 Pollen grains develop within the anther by microsporogenesis and

microgametogenesis.
 Microsporogenesis involves the division of the microspore mother cells
by meiosis, followed by cytokinesis to form the haploid microspores.
 Callose is deposited around each microspore mother cell at the onset of
meiosis.
 Synchrony in meiosis is established due to the formation of a meiotic
syncytium between the microspore mother cells of each
microsporangium.
 Isolation of the individual microspore mother cells by the callose wall is
necessary for the transition from a sporophytic phase to a gametophytic
phase, for expression of the gametophytic genome, to provide a
template for the formation of species specific exine and for the
autonomous development of the meiocytes.
 Cytokinesis may be successive (usually in monocots) where wall
formation follows both meiotic divisions I and II or simultaneous
(usually in dicots) where meiotic divisions I and II are completed prior
to wall formation.
 Tetrahedral tetrads are generally produced when cytokinesis is
simultaneous whereas successive cytokinesis results in the formation of
isobilateral and decussate tetrads or less frequently linear and T-shaped
tetrads.
 The exine formation around each microspore initiates within the tetrad
stage.
 Microspores are released in the anther locule by the breakdown of
callose wall by an enzyme callase secreted by the tapetum.
 The microspores represent the beginning of the male gametophyte
generation and show a thin exine. The microspores enlarge, become

vacuolated and the nucleus undergoes an asymmetric mitotic division to

form a large vegetative cell (VC) and a smaller generative cell (GC).
 The vegetative cell is a metabolically active cell that receives most of
the plastids, mitochondria and lipid bodies. The nucleus of the VC in
mature pollen is distorted with dispersed chromatin.
 The generative cell is completely enclosed within the cytoplasm of the
VC. The GC receives very few organelles. The GC is spheroidal in the
initial stages but becomes spindle shaped or lenticular to facilitate its
movement inside pollen tube. The nucleus of the GC has condensed
chromatin and undergoes DNA synthesis. The GC undergoes second
pollen mitosis to form the two sperms. The GC may divide inside the
pollen grain (tricellular pollen) or in the pollen tube after pollen
germination (bicellular pollen)
 Male Germ Unit (MGU) is a stable physical connection between the two
sperm cells and the vegetative cell nucleus. MGU forms an effective unit
in which all the DNA, both nuclear and cytoplasmic of male heredity is
linked together and travels as a single unit through the pollen tube. The
two sperms may be dimorphic in size, shape and organelle content. One
of the sperm cell is consistently associated with the vegetative nucleus
through a cytoplasmic projection.
 MGU appears to be a universal feature among the flowering plants and
may be organized before or after pollen germination.
 MGU linkage is necessary to direct the movement of sperms in the
pollen tube.
 MGU may dictate the ordered arrival of each sperm cell to the embryo
sac and may ensure targeted fusion with the egg and the polar nuclei.
 Sperm to sperm linkage may also reduce the incidence of
heterofertilization.
 The cytoplasmic projection and MGU may facilitate close communication
between the vegetative cell nucleus and the sperm cells via RNA and/ or
protein-transport.
 Maturation of pollen involves dehydration, elaboration of pollen wall and
accumulation of storage material.
 A characteristic feature of the members of Cyperaceae is the presence
of pseudomonads. Pseudomonads are wedge shaped and are recognized
by one viable microspore and three degenerated microspores following
meiosis in the microspore mother cells.

 Pollen grain embryo sacs were first seen by Nemec in the petaloid
anthers of Hyacinthus orientalis. An increase in the number of nuclei in
male gametophyte leads to the formation of such abnormal embryo sacs
and this is called as the Nemec phenomenon.
Practice Questions
Q1. What is the significance of callose in microsporogenesis?

Q.2. What is meiotic syncytium and where is it established?
Q.3. Differentiate between the vegetative and the generative cell.
Q.4. Explain the series of events in microsporogenesis with the help of suitable
diagrams.
Q.5. Describe the process of microgametogenesis with suitable diagrams.
Q.6. Define the Male Germ Unit. What are the potential roles of the MGU in
flowering plants?
Q.7. Explain the male Germ Unit of Plumbago zeylanica with suitable illustrations.
Which sperm fertilizes the egg cell in Plumbago?
Q.8. What type of cytokinesis is common in the monocots?
Q.9. What are the three major changes that occur during maturation of pollen?
Q.10. What is unique about the pollen development in Cyperaceae?
Q.11. What is Nemec phenomenon?
Glossary
Microspore Mother Cell (MMC)

Microspore Mother Cell is a diploid cell that develops within the microsporangium
and undergoes meiosis I and II to form a group of four haploid microspores
referred to as a tetrad.
Cytokinesis
The process that cleaves the MMC into separate microspore cells by wall
formation.
Vegetative Cell (VC)
Large metabolically active cell that is formed during microgametogenesis by an
asymmetric division of the microspore. It receives many plastids and
mitochondria and accumulates reserve food material in the form of lipids and
starch in the mature pollen grain.

Generative Cell (GC)

Small cell of the pollen grain completely enclosed within the vegetative cell
cytoplasm. GC receives few organelles, especially plastids. The GC divides to
form the two sperm cells (male gametes).
Male Germ Unit (MGU)
Male Germ Unit (MGU) is a stable physical connection between the two sperm
cells and the vegetative cell nucleus. MGU dictates the ordered arrival of each
sperm cell to the embryo sac and may ensure which sperm will fertilize the egg
and which sperm fuses with the polar nuclei.
Pseudomonads:
Pseudomonads characterize the family Cyperaceae (sedges) and are abnormal
pollen with one viable and three degenerated microspores.
Pollen grain embryo sacs:
Abnormal male gametophytes with eight nuclei similar to the female embryo sac.
Suggested Readings
1. Furness CA and Rudall PJ ‘Microsporogenesis in Monocotyledons’ (1999)

Annals of Botany 84, 475–499
2. Owen and Makaroff ‘Ultrastructure of microsporogenesis and
microgametogenesis in Arabidopsis thaliana (L.) Heynh. ecotype
Wassilewskija (Brassicaceae)’ (1995) Protoplasma 185:7-21
3. Mc Conchie CA, Jabsons, Knox RB (1985) Computer-assisted
reconstruction of the male germ unit in pollen of Brassica campestris.
Protoplasma 127:57-63
4. Russel SD (1984) Ultrastructure of the sperm of Plumbago zeylanica II.
Quantitative cytology and three-dimensional organization Planta162:385-
391
5. SS Bhojwani, SP Bhatnagar and PK Dantu. The Embryology of
Angiosperms. 6th ed. New Delhi. Vikas publishing house Private limited
(2014)
6. Shivanna KR. Pollen Biology and Biotechnology 1st ed. New Hampshire,
USA, Science Publishers Inc. (2003) ISBN 1-57808-241-2(PB)
7. Yu HS, Hu SY, Zhu C (1989) Ultrastructure of sperm cells and the male
germ unit in pollen tubes of Nicotiana tabacum. Protoplasma 152: 29–36
8. Brown Roy C. and Lemmon B.E. (2000) The cytoskeleton and
polarization during pollen development in Carex blanda (Cyperaceae).
American Journal of Botany 87(1):1-11

Pollen Development

Uploaded by

Copyright:

Available Formats

Pollen Development

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Pollen Development

Uploaded by

Copyright:

Available Formats

Pollen development and maturation

Paper: Reproductive Biology of Angiosperms

Institute of Lifelong Learning, University of Delhi

Institute of Lifelong Learning, University of Delhi