INTRODUCTION

Pollen is a fine to coarse powdery substance comprising pollen grains which

are male microgametophytes of seed plants, which produce
male gametes (sperm cells). Pollen grains have a hard coat made
of sporopollenin that protects the gametophytes during the process of their
movement from the stamens to the pistil of flowering plants, or from the
male cone to the female cone of coniferous plants. If pollen lands on a
compatible pistil or female cone, it germinates, producing a pollen tube that
transfers the sperm to the ovule containing the female gametophyte.
Individual pollen grains are small enough to require magnification to see
detail. The study of pollen is called palynology and is highly useful
in paleoecology, paleontology, archaeology, and forensics. Pollen in plants is
used for transferring haploid male genetic material from the anther of a single
flower to the stigma of another in cross-pollination. In a case of self-
pollination, this process takes place from the anther of a flower to the stigma
of the same flower. Pollen is commonly used as food and food supplement.
However, because of agricultural practices, it is often contaminated by
agricultural pesticides.
Pollen itself is not the male gamete. Each pollen grain contains vegetative
(non-reproductive) cells (only a single cell in most flowering plants but
several in other seed plants) and a generative (reproductive) cell. In flowering
plants the vegetative tube cell produces the pollen tube, and the generative
cell divides to form the two sperm cells.

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STRUCTURE AND FORMATION

Triporate pollen of Oenothera speciosa

Pollen of Lilium auratumshowing single sulcus (monosulcate)

Arabis pollen has three colpi and prominent surface structure.

Pollens/Microspores of Lycopersicon esculentum at coenocytic tetrad stage of development observed through oil
immersion microscope; the chromosomes of what will become four pollen grains can be seen.
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Apple pollen under microscopy

FORMATION

Pollen is produced in the microsporangia in the male cone of a conifer or

other gymnosperm or in the anthers of an angiosperm flower. Pollen grains
come in a wide variety of shapes, sizes, and surface markings characteristic of
the species (see electron micrograph, right). Pollen grains of pines, firs,
and spruces are winged. The smallest pollen grain, that of the forget-me-
not (Myosotis spp.), is around 6 µm (0.006 mm) in diameter. Wind-borne
pollen grains can be as large as about 90–100 µm.
In angiosperms, during flower development the anther is composed of a mass
of cells that appear undifferentiated, except for a partially differentiated
dermis. As the flower develops, four groups of sporogenous cells form within
the anther. The fertile sporogenous cells are surrounded by layers of sterile
cells that grow into the wall of the pollen sac. Some of the cells grow into
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nutritive cells that supply nutrition for the microspores that form by meiotic
division from the sporogenous cells.
In a process called microsporogenesis, four haploid microspores are
produced from each diploid sporogenous cell (microsporocyte, pollen mother
cell or meiocyte), after meiotic division. After the formation of the four
microspores, which are contained by callose walls, the development of the
pollen grain walls begins. The callose wall is broken down by an enzyme
called callase and the freed pollen grains grow in size and develop their
characteristic shape and form a resistant outer wall called the exine and an
inner wall called the intine. The exine is what is preserved in the fossil record.
Two basic types of microsporogenesis are recognised, simultaneous and
successive. In simultaneous microsporogenesis meiotic steps I and II are
completed prior to cytokinesis, whereas in successive microsporogenesis
cytokinesis follows. While there may be a continuum with intermediate forms,
the type of microsporogenesis has systematic significance. The predominant
form amongst the monocots is successive, but there are important exceptions.
During microgametogenesis, the unicellular microspores undergo mitosis and
develop into mature microgametophytes containing the gametes. In some
flowering plants, germination of the pollen grain may begin even before it
leaves the microsporangium, with the generative cell forming the two sperm
cells.

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STRUCTURE

Except in the case of some submerged aquatic plants, the mature pollen grain
has a double wall. The vegetative and generative cells are surrounded by a
thin delicate wall of unaltered cellulose called the endospore or intine, and a
tough resistant outer cuticularized wall composed largely
of sporopollenin called the exospore or exine. The exine often bears spines
or warts, or is variously sculptured, and the character of the markings is often
of value for identifying genus, species, or even cultivar or individual. The
spines may be less than a micron in length (spinulus, plural spinuli) referred
to as spinulose (scabrate), or longer than a micron (echina, echinae) referred
to as echinate. Various terms also describe the sculpturing such as reticulate,
a net like appearance consisting of elements (murus, muri) separated from
each other by a lumen (plural lumina).
The pollen wall protects the sperm while the pollen grain is moving from the
anther to the stigma; it protects the vital genetic material from drying out and
solar radiation. The pollen grain surface is covered with waxes and proteins,
which are held in place by structures called sculpture elements on the surface
of the grain. The outer pollen wall, which prevents the pollen grain from
shrinking and crushing the genetic material during desiccation, is composed
of two layers. These two layers are the tectum and the foot layer, which is just
above the intine. The tectum and foot layer are separated by a region called
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the columella, which is composed of strengthening rods. The outer wall is
constructed with a resistant biopolymer called sporopollenin.
Pollen apertures are regions of the pollen wall that may involve exine thinning
or a significant reduction in exine thickness. They allow shrinking and
swelling of the grain caused by changes in moisture content. Elongated
apertures or furrows in the pollen grain are called colpi (singular: colpus) or
sulci (singular: sulcus). Apertures that are more circular are called pores.
Colpi, sulci and pores are major features in the identification of classes of
pollen. Pollen may be referred to as inaperturate (apertures absent)
or aperturate (apertures present). The aperture may have a lid (operculum),
hence is described as operculate. However the term inaperturate covers a
wide range of morphological types, such as functionally inaperturate
(cryptoaperturate) and omniaperturate. Inaperaturate pollen grains often
have thin walls, which facilitates pollen tube germination at any
position. Terms such as uniaperturate and triaperturate refer to the
number of apertures present (one and three respectively).
The orientation of furrows (relative to the original tetrad of microspores)
classifies the pollen as sulcate or colpate. Sulcate pollen has a furrow across
the middle of what was the outer face when the pollen grain was in its
tetrad. If the pollen has only a single sulcus, it is described as monosulcate,
has two sulci, as bisulcate, or more, as polysulcate. Colpate pollen has
furrows other than across the middle of the outer faces. Eudicots have pollen
with three colpi (tricolpate) or with shapes that are evolutionarily derived
from tricolpate pollen. The evolutionary trend in plants has been from
monosulcate to polycolpate or polyporate pollen.
Additionally, gymnosperm pollen grains often have air bladders, or vesicles,
called sacci. The sacci are not actually balloons, but are sponge-like, and
increase the buoyancy of the pollen grain and help keep it aloft in the wind, as
most gymnosperms are anemophilous. Pollen can be monosaccate,
(containing one saccus) or bisaccate (containing two sacci).
Modern pine, spruce, and yellowwood trees all produce saccate pollen.
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POLLEN VIABILITY
Pollen viability refers to the ability of the pollen to perform its function of
delivering male gametes to the embryo sac. This functional property of the
pollen after their release from the anther varies greatly from species to
species and its quality is assessed on the basis of its viability. Pollen viability is
an index of its quality and vigour.

Pollen viability varies between minutes and years, and which primarily
depends on the taxonomic status of the plant and on the abiotic
environmental conditions. In order to maintain the viability and fertilizing
ability of the pollen for a long period of time special storage conditions are
needed.

Reports on the storages and transportation of date palm pollen were among
the earliest concern with pollen viability. The male inflorescence of Phoenix
dactylifera was prominently mentioned in trade contracts of the Hammurabi
period about 2000 BC when storage of male flower in a dark, dry place was
first recognized as prolonging fertilization capacity.

Cryopreservation is the most efficient method for long-term preservation of

partly dehydrated pollen grains. In vitro biotechnological techniques like
isolation and fusion of reproductive cells, and DNA transformation of
artificially produced zygotes and embryos, have opened new prospects for
germplasm storage.

Sophisticated methods such as nuclear magnetic resonance (NMR)

spectrometry, Fourier transform infrared spectroscopy (FTIR), and different
ultra-micro techniques for electron microscopy have helped to carry our
precise with molecular changes occurring in membranes during pollen
dehydration and rehydration.

Several reasons have been assigned for the loss of viability, like deficiency of
respiratory substrate, inability to withstand desiccation and the loss of
membrane integrity.
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VARIATIONS IN VIABILITY OF POLLEN
The life span of pollen is primarily determined by the plant genome but is also
influenced by external environmental conditions.

Harrington (1970) on the basis of pollen viability has classified the

examined plant taxa into three main groups, viz.:
a) Long Lived Pollen (six months to a year), example, Ginkgoaceae, Pinaceae,
Arecaceae, Saxifragaceae, Rosaceae, Fabaceae, Anacardiaceae, Vitaceae and
Primulaceae.

b) Pollen with a medium life span (approximately 1-3 months), examples,

Liliaceae, Amaryllidaceae, Salicaceae, Ranunculaceae, Brassicaceae, Rutaceae,
Scrophulariaceae, and Solanaceae.

c) Short Lived Pollen (from few minutes to a couple of days), examples,

Alismataceae, Poaceae, Cyperaceae, Commelinaceae and Juncaceae.
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CAUSES FOR THE LOSS OF POLLEN VIABILITY
It has been extremely difficult to access the exact reasons behind the loss of
viability among pollen grains within a span of short or long period. Studies of
Stanley and Linskens (1974) suggest that it is the deficiency of respiratory
substrates or/and inactivation of certain specific enzymes or growth
hormones that are likely to affect the viability of the pollen.

This idea is however, untenable when it is seen that the pollen of cereals
(short lived) inspite of having abundant metabolites quickly lose their
viability. Similarly changes in amino acid composition of stored pollen fail to
explain the loss of viability. There are variable reasons to explain such
inactivity as stated below.

i. Biochemical Alteration in Pollen:

The major biochemical cause for the loss of viability during storage is basically
due to the deficiency of respiratory metabolites, which is the result of
continuous metabolic activity by the pollen. As a result of long term storage
there are reports of considerable changes in the amount of carbohydrate,
amino acids and organic acid level in the pollen of different species.

A higher respiratory rate in the three- celled pollen leads to the scarcity of
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respiratory substrate that strongly contribute to their rapid loss of pollen

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viability. It is reported that stored pollen grains require a higher
concentration of sugar for germination in vitro than fresh pollen. Higher
relative humidity also plays an active role in decreasing germinability by
rapidly degrading endogenous substrates essential for germination.

The content of amino acids has also been reported to change in course of
storage. An investigation into sixteen amino acids in Zea mays has recorded a
consistent increase in aspartic acid, aminobutyric acid, ethanolamine,
isoleucine, leucine, lysine, and phenylalanine, while alanine, glycine, glutamic
acid, and proline decreased gradually.

A similar correlation in the endogenous level of proline and germinability has

been noted in Lilium longiflorum, which however, increased with exogenously
supplied proline. The other possibility of decrease in germinability due to
deficiency of respiratory substrate might be the inactivation of enzymes like,
amylase and phosphatases associated with degradation of reserves stored in
pollen grains.

ii. Desiccation and Loss of Membrane Integrity of Pollen:

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The regulation of pollen water content is an important adaptive mechanism

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for survival after pollen dispersal and accordingly pollen grains that remain
viable after dehydration are called desiccation tolerant, and those that lose
viability parallel to dehydration are called desiccation sensitive.

The water content of living pollen grains in different families vary between
15% and 35% of fresh weight at the time of shedding, which is however, very
high in Poaceae pollen between 35-60 percent.

The original pollen moisture to some extent depends upon temperature, air
humidity, and the water supply to the pollen donor plant. This water content
is measured accurately with nuclear magnetic resonance (NMR)
spectrometry.

An investigation on the membrane state of pollen grains (using fluorescein

diacetate test) from different taxa exposed to dry conditions indicated that
most of the samples had lost their membrane integrity. It has also been
observed that the plasma membrane may undergo gel- phase transition
during water loss by increasing van der Waals interaction or free-radical
damage.

Thus water plays an important role in maintaining the structural integrity and
the stability of the pollen membrane, by acting through hydrophobic and
hydrophilic interactions. A positive correlation has been established between
the loss of viability and a reduction in the amount of membrane phospholipids
irrespective of the storage conditions.

The studies of Simmon (1974, 1978) on desiccated seeds have shown that
when the moisture level of the membrane falls below 20%, the membrane
loses its lamellar structure and permeability properties leading to
imbibitional leakage, rehydration however, restores membrane integrity.

In most of the desiccated pollen systems, gradual hydration in humid air (ca
95% RH) is favourable for restoration of membrane integrity. This gradual
rehydration causes a shift in membrane lipids from, the gel phase to the liquid
crystalline phase, which has been explained by Crowe (1989) as phase
transition changes in membrane phospholipids following desiccation and
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hydration.
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The pollen grains of grasses are highly sensitive to greater degrees of
desiccation and restoration of normal function by controlled rehydration
becomes extremely difficult. It has been seen that dehydration after dispersal
rapidly disrupts the actin cytoskeleton in wheat pollen and leads to a loss of
germination capacity. Thus for a successful preservation of pollen that loses
its viability in a short time, special condition should be provided before and
during storage.

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FACTORS AFFECTING POLLEN VIABILITY
i. Pollen Cytology:

There exists a close relation between the cytology of pollen and its viability.
Studies on pollen morphology and physiology have shown that the binucleate
and trinucleate pollen grains show differences in their physiological and
structural characters at the time of pollen dispersal.

The two celled pollen grains have a longer life span because of their more
resistant wall structure, low plasma water content and reduced metabolic
activity, whereas the trinucleate pollen grains are short-lived due to their less
resistant wall and high moisture content, which can easily be lost by
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desiccation. This trinucleate pollen has a high rate of metabolism, respiring

two to three times more than the binucleate pollen.
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ii. Humidity and Temperature:

Environmental factors especially humidity and temperature greatly affects

pollen viability. This relationship have been investigated by many authors and
it transpired that pollen of majority of the species retained viability best at
low relative air humidities (0%-30% RH) and temperature (between 0 and 10
°C) (Table 7.1). In most of the cases it is possible to standardize the conditions
(low temperature and/or low humidity) for extending pollen viability of two
celled taxa.

However, on the other hand little progress has been made with the
preservation of the three celled pollen taxa under Poaceae, Brassicaceae,
Caryophyllaceae, Apiaceae, and Chenopodiaceae families.

The longevity of Poaceae pollen appears to be short under all conditions. Low
relative humidities are harmful and pollen stored at 0° – 10°C remains viable
only for a couple of days. Under high relative humidity (80% to 100%) also
the viability can be prolonged to 1 – 3 weeks at the most.
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POLLINATION

Pollination is the transfer of pollen from a male part of a plant to a female

part of a plant, later enabling fertilisation and the production of seeds, most
often by an animal or by wind. Pollinating agents are animals such as insects,
birds, and bats; water; wind; and even plants themselves, when self-
pollination occurs within a closed flower. Pollination often occurs within a
species. When pollination occurs between species it can
produce hybrid offspring in nature and in plant breeding work.
In angiosperms, after the pollen grain has landed on the stigma, it develops
a pollen tube which grows down the style until it reaches an ovary. Sperm
cells from the pollen grain then move along the pollen tube, enter an ovum cell
through the micropyle and fertilise it, resulting in the production of a seed.
A successful angiosperm pollen grain (gametophyte) containing the
male gametes is transported to the stigma, where it germinates and its pollen
tube grows down the style to the ovary. Its two gametes travel down the tube
to where the gametophyte(s) containing the female gametes are held within
the carpel. One nucleus fuses with the polar bodies to produce the endosperm
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tissues, and the other with the ovule to produce the embryo Hence the term:
"double fertilization".
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In gymnosperms, the ovule is not contained in a carpel, but exposed on the
surface of a dedicated support organ, such as the scale of a cone, so that the
penetration of carpel tissue is unnecessary. Details of the process vary
according to the division of gymnosperms in question. Two main modes of
fertilization are found in gymnosperms. Cycads and Ginkgo have motile sperm
that swim directly to the egg inside the ovule, whereas conifers
and gnetophytes have sperm that are unable to swim but are conveyed to the
egg along a pollen tube.
The study of pollination brings together many disciplines, such
as botany, horticulture, entomology, and ecology. The pollination process as
an interaction between flower and pollen vector was first addressed in the
18th century by Christian Konrad Sprengel. It is important in horticulture
and agriculture, because fruiting is dependent on fertilization: the result of
pollination. The study of pollination by insects is known as anthecology.

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Types: Self-Pollination And Cross-Pollination
An egg cell in an ovule of a flower may be fertilized by a sperm cell derived
from a pollen grain produced by that same flower or by another flower on the
same plant, in either of which two cases fertilization is said to be due to self-
pollination (autogamy); or, the sperm may be derived from pollen originating
on a different plant individual, in which case the process is called cross-
pollination (heterogamy). Both processes are common, but cross-pollination
clearly has certain evolutionary advantages for the species: the seeds formed
may combine the hereditary traits of both parents, and the resulting offspring
generally are more varied than would be the case after self-pollination. In a
changing environment, some of the individuals resulting from cross-
pollination still may be found capable of coping with their new situation,
ensuring survival of the species, whereas the individuals resulting from self-
pollination might all be unable to adjust. Self-pollination, or selfing, although
foolproof in a stable environment, thus is an evolutionary cul-de-sac. There
also is a more direct, visible difference between selfing and outbreeding
(cross-pollination): in those species where both methods work, cross-
pollination usually produces more, and better quality, seeds. A dramatic
demonstration of this effect is found with hybrid corn (maize), a superior
product that results from cross-breeding of several especially bred lines.

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HISTORY
German chemist and botanist Carl Julius Fritzsche observed and depicted
these grains of pollen from flowering plants using a microscope set at around
500x magnification. Fritzsche cataloged pollen grains from different angles,
trying to understand their structure, and published his findings in an 1837
book, Ueber den Pollen (About Pollen).

In 1830, English opticist Joseph Jackson Lister developed a lens that reduced
an aberration that had previously plagued people using microscopes: the
persistent apparation of a colored edge around an image. Fritzsche, who
was then working in St. Petersburg, Russia, was one of a group of scientists
across Europe who took advantage of these technological improvements to
advance studies in pollen morphology.

Fritzsche coined the terms exine (the outer wall of pollen grains and spores)
and intine (the inner wall).

Observations of pollen by Grew and Malpighi are recorded from shortly

after the invention of the microscope in the mid 17th Century. One of the
very earliest practical applications of preserved pollen in the reconstruction
of changing environments was by the Swedish palynologist Von Post in
1917. He studied tree pollen preserved in peat to build a picture of
fluctuating climatic conditions during the Quaternary. In Britain during the
1930's Raistrick used spores he recovered from coals to recognise different
coal seams, but he did not name the spores he found but assigned them an
alpha-numeric code. Perhaps the greatest contribution made by a single
person was that made by another Swede, Gunnar Erdtman, who from the
1950's to the 1970's produced several classic books and papers which
remain required reading today.
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Range

The earliest terrestrial plants are recorded from the late Silurian, and these
were homosporous (all spores produced are of the same kind). By the end of
the Devonian heterospory had appeared, this still involves dispersal by
spores only but both microspores (held in a microsporangium) and
megaspores (held in a megasorangium) are produced. Both these forms of
plants relied on water (or at least damp conditions) to allow transport of the
spermatozoid to the egg. The earliest gymnosperms appear in the very
latest Devonian and rapidly become diverse and important during the
Carboniferous. The angiosperms did not appear untill the early Cretaceous
and diversified rapidly from the mid Cretaceous.

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EXPERIMENT

AIM: To study pollen structure and calculate pollen viability.

THEORY: Microspores or pollen grains are male reproductive bodies of

seed bearing plants. They are produced in sac like structure known as
microsporongium. Commonly the pollen grains are globular in outline,
though several other shapes are also found. A pollen grain is single celled in
the beginning but it becomes 2 celled at the time of liberation. It has two
layered wall. The outer is known as exine and the inner is termed intine.
Intine is plecto-cellulosic in nature. Exine is made up of a highly resistants
fatty substance called sporopollenium. The exine provides a characteristic
sculpturing or designs over the surface of pollen grain. It helps in
identification of the species to which a pollen grain belongs. Palynology is
the branch of study of pollen grains. The insect pollinated grains have a
yellowish sticky and oily covering over the exine called pollenkitt. The exine
is thin or absent at certain places. These areas are known as germ pores. A
mature pollen grain has two cells, a large cell called vegetative cell or tube
cell and a small cell called generative cell. The vegetative cell produces
pollen tube, while the generative cell divides into two male gametes after
moving into the pollen tube. Pollen viability means the ability of a pollen
grain to germinate and produce male gametes. Some pollen grain may not
be viable either due to some abnormality or lack of stored food in them.

MATERIALS REQUIRED: Reagent bottle, cavity slides, beakers, measuring

cylinder, sucrose, boric acid, magnesium sulphate, microscope, flowers of
different plant species, potassium nitrate, plain slides coverslips.
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METHOD:

A. Pollen structure

1. First of all take a clean slide and put a drop of glycerine on it.

2. Dust a few pollen grains from the anther of a flower in the glycerine drop.

3. Now place a coverslip and observe the slide under low power and then
under high power of the microscope.

4. Observe the structure of the pollen grain carefully and draw its diagram.

5. In the similar manner study the structures of pollen grains of flowers of

different plant species.

B. Pollen viability

1. Firstly prepare a nutrient solution by dissolving 10 g. sucrose, 10 g. Boric

acid, 10 mg KNO3 , 10 mg MgSO4 in 100 ml of distilled water.

2. Stock this solution in a reagent bottle. This solution acts as a nutrient for
the developing pollen grains.

3. Take a few drops of this solution on a clean cavity slide and dust pollen
grains from mature anther of the flowers over this solution.

4. Now observe the slide under dissecting or compound microscope after 5

minutes and then regularly after every minute.

5. Perform experiment with different types of flowers in the similar manner

and record the germination of pollen grains of each species.
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OBSERVATION:

IXORA COCCINEA

MORINGA OLEIFERA

PISUM SATIVUM
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HIBISCUS ROSA-SINESIS

MOMORDICA CHARANTIA

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 1. The pollen grains of different plant species show different types of
sculpturing on the exine.

2. The pattern and distribution of germ/pores is also vary in the pollen

grains of different types of flowers.

3. The rate of germination and viability of pollen grains of different species

also differ very much. Record the observation in the following table.

S. NO. NAME OF TIME TAKEN NUMBER OF NUMBER OF PERCENT

FLOWER IN VIABLE NON-VIABLE VIABILITY
GERMINATION POLLEN POLLEN
OF POLLEN GRAINS GRAINS
GRAIN
1. Ixora 10 5 15 36%
coccinea

2. Moringa 10 10 25 40%
oleifera

3. Pisum 10 11 20 55%
sativum

4. Hibiscus 10 3 13 23%
rosa-
sinensis

5. Momordica 10 12 25 48%
charantia

CONCLUSION: Viable pollen grains germinate in the nutrient medium but

the non-viable pollen grains do not germinate. The percentage of viability
vary in different flowers.
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BIBLIOGRAPHY

http://www.biologydiscussion.com/palynology/pollen-viability-in-plants-variations-and-
factors/64581

https://www.fs.fed.us/wildflowers/pollinators/What_is_Pollination/

https://slate.com/human-interest/2015/05/pollen-scientific-history-carl-julius-fritzsche-s-portraits-
of-pollen-grains.html

https://www.britannica.com/science/pollen/media/1/467883/375

https://en.wikipedia.org/wiki/Pollen

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