Anther and pollen biology

Anther wall; Structure and function, Microsporogenesis ;callose diposition; pollen wall structure;NPC system;Palynology and scope;Pollen Viability; Storage and germination ;Abnormal features etc

1. Anther and pollen
biology
Prof. Subir Pramanik
Bajkul Milani Mahavidyalaya

2. Anther Wall:

Epidermis, endothecium, middle wall layers (2-3 layered),
and the tapetum (outer and inner layers) constitute the anther wall.



i. Epidermis:
The epidermis is a single layered protective sheath of the anther. It
divides anticlinally and tries to keep space with the enlarging internal

3. tissues of the anther. As a consequence they undergo considerable
stretching in surface area. It provides the structural integrity to the
anther, assists in gaseous diffusion, prevents moisture loss, and in the
dehiscence of the anther lobes.

ii. Endothecium:
The outer most layers of the descendants of the parietal cell located
immediately below the epidermis are called the endothecium. It
attains the maximum development before the dehiscence of the
anther.
The cells are radially elongated and decorated with fibrous bands
(absent in certain members of Hydrocharitaceae and some
cleistogamous flowers) that run upward from the inner tangential
walls, ending near the outer wall of each cell as an incomplete ring.
The outer tangential walls remain thin. The endothecium is associated
with high proportion of α-cellulose and small amount of lignin at
maturity. The specialized nature of the endothecium together with the
stomium helps in the dehiscence of the anther.

iii. Middle Layer:
The cells of the middle layer are usually ephemeral and become
flattened and crushed by early meiosis in the pollen mother cell.
However, the layers persist in Ranunculus and Lilium, and the layer
adjacent to the endothecium may even develop fibrous thickenings. In
few instances it also serves to store starch that is later mobilized to the
developing pollen. (Table 1.1)

iv. Tapetum:
The tapetum is the innermost layer of the anther wall and is usually
derived from the parietal layer. However, it may have a dimorphic
origin in a few species, viz., in Alectra thomsoni the inner tapetum
consists of larger cells that is derived from the cells of the connective,

4. whereas, the outer tapetum of smaller cells is derived from the parietal
layer.
Further in Antirrhinum majus and Impatiens glandulifera the tapetum
has its origin from the peripheral cells of the sporogenous tissue.
Maheshwari (1950) and Echlin (1971) believe in the parietal origin of
the tapetum as obligatory.
The tapetum surrounds the sporogenous tissue and attains maximum
development when the microspores are in the tetrad stage, after which
they go into decline that results in the collapse of the cells. Generally it
is single layered but it may divide and become biseriate as in
Pyrostegia, Tecoma and Magnolia. While a multiseriate condition is
known in Combretum grandiflorum and Oxystelma esculentum.

Function
1. Since it is predominantly made of sporopollenin, it may resist the
free passage of materials into and out of the spore mass.
2. The peritapetal membrane is considered to form a kind of
impermeable “culture sac”, enclosing the young spores and the tapetal
Plasmodium during the period of sexine growth.
3. In members of the subfamily Cynanchoideae of Asclepiadaceae, the
tapetal membrane holds the pollen together in aggregate and assist in
their collective dispersal.

5. Microsporogenesis and
Microspore

During the development of the microsporangium, the anther is seen at
first as a homogeneous mass of meristematic cells, oblong in cross-
section and surrounded by an epidermis (Fig. 404A).
It then becomes more or less four-lobed and four longitudinal rows of
archesporial cells are differentiated. The archesporial cells are marked
off from the surrounding cells by their more deeply staining cytoplasm
and conspicuous nuclei.
There may be only one such archesporial cell in each of the four lobes
(fig. 404B) as in Boerhaavia, etc., or there may be more of them
forming a plate (Ophiopogon, etc.).
Longitudinally, also, there may be one to many of them. Each
archesporial cell now cuts off a primary parietal cell towards the
epidermis and a primary sporogenous cell on the inner side (Fig.
404C).
The parietal cell now divides by periclinal and anticlinal walls giving
rise to several layers of cells forming the wall of the anther while the
sporogenous cell usually divides a few times giving rise to a number of
microspore or pollen mother cells (Fig. 404D).
The innermost layer of wall cells directly abutting on the sporogenous
tissue forms the tapetum which is a nutritive tissue nourishing the

6. developing microspores (Fig. 404E). The wall cells just below the
epidermis form the endothecium which later loses the cell contents,
usually becomes fibrous, and forms the dry coat of the mature anther
in which the epidermis becomes rather inconspicuous.
Between the tapetum and the endothecium there are one to three
middle layers of cells. The middle layers and the tapetum are usually
crushed by the time actual meiosis occurs in the sporogenous cells.
During microsporogenesis (i.e., development of microspores or
pollens) the nucleus of each microspore mother cell undergoes meiosis
or reduction division ultimately giving rise to four haploid (i.e., pos-
sessing ‘n’ number of chromosomes) nuclei (Fig. 404—F & G).
These four nuclei are arranged tetrahedrally (Fig. 404—H & I) and are
soon invested with cell walls. Many variations are known of this
typical pattern of meiosis, e.g., in maize a wall is formed across the
dyad (2-nucleated condition).
They are now the microspores or pollens which soon dry up and
become powdery while the tapetum becomes absorbed.
The anther now becomes a dry structure, the partition walls between
the sporangia (i.e., loculi) are usually destroyed (Fig. 404J) and the
microspores (pollens) are soon liberated by dehiscence of the anther.

7. The tapetal cells often become multinucleate and play a great part in
the nutrition of the pollens. Sometimes they develop a Plasmodium
after disintegration and play a part in the development of the exine of
the pollen. Even a part of the sporogenous tissue may break down and
serve for nutrition instead of developing spores.
While the pollens are dry and powdery in most flowers, peculiar
conditions are often let with. In Annona, Elodea, Typha, etc., the four
spores in a tetrad never separate but form compound pollen grains.
In the Mimoseae 8 to 64 pollens often aggregate together while in the
gynostegium of Calotropis and the gynostemium of orchids all the
pollens of each anther lobe from a characteristic mass called pollinium

8. (Fig. 405). Each pollinium is provided with a stalk called caudicle and
a sticky base called disc or corpusculum.
The pollen or the microspore is a very minute structure (0.025 to
0.125 mm in diameter). It is unicellular and usually round although it
may be oval, pyramidal, polyhedral, etc. It is provided with two coats—
an inner, delicate cellulose layer called intine and an outer tough,
cutinised layer called exine or extine. The exine is often sculptured or
provided with spines, warts, etc. Occasionally, it is smooth.
The exine may have a waxy coating to render the pollen more or less
waterproof. Very often, there are some definitely thinner, circular
spots or slits in the exine called germ pores or slits.
These weak spots are utilised during the germination of the pollen.
The pores are sometimes provided with lids (Fig. 409 B) which open
out like valves during germination. Very often, before the pollen is
discharged from the anther it becomes binucleate (Fig. 406), the
original single nucleus dividing-into a tube nucleus and a generative
nucleus.

The latter, with some cytoplasm surrounding it, becomes the
generative cell (described later). Sometimes the pollen may even
become trinucleate (Fig. 410), as in most cereal crops, by complete
development of the male gametophyte even before it is shed. Fig. 407
shows some different forms of mature pollens. Palynology is the
science involving the study of pollens.

9. Development of The Male Gametophyte:
The nucleus of the microspore begins to divide very soon after it is
formed. Its division is usually quicker in the warmer climate than in
the cooler regions. Such division may begin even before the
microspores dissociate from the tetrad condition.
When the pollen is lodged on the stigma, usually its nucleus has
already divided. The microspore cell divides into two very equal cells
with the smaller cell attached to the wall (Fig. 408A).

The latter soon loses contact with the wall (Fig. 408 B) and becomes
the lens-shaped generative cell floating freely in the cytoplasm of the
large vegetative or tube cell (Fig. 409A).

10. It is then in the binucleate (or two-celled) stage. As the pollen
germinates and the pollen tube comes out through a germ pore (Fig.
409B) the vegetative nucleus precedes the generative cell (Fig. 409C).
The generative cell soon divides mitotically to form two male gametes
(Fig. 409 D) and this act is known as spermatogenesis as the male
gametes of Angiosperms are equivalent to the ciliated sperms of the
lower groups of plants. The gamete nuclei are enveloped in
cytoplasmic sheaths, this forming gamete cells. At a later stage the two
male gamete cells are seen to follow the vegetative ‘nucleus (Fig.
409E).
Soon the vegetative nucleus, which seems to be of no importance being
a mere vestigial organ, disappears. As the pollen is the microspore, the
contents within the pollen and the pollen tube formed by the
germination of the microspore is the male gametophyte of
Angiosperms.

11. While the male gametophyte, thus, usually develops after the pollen
has germinated, there are many instances where it develops within the
pollen before the pollen tube is formed and, in some cases, even before
pollination. Trinucleate pollens with tube nucleus and two gamete
cells are observed in such cases (Fig. 410). Such trinucleate pollen
formation is the rule in many plants (e.g., Portulaca), specially the
cereal crops like wheat, rice, maize of Gramineae and sugarcane of the
same family.

12. Callose deposition and its significance

After the discontinuity of the plasmodesmata connection among the
microsporocytes, it is covered by a primary wall made up of cellulose,
and shortly before meiosis this wall disintegrates and is replaced by a
massive deposit of callose (β-1,3- glucan), outside the plasma
membrane.
Callose deposition starts at the corners of the cells between the plasma
membrane and the original wall. However, the primary wall persists in
Allium tuberosum and Cyclamen persicum until late tetrad stage. The
possible reason for the delay is to form a barrier in the entry of
macromolecules in the microsporocytes, thus ensuring autonomous
development of microspores.

13. The deposition of the callose is initially incomplete, leaving many gaps
through which there is an establishment of massive cytoplasmic
channels between the microsporocytes. These channels are 1 -2 pm in
diameter and attain their maximum development in the zygotene-
pachytene stage. Thus at this stage the highly interconnected mass of
microsporocytes in the locule, form a large meiocytic syncytium (Fig
1.7).

14. This massive coenocyte provides a channel for the transport and
distribution of metabolites. Further it imposes a mutual influence of
one cell over the other, thus helping to maintain a close synchrony
during meiosis among the large number of microsporocytes in the
anther locule.
At the close of meiotic prophase the callose walls of the
microsporocyte lock up, and the cytoplasmic channels are cut off and

15. now the microsporocytes go through the rest of the meiosis as isolated
cells.
At the end of metaphase I or II, the callose wall around microspore
mother cells become continuous. The isolation of the microspore
mother cells and then the microspores, by a callose wall seems
essential for the normal development of the pollen grains.
Failure of callose wall development or its early breakdown results in
pollen sterility. Table-1.2 shows the formation of walls and
intercellular connections in tapetum, meiocytes and meiospores
during microsporogenesis.
The accurate determination of the timing and pattern of DNA
synthesis during microsporocyte meiosis is markedly complex. Studies
involving autoradiography and microspectro- photometry have
established that the main DNA synthesis period for the
microsporocytes begins shortly before leptotene and continues into
leptotene.
For example in Lilhim longiflorum autoradiography of
32P
incorporation, followed by enzyme digestion and acid hydrolysis, for
eliminating RNA and phosphoproteins, identified a preleptotene
period of DNA synthesis.
Whereas microspectrophotometric measurement showed a 4C DNA
amount in the microsporocytes before their entry into meiosis, a
transient 2C amount after meiosis I, and a 1C amount in the
microspores.
What prompts sporogenous cells to cease mitotic divisions and enter
the meiotic cycle is not known. There is a prediction that meiosis is
triggered by the synthesis of some factors in tissues other than the
sporogenous cells. These substances have trival names as meiosis
determinants, or meiosis-inducing substances.
Few attempts have been made to clone meiosis specific genes from
microsporocytes and identify the protein products they encode.

16. However, none of the genes so far characterized can be considered to
be meiosis specific because they encode common proteins associated
with cell metabolism such as HSP (heat shock proteins), serine
proteases, proteins involved in DNA repair, and leucine zipper
proteins.
Thus the action of specific genes that control the entry of cells into and
their exit from meiosis is still a matter of distant dream. The
cytoplasm of meiocytes undergoes profound changes during meiosis
and there is a significant fall in the cytoplasmic RNA.
Functions of Callose Wall:
The possible functions of the callose envelope are:
i. To control major features, such as the arrangement of apertures,
which are probably related to the geometry imposed by the callose
wall.
ii. To isolate the young microspore from influences of tapetal cells
during early stages of development. This isolation enables the young
microspores to deposit a primexine without interaction between them
and the tapetum. Knox (1984) thus emphasized that the callose wall
serves to separate the gametophyte from the sporophyte.
iii. Participates in the development of wall ornamentation. Godwin
(1968) described the action of callose wall as a template, defining the
position of apertures and the deposition of the primexine in Ipomoea.
iv. Protects the meiocytes from dehydration under condition of
deficient water supply.
v. It isolates and insulates meiocytes for the normal completion of
meiosis. This isolation provides avenues for two major events, viz.,
transition from sporophytic phase to gametophytic phase, and the
expression of the gametophytic genome which is essential for
achieving the limited function of gamete formation and their discharge
in the embryo sac.

17. vi. After its break down it serves as a source of soluble carbohydrates
for the developing pollen.



Microgametogenesis (Development of
Male Gametophyte):


Microspore i.e., the pollen grain, is the first cell of the male
gametophyte, which contains only one haploid nucleus (Fig. 3.5A).
During early stage of development, it remains within the
microsporangium. The cell undergoes unequal division and forms a
small generative cell and a large vegetative or tube cell (Fig. 3.5B).
Initially the generative cell remains lying at one corner of the spore
wall.
Within short time, it gets detached and becomes ellipsoid or fusiform
in shape (Fig. 3.5C) and remains suspended in the cytoplasm of the
vegetative cell (2-celled stage i.e., vegetative cell and generative cell).
Later on, the generative cell divides and gives rise to two ellipsoidal or
lenticular or spherical cells — the male gametes (3-celled stage i.e.,
vegetative cell and two male gametes, Fig. 3.5D).

18. The second division i.e., the division of generative cell, may take place
either in the pollen grain or in the pollen tube which develops through
germ pore after pollination.
The nucleus of the vegetative cell is commonly known as tube nucleus
(Fig. 3.5D). It usually shows sign of degeneration with the maturation
of generative cell. Finally the tube nucleus remains within spore or
may enter the pollen tube (Fig. 3.5E, F and G). Sooner or later it may
be degenerated completely.
Significance of tube nucleus:
Earlier workers thought that the tube nucleus had great significance in
the direction of growth of the pollen tube, as it is usually present just
behind the growing point within the pollen tube.
However, recent workers differ with the above opinion and
consider it as a purely non-functional vestigial structure,
based on the following facts:
1. In branched pollen tube, the tube nucleus remains in one tube, but
all the tubes grow normally.

19. 2. It does not always occupy the position behind the growing point
within the pollen tube, but in many cases it lies behind the male
gametes.
3. In some cases, the growing pollen tube does not have any tube
nucleus as it degenerates prior to the development of pollen tube.



Pollen wall structure
The pollen grains are covered with two concentric wall layers, the
outer exine and the inner intine. The stratification of pollen wall is
primarily based upon optical microscopy of whole as well as sectioned
grains and upon staining reactions of the strata. Recently such
stratification is complemented by the biochemical analysis of the
various pollen wall layers.
The intine is pectocellulosic in nature and acid degradable, while the
exine is composed of sporopollenin which is found to be resistant to
both physical and chemical decay.
The pectocellulosic intine can again be divided into two layers the
outer pectic polysaccharide layer, called exintine and the inner
cellulosic layer, called endintine. Cytochemically these two layers are
distinguishable.
The outer exintine layer stains positively with alcian blue, while the
inner endintine reacts strongly to the PAS test and calcoflour white. In

20. some graminaceous pollen a pectic polysaccharide rich middle layer
(called Z layer) is distinguishable which is thickened at the germinal
aperture and termed “Zwischenkörper”.
This layer is comparable to the exintine. In some members (e.g.,
Amphobolis) intine shows uniform staining reaction, hence cannot be
distinguished. Intine of different species vary greatly in thickness. In
some species, the cellulosic layer is too thin to be observed under
microscope, while in others it occludes practically the whole lumen of
the pollen grain.
The cellulose of the intine shows a microfibrillar structure and the
microfibrils are oriented parallel to the surface of the grain showing
random deposition except near the apertural areas. Hence the intine
depicts negative spherical birefringence in polarizing microscope
where maximum extinction plane is tangential. In electron micrograph
the intine often appears lamellated because of the interbedding of the
cellulose layers with the layers of protein.
The exine is further divided into two layers, the outer ektexine (sexine
and foot layer) and the inner endexine which is often well developed in
dicots, but virtually absent in monocots. These two layers can be
demarcated on the basis of their different staining property. Ektexine
is generally dissolved with 2- and 3- ethanolamine and stains deeply
with basic fuchsin and auramine 0.
Endexine is resistant to hydrolysis with 2- and 3- ethanolamine and
does not stain or often stains weakly with basic fuchsin and auramine
0. The homogenous ektexine is formed early.
It shows positive spherical birefringence where submicroscopic
structural elements are radially arranged. The ultrastructure of
endexine is comparable to intine showing negative spherical
birefringence. This layer is formed later by the apposition of
tangentially oriented lamellae.

21. NPC Classification of Pollen and
Spore Wall



NPC is an artificial system of classification of pollen and spore based
on the three features of aperture only, i.e. number, position and
character. Erdtman and Straka (1961) proposed NPC classification and
palynologists all over the world accepted it.
According to NPC system each pollen grain has an arithmetic cardinal
number consisting of three digits. The first digit reveals the absence or
presence of aperture, and when present it mentions the total number
of aperture(s) present in a pollen grain.
The second digit illustrates the position of aperture(s), i.e. distal,
proximal, and latitudinal, meridonial, equatorial etc. The microspores
reveal the position of aperture(s) with full clarity when they are in
tetrad. The third digit explains the character of an aperture, i.e.
circular/oval or elongated, simple or compound etc. ‘N’ from number,
‘P’ form position and ‘C’ from character of aperture compose the NPC-
classification.
i. Classification of Aperture Based on Number(s):
In NPC system ‘N’ denotes the number (N; L. numerus) of aperture(s)
present in a pollen grain. Aperturate pollen, i.e. pollen having
apertures are divided into seven groups. The groups are mentioned as
N1 to N7. Each group has characteristic number of aperture, i.e. N1 has
one aperture and N2 has two apertures and so on. The N7 group has
seven or more apertures.
N1 to N7 groups are also referred to respectively as monotreme,
ditreme, tritreme, tetratreme, pentatreme, hexatreme, and polytreme
(Greek trema means hole, opening, aperture; pl. tremata).

22. There are pollen grains where apertures are absent. Such pollen grains
are termed as inaperturate or atreme and they are placed in N0 group.
Another special group N8—termed anomotreme is created where the
pollen grains and spores have one or several irregular or irregularly
spaced apertures.
ii. Classification of Aperture Based on Position (Fig. 4.26):
In NPC system ‘P’ denotes the position (P; L. positio) of aperture in a
pollen grain and spore. The position may be proximal, distal and
equatorial. There are seven groups of aperture based on position
namely –P0 to P6. Pollen grains having P0 group have uncertain or
unknown position of aperture. P1 groups of pollen and spores are
catatreme (Gr. Kata = down; -treme is suffix used as a synonym of
aperture).
Catatreme pollen grains have one aperture that occurs on the proximal
part of a grain. The proximal (L. proximus, nearest) part is the face of
a pollen grain or spore that faces inward/nearest or toward the centre
of tetrad (Fig. 4.31). P2 groups of pollen and spores are anacatatreme
(Greek ana = up). Anacatatreme pollen and spores have two apertures.
One aperture with its centre occurs at the proximal pole. The other
aperture with its centre occurs on the distal pole. The distal (L.
distalis, remote, outer) part is the face of a pollen grain and spore that
faces outward, i.e. away from the centre of tetrad and opposite the
proximal part (Fig. 4.31). P3 groups of pollen and spores are anatreme,
i.e. the aperture is distal in position.
P4 groups of pollen and spore are zonotreme. A zonotreme (zono-a
prefix used to indicate the equatorial/subequatorial region) pollen
grain is characterized in having apertures on equator or sub-equator.
The equator is the part of a pollen grain or spore that runs midway
between the proximal and distal poles and perpendicular to polar axis.
P5 groups of pollen and spore are dizonotreme.
Dizonotreme pollen grains have apertures arranged in two or more
zones. The apertures occur parallel to equator. P6 groups of pollen and
spore are pantotreme (Greek pan, gen. Pantos, all, wholly).
Pantotreme pollen grains have apertures scattered over the whole
surface uniformly. As a rule, pantotreme pollen grains are spheroidal.

23. iii. Classification of Apertures Based on Character (Fig.
4.27):
In NPC-system ‘C’ denotes the character (C; L. character) of an
aperture in a pollen grain and spore. The character groups of pollen
and spore are seven and they are mentioned as C0 to
C6. C0 groups have
apertures whose character cannot be established with certainty.
C1 groups of pollen and spore have leptoma (Greek leptoma means
thin place).
Leptoma is a thin area, aperture like and functions like an aperture.
Pollen grains having one leptoma are termed as monlept. The leptoma
may occur on either proximal-or distal face of a pollen grain and spore
and accordingly termed as catalept and analept. C2 groups are
trichotomocolpate (Gr. Tricha, in three parts; tome, cut; kolpos,
depression, furrow).
Trichotomocolpate is a three- branched aperture, the branches of
which are more than two times longer than breadth.
Trichotomocolpate pollen and spores having aperture on porximal
face are termed as trilete.
The group C3 has colpate grains. The group C4 comprises porate pollen
grains. The group C5 comprises colporate pollen. The group
C6 comprises pororate pollen. C3, C4, C5 and C6 groups of aperture are
previously discussed under aperture.

24. In NPC classification a grain is mentioned in three-digit number (Figs.
4.28, 4.29 & 4.30), e.g. 343 instead of N3P4C3. Pollen grains having
NPC 343 are tritreme zonocolpate, which is also described as
tricolpate pollen.
NPC 764 characterizes those pollen grains that are polytreme
pantoporate, which are also described as pantoporate or polyporate.
Pollen grains of Amaranthaceae, Chenopodiaceae etc. have NPC 764.
Examples of tricolpate pollen grain, i.e. NPC 343 are Rumex, Vitex,
Tectona, Argem one etc.
Merits of NPC classification:
1. It is a simple system of classification and illustrates the apertures of
a pollen grain and spore.
2. NPC makes the description of apertures precise.
3. With the aid of NPC pollen grains and spores of pteridophyta,
monocotyledon and dicotyledon, to some extent, can be differentiated.
Most of the spores of pteridophyta are monolete or trilete. Monocots
are characterized by inaperturate, monosulcate and monoporate
pollen grains. Dicots, with a few exceptions, have pollen grains that
are mostly with three meridonial furrows and polyaperturate. Thus
NPC narrows the search list of identification of unknown
sporomorphs.

25. 4. NPC is supposed to be of primary classificatory character because
apertures are most conservative. It is supplemented by surface
ornamentation, size and shape etc. of a pollen grain. Sometimes it
becomes possible to identify the family or genus or even species of a
pollen grain with the aid of NPC in combination with other
morphological characters.
5. Palynologists all over the world accepted NPC-classification as it is
basically simple and consistent where pollen grains and spores could
be arranged easily. This helps to identify unknown sporomorphs.

26. 6. NPC, sporoderm stratifications, exine patterns, size and shape etc.
of a pollen grain are genetically stable. This property is utilized for
various purposes and the followings are a few illustrations. With the
aid of NPC and other characters a key can be formulated that helps to
identify unknown pollen and spores.

27. Identification of pollen and spores is the essential prerequisite in the
applied aspects of palynology, i.e. aeropalynology, melissopalynology,
forensic palynology and palaeopalynology etc.
The interfamily and intra-family affinities of taxa, to some extent, can
be determined with the aid of NPC. As for example the family
Gramineae seems to be closely related to Restionaceae,
Centrolepidaceae and Flagellariaceae, because pollen grains of above
taxa are monoporate.

28. 7. NPC and the various types of exine patterns and ornamentation
provide characters of taxonomic significance and thus become one of
the sources of alpha taxonomy.

29. Bombacaceae is segregated from Malvaceae; Zingiberaceae,
Cannaceae and Musaceae are amalgamated into Scitamineae.
Demerits of NPC classification:
1. It is an artificial system of classification.
2. Syncolpate and parasyncolpate pollen grains do not fit neatly in
NPC system.

30. 3. Pollen grains that are characteristically present as aggregates in
tetrads, e.g. Ericaceae, Typhaceae and polyads, e.g. Orchidaceae,
Mimosa etc. are not grouped in NPC system.

31. NPC-system of classification is always compared with Linnaeus’s
system of classification, because the latter is also an artificial system of
classification. The characters of stamen were the basis of classification.
‘Linnaeus accepted the weakness of his classification but claimed that
it was propounded mainly as an aid to identification’. This is also
applicable to Professor Erdtman. Palynologists from every discipline
of palynology utilize Erdtman’s NPC classification and other
characters related to pollen morphology as an aid to the identification
of unknown sporomorphs.

32. Palynology, scientific discipline concerned with the study of plant pollen,
spores, and certain microscopic planktonic organisms, in both living and fossil form. ...
Palynology also has applications in archaeology, forensic science and crime scene
investigation, and allergy studies.

What is Palynology ?
The science concerning the study of pollen and spores is called
‘palynology”, and this term was coined by Hyde and Williams in 1945.
“Pollen grains” or microspores are the male reproductive bodies of the
flowering plants, while the term “spore” is very loosely applied to
several types of reproductive bodies in algae (e.g. zoospores,
exospores, endospores, akinetes, etc.), fungi (e.g. conidiospores,
ascospores, uredospores, basidiospores, chlamydospores, etc.) and
pteridophytes. Pollen grains develop in the sporogenous tissue of
anthers or microsporangia in angiosperms.
According to Zetzsche and Vicuri (1931) the outer walls of pollen and
spores are made up of a pectinous substance called “pollenin”. Its
chemical formula is C90H129 (OH)5. The protoplasm of pollen grains
contains proteins, carbohydrates, lipids, vitamins, hormones and
enzymes. It also contains traces of some inorganic substances such as
Mg, K, Ca, Cu, Fe, Si, P, S and CI.
Some Common Terms:
1. Proximal Pole:
The end of the pollen grains towards the centre of the tetrad is called
proximal pole. 2. Distal Pole:
The end of the pollen grains towards the outside of the tetrad is called
distal pole.
3. Polar Axis:
It is a hypothetical line which connects proximal and distal poles.

33. 4. Equatorial Axis:
A hypothetical line perpendicular to the polar axis is called equatorial
axis.
5. Colpi and Colpate:
The elongated or furrow-like apertures on the pollen grains are called
colpi (sing., colpus) and such grains are called colpate (Fig. 4).

6. Port and Porate:
The circular apertures on the pollen grains are called pori and such
grains are called porate (Fig. 5).

34. In colpi and pori both, the outer face of the aperture is congruent with
the inner face.

7. Colporate:
When the outer and inner faces of apertures are incongruent (Fig. 5),
the apertures are called colporate.
(In most of the gymnosperms and monocots the apertures are known
to be distal while in pteridophytes they are proximal).
8. Zonocolpate and Zonoporate:
When the colpi or pori are zonal in position, they are called
zonocolpate or zonoporate, respectively.
9. Pantocolpate and Pantoporate:
When the colpi or pori are uniformly distributed on the exine surface,
they are called pantocolpate or pantoporate, respectively.
10. Crassimarginate:

35. The apertures (colpi or pori) with thickened margins are called
crassimarginate.
11. Syncolpate:
When ends of the colpi unite at the poles, die grains are called
syncolpate.
12. Intine and Exine:
The protoplasm of the pollen grains is enclosed by a wall made of
intine and exine. The intine is a hyaline layer. The exine (Fig. 6)
consists of an inner homogeneous layer (called endine or nexine and
an outer heterogeneous layer (called ectine or sexine).

13. Columellae and Tegillum:
The radial rods which form the ectine are called colurnellae. The
colurnellae are either free at their tips or are fused to form a layer
called tegillum.
14. Inaperturate:
A pollen grain without any aperture (Fig. 5).

36. Various types of ornamentation patterns are shown by exine surface.
As mentioned above, the ectine of exine is composed of radial rods or
colurnellae. When the distal surfaces of colurnellae are bright and the
intervening regions are dark, the pattern is called pilate (Fig. 6). The
colurnellae in most of the grains are fused to form different types of
patterns having depressed areas (called lumina) and the intervening
areas between lumina (called muri).
When a network is produced by lumina and muri it is called reticulate.
In the reticulate pattern, if there is the incomplete fusion of
colurnellae, it is called retipilate, if there are circular and closely
placed lumina it is called foveolate, if there are circular but distantly
placed lumina it is called scrobiculate, if lumina are elongated it is
called fossulate, if lumina are parallel it is called striate and; if lumina
are anastomosing it is called rugulate.
The exine ornamentation is called areolate when luminoid network
surrounds islands of raised areas. If the excrescences (outgrowths) on
the exine are in the form of very minute granules the pattern is called
granulose, if the excrescences are in the form of spinules with pointed
or blunt ends it is called spinulose, if the excrescences are in the form
of rounded warts with constricted base it is called gemmate, and if the
bases are not constricted it is called verrucate.
When the outgrowths on the exine are in the form of tubercles it is
called tuberculate, if these are long with pointed ends the pattern is
called spinose, if outgrowths are rod shaped it is called baculate, and
when they are club shaped it is called clavate.
16. LO- or OL- Pattern:
In LO or OL pattern ‘L’ (lux) stands for “light” and O (obscuritas)
stands for darkness. At different foci, a varying pattern of bright and
dark islands is observed. At the uppermost focus, the depressed ends
on the surface of pollen grains appear dark, and this darkness changes
into brightness when focused down.
17. OLO- Pattern:

37. A succession of three patterns, i.e., dark (O), bright a) and dark (O) is
called OLO-pattern.
18. Ectine and Endine:
Ectine is the outer layer of exine while endine is the inner layer of
exine.
19. Ectocolptum and Endocolpium:
The outer and inner faces of a colpus are called ectocolpus and
endocolpus, respectively.
20. Tenuimarginate:
The apertures (colpi or pores) with thin margins are called
tenuimarginate.

Proteins in Pollen wall

T. Singer and Petrovskya – Baranova (1961) were the first to show the
existence of proteins with enzymatic properties in certain layers of the
pollen walls of Paeonia and Amaryllis. Subsequently, Makinen and
McDonald (1968) demonstrated the pollen enzymes and isoenzymes
in flowering plants.
The details of the pollen wall proteins and their significance are
highlighted through the works of J. Heslop- Harrison and others
Certain proportion of the total protein of the pollen grain
occurred in the wall in three sites:

38. i. Many proteins are situa ted most prominently in the cellulosic intine
(endintine) specially at the apertural part in the form of radially
arranged tubules or tangentially oriented leaflets.
ii. Exine proteins are present in the cavities between the baculae of the
tectate grains or in the surface depressions of the intectate or
subtectate grains.
iii. Some proteins are located in the superficial materials (e.g. pollen
kitts) der. ed from tapetum.
The major portions of these proteins are enzymes, mostly hydrolytic
enzymes and the wall proteins are quickly lost when the pollen grain is
moistened. All the six classes of enzymes namely, dehydrogenases,
oxidases, transferases, hydrolases, lyases and ligases are reported to
be present and remain active in pollen grains of higher plasts (Table
3.6).

Esterases are the predominant enzymes of the exine layer, while acid
phosphatases are the chief enzymes of the intine. Therefore, in general

39. esterases and acid phosphatases can be used as marker proteins for
exine and intine layers respectively.
Some exceptions are there, for example Helianthus pollen contains
esterases and acid phosphatases in both exine and intine. In the
mature, dormant pollen the hydrolase activity is found to be
associated with the wall, with very little in the protoplast of the
vegetative cell.
In some species with exine absent or highly reduced, the wall protein
comprising of acid phosphatase activity are exclusively confined to
intine. These proteins are surrounded by a layer of mucilage with
glycoprotein fractions originate from tapetum which help to prevent
the release of wall protein into water.
In inaperturate pollen of Crocus vernus, with a thick intine and
relatively thin exine, the Site of esterase and acid phosphatase activity
is found in the central zone of the intine over the whole wall. A very
low activity of such enzymes are also observed within the
plasmalemma and in the superficial pollen-kitt.
In monoporate grains (e.g., grass pollen) hydrolase activity is confined
to the intine underlying the pore. In pantoporate grains (like
Malvaceae, Convol- vulaceae) such activity is mainly confined in the
intine at each pore, though a slight activity is also observed in
interporal intine. Sometimes, intense esterase activity is observed in
pollenkitt of such grains (e.g., Cobaea).
In monosulcate (Liliaceae, Amaryllidaceae) or triporate (Urticaceae,
Ulmaceae, Juglandaceae) or tricolpate (Lamiaceae, Brassicaceae)
pollen grains, intine is the site of hydrolase activity, but much less
activity is noticed in such grains having thin intine. In all cases such
activity is concentrated in the intine of apertural zone.
Among gymnosperm (Pinus, Abies, Cryptomeria) acid phosphatase
and RNase activity is confined to intine especially in the apertural
region. Among pteridophytes only Equisetum palustre shows

40. enzymatic activity in the entire intine. But spores of several ferns and
six species of Bryophytes examined showed no detectable wall enzyme.
It is evident from the isoenzymic analysis that zymograms of pollen
are rarely identical to those of tapetal and pistillate tissue. Pollen
grains exhibit few of the isoenzymes that are common to seed or
vegetative tissue, but the zymograms of dehydrogenase, peroxidase
and esterases have revealed bands apparently unique to pollen grain
supporting unique physiological properties of pollen.
Pollen grains are normally devoid of catechol oxidases (Polyphenol
oxidase) and the enzymes associated with plastid and plant pigments
(Though many pollen grains are pigmented).
Other enzymes namely, maltase, lipase, [3- glucoronidase,
arylsulphatase, p-diphenyl oxidase (laccase), several
pyrophosphorylases and zymase have not yet been reported from
pollen. The activity of some enzymes like alkaline phosphatase,
ATPase, DNA synthetase and β-1,3-gluconase are reported to be very
low or absent in pollen grains.
Pollen enzymes are freely and rapidly diffusible from pollen grains.
Enzymatic activity for many enzymes like amylase, P-fructofurano-
sidase, phosphorylase, transaminase is increased during germination
It is noticed that most of the pollen enzymes are synthesized
subsequently to meiosis, under control of the haploid genome. Pollen
slowly looses certain types (peroxidases, phosphatases) of enzymatic
activity during storage but these cannot account for the rapid loss of
viability.
i. Formation of intine proteins:
After the release of monad grain from tetrad, the plasmalemma of
pollen cytoplasm puts out radially oriented tubules with their protein
addition into the developing intine (Fig.3.1). Subsequently these
tubules disconnect from plasmalemma and are sealed off from the cell

41. surface with the deposition of a further layer of intine free from the
tubules.
In inaperturate grains proteins are dispersed throughout the intine,
while in aperturate grains intine proteins are mostly condensed in the
apertural region. In some taxa (e.g. Cosmos bipinnatus) in place of
tubular invagination, leaflets of plasmalemma with their protein
inclusions get incorporated into the intine as a series of tangential
lamellae after parting from the cytoplasm. Intine proteins are the
product of pollen cytoplasm, thus gametophytic in origin.

ii. Formation of exine proteins:
Exine proteins originate from the sporophytic tapetum tissue. During
the meiotic division of pollen mother cell, proteins in single
membrane-bound vesicles derived from rough ER and lipids resultant
from plastids get accumulated in tapetal cells (Fig.3.1). At the end of
pollen development these proteins and lipids are free into thecal cavity
after breaking down of tapetal cells.

42. These proteins are deposited in the surface depression of exine in
intectate or sub-tectate grains, but in tectate grains the protein
fraction passes through the micropores of the tectum and finally
deposited in the spaces between the baculae. In all the pollen types
lipids remain in the surface of the exine. Exine proteins are the
product of tapetal cells, hence they are sporophytic in origin.

43. Pollen Viability in Plants:


Meaning of 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.

44. 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.
Variations in the 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.
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.

45. 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 respiratory substrate that strongly contribute to their rapid
loss of pollen 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:
The regulation of pollen water content is an important adaptive
mechanism 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.

46. 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 hydration (Fig. 7.1).

47. 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.
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

48. 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 desiccation. This trinucleate pollen has a high rate
of metabolism, respiring two to three times more than the binucleate
pollen.
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.

49. Pollen Storage in Plants:
Methods and Significance


Systematic research on pollen storage started at the end of the
19
th century. There is large number of crop species, including
vegetables, fibre and fruit crops, forage and cereals, for which pollen
storage strategies are desirable. Genetic conservation through pollen
storage is desirable for a variety of horticultural plant species, since
pollen is known to transmit important genetically heritable characters.

Pollen is a product of genetic recombination and can provide a reliable
source of nuclear genetic diversity at the haploid stage. Although
genetic conservation through pollen storage does not accomplish the
whole genome conservation, a plant breeder involved in genetic
enhancement of a given horticultural crop could have access to a
facility called ‘Pollen Cryobank’, from where he can draw pollen
parents of his choice in the process of breeding a new cultivar.

Keeping the viability and vigour intact the pollen grains can be
suitably stored in appropriate containers like, glass or plastic vials for
an extended period of time. Such containers are stored in desiccators
with dehydrating agents to control humidity. Saturated solutions of
different salts are used to obtain the required humidity.
Lycopodium spores are used as diluents before storage to increase the
bulk of pollen and prevent wastage of pollen sample during artificial
pollination. This diluent has all the property of a good diluent, like non
sticky and non-hydrating and in addition it keeps the viability rate

50. quite high. It also provides its own growth factors, which leads to
higher percentage of germination.
Comprehensive studies have been done to assess the different storage
conditions that can prolong the viability of pollen grains. This storage
can be conveniently grouped as short term and long term storage
methods.
Method of Pollen Storage:
I. Short Term Pollen Storage:
It includes the effect of temperature and humidity, and pollen storage
in organic solvent.
i. Effects of Temperature and Humidity:
In general low temperature and relative humidities are favourable for
most taxa. However, pollen of a large number of taxa can be
successfully stored for a limited period of time through the
manipulation of storage temperature and humidity. Pollen grains can
be suitably stored in glass or polythene vials or in appropriate
containers.
In case of unsealed containers suitable dehydrating agents like silica
gel, various concentrations of sulphuric acid or saturated solution of
different salts are used to maintain required relative humidity.
Sticking together of the pollen grains can be prevented by using
Lycopodium powder, talc, or corn, or wheat flour. Tricellular pollen of
Gramineae requires sophisticated environmental conditions to
preserve viability and fertility even for a short period storage.
ii. Pollen Storage in Organic Solvent:
Iwanami and Nakamura (1972) first demonstrated the use of different
organic solvents in pollen storage. The organic solvents include
benzene, petroleum, diethyl ether, acetone, chloroform, etc., whose
efficiency varies greatly for different plant species. Table 7.2 shows the
effect of organic solvents on pollen germination in-vitro after soaking
them for 25 h in the solvent.

51. It supports the retention of viability in different organic solvents.
Pollen grains stored in non-polar organic solvents like benzene,
diethyl ether and cyclohexane retained viability and showed very little
leaching of phospholipids, sugars, and amino acids into the solvent.
On the other hand extensive leaching of substance and loss of viability
was seen in polar organic solvents, thus establishing a correlation
between the polarity of the solvents and its potency for pollen storage.
The Citrus pollen maintained viability in different organic solvents for
three months. Investigation of Liu et al. (1985) on plants like,
Armenica vulgaris, Camellia japonica, Ginkgo biloba, Juglans regia,
Malus pumila, Prunus triloba, Prunus percia, Salix babylonica, and
Zea mays shows that the insect pollinated species stored in a suitable
organic solvent at 4°C for 35-40 days exhibited the needed viability.
Chrysanthemum pacificum pollen loses viability (12%) in dry
conditions (25° C) within 60 minutes. When pollen grains stored in
such dry conditions for 30 minutes are then transferred to diethyl
ether, the viability remained at the level of 12% even after 20 days of
storage (Fig 7.2).

52. Removable from the organic solvent thereafter, lead to complete loss
of viability within 30 minutes. Literally while storage in organic
solvent there was no loss of viability which has been referred as
absolute dormancy. Inspite of the fact that the efficacy of individual
organic solvents varies greatly for different plant solvent has proved to
be better for the storage of any pollen than low temperature and
humidity.
II. Long Term Pollen Storage:
Storage at temperatures above 0°C slows down the metabolic activity
of the pollen, resulting in gradual decrease and finally total loss of
pollen viability. Thus for a long term preservation cryogenic technique
seems to be more promising. Some of the methods of long term
preservation are stated below.

53. i. Storage at Sub-Zero Temperatures:
Using a storage temperature of -10° C and – 34° C the longevity of
bicellular pollen (desiccation tolerant) and pollen with original low
content of moisture has been successfully extended between 1 and 3
years.
ii. Freeze or Vacuum Drying (lyophilization):
Pollen of different taxa especially the desiccation-tolerant pollen can
be successfully preserved for a long period of time by freeze or vacuum
dying method. Freeze-drying involves the rapid freezing of pollen to
sub-zero temperature of -60° C or -80° C using inert gas helium or
nitrogen, and then the gradual removable of water under vacuum
sublimation.
In vacuum drying the pollens are directly exposed to a vacuum and
simultaneous cooling. The moisture is later withdrawn by evaporative
cooling. In number of taxa when freeze drying is combined with
lyophilization then storage and viability of pollen has been found to be
very effective.

54. iii. Cryopreservation by Deep-Freezing:
Long term preservation can also be done by ultra low temperature,
ranging between -70° C and -196° C. Since the first half of 1950s,
several studies have been conducted on the cryopreservation of pollen.
A list of few important crop species whose pollen has been successfully
stored at cryogenic temperature is presented below in Table 7.3.

A reduction in the pollen water content below a threshold level before
low temperature exposure seems to be important for achieving
viability. Thus partially dehydrated pollen possesses less freezable
water and can survive deep freezing.
However, further studies are needed to determine the critical moisture
level of the pollen grains for a successful long-term cryostorage.
Studies on the molecular structure of the membrane using Fourier
transform infrared spectroscopy (FTIR) indicate that lipid transitions
in membranes may cause major damage during freezing or warming
after freeze-thaw has been completed.
The Graminaceous pollen contains nearly 35 to 60% water when shed,
thus immediate freezing would cause irreversible structural damage as

55. a result of ice formation. Thus the water content of the pollen needs to
be reduced before cryostorage, which is however, problematic, as there
is rapid loss of viability with decreasing water content.
Water loss without any detrimental effects in pollen viability ranges
between 50 % and 80%, this however, again depends on the species
and its genotype. Secale cereale and Zea mays can tolerate higher
degrees of desiccation than the grains of Triticale. Triticum aestivum
is however, intolerant to any degree of dehydration.
Some of the agronomically important Gramineae species shown in the
Table 7.4 have been stored successfully at cryogenic temperatures or
in deep freezer for long periods.

Significance of Storage:
Pollen storage has both its application in basic and applied
sciences. Some of the applications include:
i. The spatial and temporal isolation of parental species that enforce
cross pollination barriers can be overcome.
ii. In order to continue productivity supplementary pollinations like
pollen sprays can be implemented.
iii. In breeding programmes there is no need to grow the pollen parent
continuously.

56. iv. Genetic property can be conserved and can be a source for
germplasm in international exchange programmes.
v. In the study of pollen allergy and pollen biology it can serve as a
continuous source of pollen.
vi. Exotic nuclear genetic diversity can be easily received and
exchanged through pollen, thereby eliminating the need to go through
a long juvenile phase, common in most fruit trees to produce pollen
for hybridization at a desired location. Thus, stored pollen can be used
to improve breeding efficiency.
vii. Fruit tree pollen is generally required to be stored for controlled
crossings, either to achieve a desired breeding objective, or to
overcome a constraint involved in commercial fruit production.
viii. Pollen storage has come to the rescue, where stored pollen
indexed as viable can be used in crossing with the desired female clone
so as to accomplish the breeding objective.