UNIT 2

POLLEN WALL & EXTRAEXINOUS

WALL MATERIALS

Sporoderm stratification and sculptures; LO-

analysis; Sporopollenin; Pollen Wall Development;
Ubisch Body; Pollen Connecting Threads; Perine;
Pollen-Kitt
 SPORODERM
(the wall or covering of a spore)

STRATIFICATION
Sporoderm: generally stratified with two basic layered structure
i. Intine - composed of cellulose
ii. Exine – composed of sporopollenin
The exine covers the entire pollen surface except germinal apertures
where it is absent or greatly reduced.

Exine of pollen grains: Outer sculptured SEXINE

Inner unsculptured NEXINE

Sexine : ECTOSEXINE Tectum

ENDOSEXINE [EKTEXINE] Columella
Nexine : NEXINE I Foot layer

NEXINE II [ENDEXINE]

Sexine is structured as a set of radially arranged rods

(Columella-supported by tectum/bacula- not supported by tectum) either
supported by a roof-like structure (tectum/tegillum) or not. If
tectum present, it may be partially perforated.
 TECTUM (synomym Tegillum)
The layer of sexine, which forms a roof over the columellae, granules or
other infratectal elements.
Tectate: pollen grains that have tectum
Imperforate or solid
Perforate

Infratectal
elements
collumellae,
granules,
alveoli etc.
Semitectate: A partially
discontinuous tectum in which the
tectal perforations are equal to or
wider than the muri (ornamentation elements
forming the meshes in reticulum) and usually
larger than 1 μm in diameter

Atectate: pollen grains that have an

exine with little or no internal
structure. Example: Degeneria
(Degeneriaceae).

Intectate: pollen grains without a

tectum, but with sculpturing.
Examples: Viscum (Loranthaceae),
Ilex (Araliaceae).

Etectate: pollen grains interpreted as

having lost their tectum during
evolutionary development
 LO-analysis (Erdtman, 1952)

A method for analyzing patterns of sexine

organization by means of light microscopy.

This method is valuable for elucidating exine

patterns. When focused at high level (H),
raised sexine elements appear bright (Lux),
whereas holes in the tectum are relatively
dark (Obscuritas). At lower focus (L) holes
become lighter and the sexine elements
become darker.
Identification key of sculpturing (ornamentation of exine surface)
A. Sculpturing elements absent
B. Surface even or diameter of pits <1µm Psilate
BB. Surface pitted, diameter of pits >=1µm Foveolate
BBB. Surface with grooves Fossulate

AA. Sculpturing element present

B. Radial portion of sculpturing elements ± isodiametric (sphaerical or polyhedral)
C. No dimensions >=1µm Scabrate
CC. At least one dimension >=1µm
D. Sculpturing elements not pointed
E. Lower part of element constricted
F. greatest dia of radial Gemmate
projection is more or
equal to height of element
FF. height of element is Clavate
more than greatest dia of
projection, club shaped
 Identification key of sculpturing

EE. Lower part of element not constricted

F. greatest dia of radial projection Verrucate
>=height of element; wart like
FF. height of element >greatest dia Baculate
meter of projection, cylindrical

DD. Sculpturing element pointed

E. Spines long, > 3µm Echinate
EE. Small spines, <3µm in length Spinulate

BB. Radial projection of sculpturing elements elongated

C. Elements regularly distributed Rugulate
CC. Elements parallel Striate

BBB. Sculpturing elements forming a reticulate pattern Reticulate

Punctate Reticulate
Striate
Psilate Spine Striate Reticulate

Clavate Verrucate Scabrate Perforate

Different wall
sculptures under
Scanning Electron
Echinate
Echinate Microscope (SEM)
 SPOROPOLLENIN
• The exine of spores & pollen grains is chemically
composed of a substance named sporopollenin-a
unique and novel biopolymer of ß-carotene,
Xanthophyll (Antheraxanthin) and fatty acid. Highly
resistant to deteriorate other than oxidation by strong
oxidants such as H2O2 or CrO3.

• First observed and named as ‘sporonin’ by John (1814)

and later characterized by Berzelius (1830).

• The oldest sporopolleninous acritarchs (non-acid soluble

i.e. non-carbonate, non-siliceous organic-walled microfossils
consisting of a central cavity, and whose biological affinities
cannot be determined with certainty) were found in Pre-
Cambrian rocks, 1.2-1.4 billion years old.
 WHERE DOES SPOROPOLLENIN OCCUR?

• It forms the basic structure of the resistant wall of most

palynomorphs, like spores, pollen grains, dinoflagellates
and acritarchs mainly spores of Bryophytes,
Pteridophytes, pollen grains of Gymnosperms and
Angiosperms.
• It has also been recorded from spores of Aspergillus niger,
sexual spores of Mucor mucedo, asexual spores of Pithophora
oedogonia, cell wall of some Phycopeltis epiphyton, Chara
corallina, cyst of Presinocladus marinus
• Besides exine, it is found in perispore, viscin threads,
elaters, and Ubisch bodies.
 SPOROPOLLENIN: Chemical Nature
• It is highly inert compound having empirical formula
C90H142O27 in spores of Lycopodium. It resists acetolysis, but
degrades in strong oxidants like H2O2 or CrO3 and exhibits
secondary fluorescence when stained with primuline.

An approximate empirical formula

C90H142O27 for Lycopodium, Picea, Pinus,
Corylus sporopollenin

• Zetzsche et al. (1937) determined that the sporopollenin is an

oxygenated hydrocarbon and contains hydroxyl and C-methyl
groups and substantial level of unsaturation.
• Shaw and Yeadon (1966) proposed that the sporopollenin is
composed of a lipid fraction of 55-65%, consisting of molecules
with a chain length up to C16 and a lignin fraction representing
10-15% of the total mass.
• Heslop-Harrison (1968) and Brooks et al. (1969) suggested that
sporopollenin is a copolymer of β-carotene and xanthophylls such
as antheraxanthin and fatty acids. By applying 13C NMR
spectroscopy, it is seen that sporopollenin contains a series of
related biopolymers derived from largely saturated precursors
such as long chain fatty acids and oxygenated aromatic rings.

• Traverse (1988) indicated that sporopollenin was close in structure

to the potassium salt of glucuronic acid.

• In 2019, researchers at MIT (Massachusetts Institute of

Technology) determined that it is primarily composed
of polyvinyl alcohol units alongside other aliphatic monomers,
all crosslinked through a series of acetal linkages by the study of
thioacidolysis degradation (solvolysis in dioxane-ethanethiol with boron
trifluoride etherate, is an acid-catalyzed reaction which results in the
depolymerization of lignins) and solid-state NMR of the molecular
structure of pine sporopollenin (Weng et al. 2019).
 POLLEN WALL DEVELOPMENT

Exine Growth Phase

Intine Growth Phase

Microsporocyte (microspore mother cells)

MEIOSIS

Tetrads of haploid microspore:s

are enveloped by a callosic wall
Callose wall can be detected around the
microspore mother cells during initiation
of meiosis; it forms a layer between the
cytoplasm and pollen mother cell wall
Callose wall surrounds the
plasmalemma
• Additional callose is formed after the second meiotic division which
isolates the young microspores from each other

• The callose is special cell envelope, persists until it is enzymatically

digested at the end of the tetrad stage
Callose: It is a polysaccharide in the form of β-1,3-glucan with some β-
1,6-branches and it exists in the cell walls of a wide variety of higher plants
EXINE GROWTH: Two widely accepted models
Primexine model & Undulation model

• Primexine model:
• Primexine is the blueprint for the exine (Heslop-Harrison, 1971)
• It has a matrix presumably made of cellulose microfibrils
• It is deposited in between the spore/pollen and the callose wall

“the primexine acts as a loose scaffold on to which sporopollenin

monomers (fatty acids and phenols) are covalently attached by
the localized action of superoxide radicals generated at the
plasmalemma” (Scott, 1994)

Beside its function as a controlled deposition of sporopollenin it

also functions as a pathway for the diffusion of other substances
such as enzymes, including those responsible for digestion of the
callose wall
 STEP 1
In the beginning, the primexine is discontinuous at certain
specific regions of the plasmalemma which marks the
position of the future germ pore.

PRIMEXINE

Deposition of primexime between the callose wall and

plasmalemma
 STEP 2
During the later period of primexine deposition, additional
gaps appear, and these are occupied by columns of electron
dense intricate tubular lamellae of 70 nm diameter, called
probaculae that rests on the plasmalemma

PRIMEXINE

PROBACULUM

Appearance and penetration of probaculae

in the primexine
The probaculae appear to condense round the plasmalemma and become more evident by the deposition
and polymerization of the sporopollenin precursors. Later during the late tetrad stages, the probaculae
become disjointedly differentiated into electron dense baculae
 STEP 3
The baculae become laterally expanded at the base to form the
foot layer

PLASMALEMMA

Laterally expanded
baculae
Depending upon the taxon-
• baculae may remain free above or
• increase in electron density due to rapid deposition of
sporopollenin, and their heads expand laterally to form the
tectum (roof), over the primexine matrix
 The foot layer represents the future
nexine 1, while the baculae and the
roof layer, the future sexine.

Further deposition of sporopollenin continues and the whole

pollen grain expands laterally and radially as the pollen
grain enlarges.
TECTUM
TECTUM BACULUM
BACULUM FOOT LAYER
Formation of baculae Formation of well
& tectum developed ektexine
TECTUM
BACULUM
FOOT LAYER
ENDEXINE
Gradual disappearance of INTINE
Fully developed wall of
callose wall mature pollen
• The chemical constituent of the probaculae
and the foot layer is not clear

• Their electron opacity is different from that

of the sporopollenin of mature pollen, for
which it has been described as
protosporopollenin

• In the initial stages these layers are not

resistant to acetolysis, however, they
become resistant with the development of
the tetrad stage.
• The presumptive germinal apertures of pollen grain are
already demarcated during the microspore tetrad stage. The
sites of pre-aperture are distinguished by the absence of the
primexine matrix and is associated with an underlying plate of
endoplasmic reticulum oriented parallelly to the plasma
membrane.

• This endoplasmic reticulum may physically prevent the

movement of membranous structures (coated with primexine
material) to the cell surface. Sheldon and Dickinson (1986)
believed that the meiotic spindle plays a role in aperture
positioning.

• Later the callose wall dissolves, thus releasing the tetrads. The
pollen now expand rapidly and the primexine matrix is largely
disrupted, and only the residue can be observed between the
baculae of the mature exine. A rapid conversion of the
protosporopollenin occurs, and the primexine acquires the
staining properties of the exine.
 In which pollen grains primexine is
absent?
In many marine angiosperms, primexine is absent
and consequently do not develop normal exine as
the nexine 2 is deposited below the sexine (nexine 1
absent)

Process:
In the initial process, several very thin electron
transparent lamellae appear to arise from the
cytoplasm and provide a locus around which
sporopollenin is deposited. With the progress of
deposition, the lamellae thicken and merge with
each other to from the nexine 2.
 UNDULATION MODEL
• Studies on exine development by Takahashi, (1989, 1993, 1995) in
Caesalpinia and Lilium gives little support to the primexine model

• It explains that the exine formation commences with the tetrad stage
by the invagination or undulation of the plasma membrane which is
possibly under the control of cytoskeleton elements

• These invaginated localized regions match to the regions of future

lumina and distensions (swelling) that correspond to the muri (A
system of ridges of the reticulum) of the mature exine

• Takahashi (1995) observed that in Lilium, the plasma membrane

assumes a reticulate pattern which matches the pattern of the mature
exine

• Fibrous threads (10-20 nm diameter) together with granules (10 nm

diameter) aggregate at the regions of protuberances of the plasma
membrane and slowly these aggregates develop into a smooth
protectum of 0.5 to 0.7 µm diameter
 Callose
Undulation of Development of
deposition
plasma membrane protectum

Callose degradation, Fully developed

probaculae formation & wall of mature
appearance of primexine pollen

• During the later part of the tetrad stage, the probaculae and the protectum are more
distinguishable beneath the callose wall and the plasma membrane assumes a
smooth outline.

• Probaculae are later formed in between the plasma membrane and the protectum. At
this time the cellulosic fibrous primexine is distinguishable in spaces between the
probaculae and the callose wall dissolves, thus releasing the microspores and further
differentiation of the exine continues. Thus the undulated plasmamembrane plays
an important role in pollen wall development and in fact the protectum is the first
exine layer that is deposited on this membrane.
Christensen (1972) outlines the following steps in the
development of the pollen wall:
• In the tetrad stage, a new wall called primexine, is deposited
around the microspore protoplast within the wall of callose.
The primexine appears to contain cellulose microfibrils
• In the transition of primexine to exine elements, primexine produces precursors
of rod like bacula, which form the sexine. As a result of rapid deposition of
sporopollenin the baculae enlarge in electron density and their heads expand
laterally to form the tectum

• This is simultaneously followed by the lateral expansion of the base of the

bacula to form the foot-layer. Deposition of sporopollenin continues and the
whole wall expands laterally and radially. It also witnesses the dissolution of
the cellulose wall & consequently the pollen grains lie free in the pollen sac

• Nexine is deposited below the sexine. This is initiated by the deposition of

number of electron transparent lamellae originating from the cytoplasm, around
which sporopollenin is deposited. Deposition is followed by the thickening of
the lamellae which unite with each other to form the nexine
• In the aperturate region, the sexine has very short bacula,
while the nexine becomes much thicker and discontinuous
in this region than the rest of the pollen wall.
• At later stage, cellulosic intine is formed inside the nexine.
 Intine Growth Phase
• The intine usually starts to develop at the vacuolated stage,
beneath the apertures. It increases in thickness under the pores
and later starts to develop under the interapertural parts as a thin
layer.

• Golgi bodies are frequent during intine synthesis e.g.,

Ranunculaceae while in others, E.R. and polyribosomes are
abundant e.g. Cosmos. The intine beneath the pore become
comparatively very thick and is provided with fibrillar material
and radially arranged membranous units.

At final period of wall development

Additional lipoidal and pigmented substances may accumulate
on or/and within the outer exine. This material called the
pollenkitt; it imparts colour or odour to the pollen and may
cause the pollen grains to adhere together during dehiscence
 Ubisch bodies (after G. von Ubisch) or Orbicules
• They are spherical structure (2-5 µm in
dia) found in the anthers of many genera
of angiosperms, both monocotyledons,
e.g. Triticum aestivum and dicotyledons,
e.g. Betula pendula and many
gymnosperms, e.g. Ephedra foliata.

• They may also oval, rounded, triangular,

rod like etc.

• They frequently fuse into large compound

aggregates as, in Euphorbia caput-medusae.
It was first discovered by
• They generally have a central core and Rosanoff (1865) in close
thick wall with microchannels. association with the tapetum.
Erdtman et al. (1961) proposed
• They are mainly a distinctive feature of the term ‘orbicules’ for these
secretory or glandular tapetum. Their bodies.
walls apparently consist of
sporopollenin.
Development Function

1. Orbicules have no specific

function; they are a byproduct
of tapetal cell metabolism.

2. They might actively

participate in lysis and
degradation of tapetal cell.

3. They prevent osmosis and

collapses of the developing
microspores.
 Pollen Connecting Threads
The selective factors in pollination biology include both structural and functional
efficiency. It is observed that anemophilous angiosperms produce mostly small, single
and dry pollen grains whereas, in zoophilous angiosperms, flower visitors transfer large
clusters of pollen grains that are certainly a positive selective factor in pollination
biology. There are three different types of pollen clusters occur in angiosperms: a. sticky
pollen grains b. polyads and c. pollen connected by threads.

Types and Function of pollen connecting threads

Previously most of the known pollen connecting threads were grouped under
the term “viscin threads”. But recent knowledge interprets that principally
there are two main types of adhesion threads, either containing sporopollenin or
not. The adhesion strings containing sporopollenin is named as ‘viscin
threads’ whereas threads lacking sporopollenin cannot be called as viscin
threads.
In modern day, four major types of pollen connecting threads are categorized on
the basis of nature and ultrastructure, which are as follows:
a. Sporopollenin threads (viscin threads): long threads; small filiform bridge like
threads
b. Non Sporopollenin threads: cell originated; dead cell secretion originated
c. Cellular threads: modified anther cells originated
d. Non cellular threads: Elastoviscin threads of tapetal origin; lipid threads
degenerated materials; slimy viscous fibres of unknown origin
 a. Sporopollenin Threads (viscin thread)

It occurs only in three zoophilous unrelated families of

angiosperm viz., Onagraceae, in many Ericaceae and in a single
genus Jacqueshuberia of Caesalpiniaceae but the nature and
function of the threads is same.

Function:
i. Strictly ektexinous viscin threads connect the neighboring
pollen grains or polyads and help in easy dispersal of large
quantity of pollen grains by flower visitors.

ii. Extremely short bridge like threads act as additional cohesion

elements in polyads and does not connect neighboring polyads.
These are formed from endexinous material.
Patel et. al. (1985) described the ultrastructure of sporopollenin threads and added some
interesting characters:
1. Often four grains are found connected by threads – this indicates that four microspores of post-
meiotic tetrads remain connected.

2. Before acetolysis the Jacqueshuberia threads and pollen grains are widely covered and sculptured
by some granular tapetal debris. After acetolysis the pollen grains and threads are found free of
debris with smooth surfaces.

3. The sporopollenin threads are very flexible but apparently not elastic, their average diameter is
similar to that of columella.

4. The columellae are pillar like and distally branched, with tangled ribs forming a network. Some
enormously elongated ribs of the columellae form the pollen connecting threads, which are
extension of ektexine.

5. The sporopollenin threads of Ericaceae and Onagraceae pollen also represent distal elongations of
sculpture elements i.e. branched columellae. The ektexine characters are strikingly similar to that of
viscin threads.

6. In Ericaceae and Onagraceae, like Jacqueshuberia, the ektexine of viscin thread pollen consists of
distally branched and vaulted columellae forming a peculiar type of tectum, while some extremely
elongated, not vaulted and not thickened columellar branches form sporopollenin threads.

7. In onagraceae, in addition to long sporopollenin threads, small filiform bridge like threads and
fragile, short non-sporopollenin fibers are also present.
 Tetrad with viscin threads
Rhododendron hirsutum (Ericaceae) Epilobium montanum (Onagraceae)

Epilobium parviflorum (Onagraceae) Ludwigia octovalvis (Onagraceae)

 b. Non Sporopollenin threads

This type of threads is formed by:

1. Cells – in only one zoophilous angiosperm family

2. Exclusively by ‘dead cell’ secretions – in most of the

zoophilous angiosperm families
 c. Cellular threads

• This type of threads is rough and massive and formed by

modified anther cells (e.g. Strelitzia of Musaceae).

• The threads are covered by thin, lipid coating and stick on the
pollen grains.

• These large, massive and rough threads connected the pollen

for carrying by heavy pollinators while flexible threads seem to
be associated with small insect pollinators.
 d. Non-Celllular threads
It is found in several zoophilous angiosperm taxa. At least two different types of
threads are present in Orchids among the followings:
1. Elastoviscin threads – These are produced by specialized tapetum cell
cytoplasm and not by plastids. Threads are elastic, non-sticky, often non
homogenous, mostly lipid materials in and outside the pollinia
connecting the individual pollen grains. When the pollen grains are physically
separated, the elastoviscin forms highly elastic threads.

2. Lipid threads – This type of threads are sticky, highly viscous but inelastic
substances originate from some immature, degenerating cells in the
transition region between the caudicle (a slender, elastic appendage, to
which the masses of pollen in orchidaceous plants are attached) and the
massulae (=polyad) (e.g. Habenaria).

3. Slimy, viscous fibres (containing protein and lipid) connect individual pollen
grains in some zoophilous angiosperm families. Very little is known about their
nature and origin. Slimy threads connecting pollen are either coated with
small droplets or resemble a rather rope like fluid (e.g. Aristolochiaceae,
Annonaceae). These slime threads may act as special pollen cement or may play
some role in pollen germination.
 Perispore/Perine
• It is a hyaline loosely organised sporopollenin envelope covering exine
and occurs in some pteridophytes (Polypodiaceae) and a few
gymnosperms (e.g. Taxodium).

• It is secondary in origin and developed after differentiation of exine. It

may be continuous or sometimes folded in various ways. Well-developed,
complex and highly ornamented perine is found in advanced groups of
ferns.

• The presence of fold and well-developed excrescences (outgrowths) is

possibly a special advancement of retention of moisture and adherence to
the substrate to enhance healthy germination.

• Sometime it is difficult to decide whether a layer is perinous or

exinous. In such case, Erdtman suggested the term ‘Sclerine’ (Gk.
Skleros, hard) which can be used as a comprehensive name for perine and
exine. Generally, perine is used for pollen, and perispore for spore.
Perine layer in monolete and trilete spores
 Pollen kitt

• In the final period of pollen wall development, additional lipoidal and

pigmented substance may accumulate on or/and within the outer exine.
This material is called pollen kitt. Originally the term was used to
describe the whole content of the mature anther. However, the term is
now referred as an oily often pigmented layer found coating the exine.

• In entomophilous flower, it is electron-dense homogenous films on the

pollen surface; in anemophilous flower it is present as small,
heterogenous lumps in the cavity of exine.

Chemical nature- It is fluid, viscous, sticky and partially with crystalline

inclusions. It contains carotenoid and other lipid; its principal pigment is
carotene 5, 6-epoxide.
Ontogeny- It is synthesized from plastids of anther tapetum. Within the
tapetal plastid, osmiophilic globuli appear as small droplets; they coalesce
upon contact and attain certain size; finally, they occupy the whole plastid
and interact with plastid membrane; after breaking the membrane they
release in cytoplasm; degeneration of cytoplasm results in discharge of
globules which is deposited as pollen kitt on surface of pollen grain.

Functions-
1. It imparts colour and odour to the pollen, as such acts as insect attractant;
2. Sticky nature would serve as adherent to insect’s body and because of its
hydrophilic nature might even be associated with dispersal of the pollen grain;
3. Its carotenoid can protect the genetic content of the pollen against radiation
(UV) damage during dispersal