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Anther and pollen biology

The document extensively discusses the structure and function of the anther wall, which comprises several layers, including the epidermis, endothecium, middle layers, and tapetum, each contributing to the development and dispersal of pollen. It details the process of microsporogenesis, including the formation of microspores, the role of callose during meiosis, and the subsequent development of the male gametophyte. Additionally, it highlights the importance of callose for isolating microspores and its protective roles during pollen development.

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Anther and pollen biology Prof. Subir Pramanik Bajkul Milani Mahavidyalaya
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
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 outermost 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 outertangential 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 mothercell. 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,
whereas, the outer tapetum of smallercells is derived from the parietal layer. Further in Antirrhinum majus and Impatiens glanduliferathe 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 togetherin aggregate and assist in their collective dispersal.
Microsporogenesis and Microspore During the development of the microsporangium, the anther is seen at first as a homogeneous mass of meristematiccells, 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 surroundingcells by their more deeply staining cytoplasm and conspicuous nuclei. There may be only one such archesporial cell in each of the fourlobes (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 numberof 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 nourishingthe
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 mothercell 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.
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
(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 unicellularand usually round although it may be oval, pyramidal, polyhedral, etc. It is provided with two coats— an inner, delicate cellulose layercalled 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 utilisedduring 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 involvingthe study of pollens.
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).
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.
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.
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 ensuringautonomous development of microspores.
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 interconnectedmass of microsporocytes in the locule, form a large meiocytic syncytium (Fig 1.7).
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
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 intercellularconnections 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 establishedthat 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, followedby enzyme digestion and acid hydrolysis, for eliminatingRNA and phosphoproteins, identified a preleptotene period of DNA synthesis. Whereas microspectrophotometricmeasurement showeda 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-inducingsubstances. Few attempts have been made to clone meiosis specific genes from microsporocytes and identify the protein products they encode.
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 functionsof 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 youngmicrospore 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 emphasizedthat 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.
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 lenticularor spherical cells — the male gametes (3-celled stage i.e., vegetative cell and two male gametes, Fig. 3.5D).
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.
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 complementedby the biochemical analysis of the various pollen wall layers. The intine is pectocellulosicin nature and acid degradable, while the exine is composed of sporopollenin which is found to be resistant to both physical and chemical decay. The pectocellulosicintine can again be divided into two layers the outer pectic polysaccharide layer, called exintine and the inner cellulosiclayer, called endintine. Cytochemically these two layers are distinguishable. The outerexintine layer stains positively with alcian blue, while the inner endintine reacts strongly to the PAS test and calcoflourwhite. In
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 cellulosiclayer 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 lamellatedbecause of the interbedding of the cellulose layers with the layers of protein. The exine is furtherdivided 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.
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 consistingof 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 numberof 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).
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 suffixused 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.
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.
In NPC classification a grain is mentionedin 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.
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.
6. NPC, sporoderm stratifications,exine patterns, size and shape etc. of a pollen grain are genetically stable. This property is utilizedfor various purposes and the followings are a few illustrations.With the aid of NPC and other characters a key can be formulatedthat helps to identify unknown pollen and spores.
Identification of pollen and spores is the essential prerequisite in the applied aspects of palynology, i.e. aeropalynology, melissopalynology, forensicpalynology 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.
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.
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.
3. Pollen grains that are characteristically present as aggregates in tetrads, e.g. Ericaceae, Typhaceae and polyads, e.g. Orchidaceae, Mimosaetc. are not grouped in NPC system.
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 ProfessorErdtman. 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.
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 floweringplants, 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 formulais 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.
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).
In colpi and pori both, the outer face of the aperture is congruent with the inner face. 7. Colporate: When the outerand 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:
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 eitherfree at their tips or are fused to form a layer called tegillum. 14. Inaperturate: A pollen grain without any aperture (Fig. 5).
Various types of ornamentation patterns are shown by exine surface. As mentionedabove, 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 fusedto form different types of patterns having depressed areas (called lumina) and the intervening areas between lumina(called muri). When a network is produced by luminaand 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 luminaare 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 focuseddown. 17. OLO- Pattern:
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 outerand 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 enzymaticproperties in certain layers of the pollen walls of Paeonia and Amaryllis. Subsequently,Makinen and McDonald (1968) demonstrated the pollen enzymes and isoenzymes in floweringplants. 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:
i. Many proteins are situa ted most prominently in the cellulosicintine (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
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 confinedto 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 plasmalemmaand 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 confinedin 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
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 isoenzymicanalysis 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 plasmalemmaof pollen cytoplasm puts out radially oriented tubules with their protein addition into the developing intine (Fig.3.1). Subsequently these tubules disconnect from plasmalemmaand are sealed off from the cell
surface with the deposition of a furtherlayer 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 plasmalemmawith 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.
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.
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 assessedon 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 taxonomicstatus of the plant and on the abiotic environmental conditions. In order to maintain the viability and fertilizingability 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 Phoenixdactylifera was prominently mentionedin 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. Variations in the Viability of Pollen: The life span of pollen is primarily determined by the plant genome but is also influencedby 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.
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 metabolicactivity 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, aminobutyricacid, 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.
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 followingdesiccation and hydration (Fig. 7.1).
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 shouldbe 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
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.
Pollen Storage in Plants: Methods and Significance Systematic research on pollen storage started at the end of the 19th 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
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 storedfor 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 unsealedcontainers 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 togetherof 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.
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 establishinga 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, Camelliajaponica, 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).
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, resultingin 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.
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 extendedbetween 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.
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, furtherstudies are needed to determine the critical moisture level of the pollen grains for a successful long-term cryostorage. Studies on the molecularstructure 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
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 freezerfor 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.
iv. Geneticproperty 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 eliminatingthe 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.