PLANT REPRODUCTION BIOLOGY - N. L. Kolyasnikova - 2017

SECTION 2. PLANT SEXUAL REPRODUCTION

The flower is the reproductive organ of angiosperms, representing a combination of sterile and fertile structures. The sterile structures include the perianth, which consists of the calyx and corolla. The fertile structures are the stamens and carpels. The boundaries between sterile and fertile parts are conventional.

Let us examine the fertile parts. The stamen is a modified microsporophyll. The collective group of stamens forms the androecium. A flower may contain one or several stamens, and their number can be indefinite or fixed. They may be arranged on the receptacle in a spiral, in whorls, or in clusters.

Stamens originate as small protuberances. First, the fertile part—the anther—develops, followed by the sterile part—the stamen filament. In some species (such as the family Magnoliaceae), some stamens are entirely sterile and are referred to as staminodes. Three periods are distinguished in anther development: pre-meiotic, meiotic, and post-meiotic. During the first period, the anther wall is formed and the microsporangium develops. The second period involves the Differentiation of the anther wall and Meiosis in microsporocytes. The third period is characterized by the maturation of pollen grains.

In the early stages, the anther consists of an epidermis and meristematic Cells. Subsequently, strands of archesporial cells differentiate beneath the epidermis. Periclinal divisions of these cells result in The formation of two layers: the parietal and the sporogenous layers. The parietal layer gives rise to the anther wall, while the sporogenous layer forms the microsporocytes. Depending on the direction of differentiation, 4 types of anther wall formation are distinguished [2, 3, 4].

1. Basic type. The parietal layer forms a secondary parietal layer. Further periclinal Cell divisions lead to the Formation of the endothecium, several middle layers, and the tapetum. This type is characteristic of primitive plants, particularly the family Winteraceae.

2. Dicotyledonous type. Through Cell Division, the parietal layer forms a secondary parietal layer and the tapetum. Subsequently, the secondary parietal layer gives rise to the endothecium and the middle layer.

3. Monocotyledonous type. Periclinal Divisions of the primary parietal layer produce the endothecium and a secondary parietal layer. Next, the secondary parietal layer cells form the middle layer and the tapetum.

4. Reduced type. The parietal layer gives rise directly to the endothecium and tapetum.

2.1 The Anther

During the early Selection/3.html">Stages of development, four cell layers are distinguished in the anther wall: the epidermis, endothecium, middle layer, and tapetum.

The epidermal cells are regular in shape and divide only anticlinally. At later stages, they become covered with a cuticle, increase in size, and sometimes acquire a dentate shape or become flattened [9]. Starch or tannin may be deposited within these cells (Figs. 6, 7).

Class="center">Fig. 6. Anther of *Galega orientalis*: 1 - transverse section of the anther; 2 - wall of a young anther at the stage of prophase I of meiosis in microsporocytes; 3 - wall of a mature anther

Fig. 7. Development of the anther wall in *Echium amoenum*: 1 - formation of the tapetum; 2 - binucleate tapetum; 3 - degeneration of the tapetum, onset of endothecium development; 4 - wall of a mature anther: т - tapetum; эн - endothecium; эп - epidermis

The Cell walls of the endothecium thicken, and fibrous bands develop. These thickenings facilitate anther dehiscence once the pollen has matured within the anther locules.

The number of middle layers can range from 1 to 6. Their increase results from additional periclinal divisions. The middle layers gradually degenerate and completely disappear by the time the pollen matures.

The tapetum is distinguished as outer (facing the anther wall) and inner (facing the connective). The inner tapetum is formed through the division of the basic tissue of the connective. Two MAIN TYPES OF tapetum are recognized: secretory and periplasmodial. The secretory tapetum maintains its cellular Structure up to the microspore tetrad stage. Tapetal cells may be multinucleate, and sometimes polyploid due to nuclear fusion. Later (following the tetrad stage), the secretory tapetum transforms into an amoeboid type: part of the cell walls dissolves, and the Cytoplasm invaginates into the anther locule. The periplasmodial tapetum forms before and during meiosis in microsporocytes; the cytoplasm fills the anther locule, and the nuclei within it divide synchronously. By the time the pollen grains mature, the tapetum undergoes lysis.

Sporogenous cells within the anther locules become microsporocytes. Microsporogenesis in flowering plants takes place inside the anthers while the bud is still closed. The microsporocytes are surrounded by a specialized callose wall. Microsporogenesis is based on meiosis, resulting in the formation of a microspore tetrad from a single microspore mother cell. Two Types of tetrad formation are distinguished: simultaneous and successive; an intermediate type is rarely observed. Tetrahedral, T-shaped, and linear tetrad types are recognized. During the post-meiotic period, the tetrads separate. The pollen grain and its nucleus increase in size, a vacuole appears, and The Nucleus shifts toward The Cell wall.

Mitosis occurs, yielding a lens-shaped generative cell, which migrates into the vegetative cell. The vacuole disappears, and the cytoplasm becomes packed with nutrient reserves. Mature pollen grains may be bicellular or tricellular (Fig. 8).

Fig. 8. Mature pollen grains: 1 - tricellular in sunflower; 2 - bicellular in alfalfa

The investigation of Structural and functional parameters of plant pollen is highly relevant both theoretically (to understand the mechanisms of plant damage and adaptation under technogenic stress) and practically (to determine the reproductive potential of plants from polluted areas for producing viable offspring), as well as for addressing biomonitoring problems, plant breeding, and seed production (Fig. 9).

Fig. 9. Pollen fertility of little-leaf linden in 2016: 1 - bank of the Sylva River; 2 - Perm

Pollen capable of Fertilization is referred to as fertile. Two main Methods are used: the acetocarmine and the iodine method.

To determine pollen grain fertility, anthers with mature pollen are fixed in Clarke's fluid (3 parts ethanol : 1 part glacial acetic acid). Fixation time ranges from 30 minutes to several hours.

The anther is placed on a Microscope slide, crushed, a drop of acetocarmine is added, and the preparation is covered with a coverslip and gently warmed over an alcohol burner. In fertile pollen grains, the granular cytoplasm and sperm cells are stained a deep carmine-red. Sterile pollen grains remain almost unstained or stain unevenly. Their contents often pull away from the wall and show various stages of degradation (Fig. 10).

Fig. 10. Pollen grains of alfalfa: 1 - fertile; 2 - sterile

For certain plants that have a thick exine and where sperm cells are difficult to observe using the acetocarmine method, the iodine method can be used. This method is based on the starch iodine reaction. Fertile pollen grains are completely filled with starch, whereas sterile ones lack it entirely or contain only traces (Fig. 11).

Fig. 11. Fertile alfalfa pollen grains

2.2 Microsporogenesis

Meiosis is a reductional division consisting of two successive divisions that result in the formation of four haploid cells from a single diploid cell (Fig. 12). In higher plants, this specialized type of division occurs prior to flowering within young anthers and ovules.

Fig. 12. Meiosis in viper's bugloss: 1 - diakinesis, 12 bivalents; 2 - telophase I; 3 - prophase II; 4 - nuclear tetrads

Meiosis proceeds through two divisions. The prophase of the first meiotic division is the most complex. DNA Synthesis, which begins during interphase, continues into prophase I.

Prophase includes five stages: leptotene, zygotene, pachytene, diplotene, and diakinesis. During the leptotene stage, thin, tangled chromosome threads appear in the nucleus. In the zygotene stage, pairing (synapsis) begins at the ends of the Chromosomes and progresses along their entire length. The paired chromosomes form a bivalent. It contains 4 chromatids, although they are not yet distinguishable under a microscope. During the pachytene stage, the chromatids of each chromosome become clearly visible. The number of bivalents is haploid. Pairing chromosomes can exchange segments of chromatids—a process known as Crossing-over. In diplotene, the four-chromatid STRUCTURE OF THE bivalents becomes distinctly evident, which is why a bivalent is also called a chromosomal tetrad. Homologues repel each other. At certain points, crossing sites resembling the Greek letter chi are visible as chiasmata. At the diakinesis stage, the number of chiasmata decreases as they shift toward the ends of the chromosomes. The bivalents move to the equatorial plane. The nuclear envelope and nucleoli disappear.

In metaphase I, bivalents align along the equatorial plane. Spindle fibers attach to the centromeres and extend from one pole to the other.

In anaphase I of meiosis, homologous chromosomes, each consisting of two chromatids, migrate toward opposite poles of the cell.

Telophase I is similar to mitotic telophase. As a result of the first meiotic division, two nuclei are formed, each containing a haploid set of chromosomes, though each chromosome still consists of two chromatids.

The second meiotic division proceeds like standard mitosis.

Prophase II is short because the chromosomes remain spiralized after telophase I.

In metaphase II, chromosomes arrange themselves along the equatorial plane of the cell. This process typically occurs synchronously in the cell dyad.

In anaphase II, chromatids (daughter chromosomes) separate and move toward the poles.

In telophase II of meiosis, following cytokinesis, cells with a haploid chromosome set are formed simultaneously (in a simultaneous type). Thus, as a result of two consecutive meiotic divisions, 4 haploid cells are produced from a single diploid cell.

2.3 Ovule

The ovule consists of the nucellus, integuments, chalaza, and funiculus (stalk).

The ovule has one or two integuments (coats). Some plants have a third one, known as an aril, which arises from the splitting of the outer integument. Another modification is the caruncle, an integument formed by the proliferation of the micropylar zone.

The micropyle may be formed by either the inner or the outer integument, or sometimes by both.

Depending on the degree of nucellus development, ovules are classified as crassinucellate or tenuinucellate. In crassinucellate ovules, the megaspore mother cells are separated from the epidermis by several layers of parietal cells. In tenuinucellate ovules, the mother cells lie directly adjacent to the epidermis. As the embryo sac matures, the nucellus undergoes lysis.

Typically, one of the nucellar cells increases in size, and its cytoplasm becomes dense. This is the primary archesporial cell, which may divide to form parietal and sporogenous cells, or directly differentiate into the megaspore mother cell. The megaspore mother cell undergoes meiosis to produce 4 haploid megaspores. The tetrad arrangement is predominantly linear, but T-shaped, decussate, or tetrahedral arrangements also occur. As a rule, the embryo sac develops from one of these four megaspores.

The types of embryo sacs are determined by 3 features:

1) the number of megaspores forming the embryo sac;

2) the number of mitoses;

3) the behavior (distribution) of the nuclei.

Embryo sac types are named after the taxa in which they occur, with 16 types currently recognized. The predominant type is the Polygonum type (Fig. 13).

Fig. 13. Embryo sacs of Caucasian goat's rue: 1 - binucleate; 2 - tetranucleate; 3 - mature embryo sac

If the embryo sac develops from a single megaspore, it is termed monosporic; if from two, bisporic; and if from four, tetrasporic.

Examples of different embryo sac types include:

Monosporic: Polygonum type (develops from the chalazal megaspore via three mitotic divisions) and Oenothera type (develops from the micropylar megaspore via two mitotic divisions).

Bisporic: Allium type (develops from a megaspore dyad, with the chalazal cell dividing 3 times).

Tetrasporic: Fritillaria type, Tulipa type, Peperonia type, etc.

Structure of the mature embryo sac.

The egg apparatus consists of the egg cell and two synergids. The Nucleus of the egg cell is larger, with a vacuole located in its basal region. A Cellulose cell wall is present only in the basal part. The synergids exhibit a reversed polarity: vacuoles are located apically, while the nucleus and filiform apparatus occupy the basal region. The filiform apparatus facilitates the penetration of the pollen tube.

The central cell is the largest and is heavily vacuolated, enclosing both the egg and antipodal complexes. Two haploid polar nuclei lie in the center of the cell. Following double fertilization, the central cell subsequently gives rise to the endosperm.

The number of antipodal cells depends on the embryo sac type, typically numbering three. Antipodals are ephemeral, though in some species their number can reach up to 300. They are entirely absent in the Oenothera type. Antipodals function as haustoria, and in certain plants, their nuclei are polyploid (Fig. 14).

Fig. 14. Embryo sac of chickweed before fertilization: e - egg cell; s - synergids; pn - polar nuclei; a - antipodals

2.4 Double Fertilization

The progamic phase of fertilization includes the following events: pollen grain adhesion to the stigma, pollen grain Hydration, activation of enzyme systems, pollen recognition, Swelling and germination of pollen grains, pollen tube growth, and the discharge of the pollen tube contents.

The exine and intine of a pollen grain are physiologically active.

By the time they are shed from the anthers, pollen grains are heavily dehydrated. Upon landing and adhering to the stigma, hydration begins as Water is taken up from the stigmatic Tissues. The pollen grain swells, and growth of the pollen tube is initiated. Stigmas are generally divided into two types: "wet," possessing a surface secretion, and "dry," which lack a secretion and feature papillae. The papillae are covered by a pellicle that facilitates interaction between the stigma and the pollen grain. The former type typically has a smooth surface and is characteristic of families such as Fabaceae, Solanaceae, and Liliaceae; the latter is found in Asteraceae, Brassicaceae, Poaceae, and others.

Styles within pistils are also classified into two types: open (hollow) and closed. In closed styles, the central region is occupied by transmitting tissue.

Pollen tube germination begins with the protrusion of the intine through the germination pore. The pollen tube wall consists of two layers: an inner callose layer and an outer pectocellulosic layer. The tip of the pollen tube houses the vegetative and generative nuclei. As growth proceeds, callose plugs form within the pollen tube, isolating its apical portion from the rest of the structure (Fig. 15).

Fig. 15. Growth of pollen tubes in the stigma and style of various alfalfa species: 1, 2 - Medicago scutellata; 3 - Medicago turbinata

Initially, the pollen tube grows using the nutrient reserves of the pollen grain itself, subsequently drawing nourishment from the substances of the stigma, style, and Ovary.

The number of growing pollen grains on the stigma significantly exceeds the number of ovules. However, their rates of growth vary. Some grains halt their growth and form club-shaped swellings. Only a single pollen tube ultimately enters the embryo sac.

In cases of self-incompatibility, pollen fails to anchor properly on the stigma, or the pollen tubes undergo abnormal thickening or branching.

The entry of the pollen tube into the embryo sac via the micropyle is the most typical pathway, known as porogamy (Fig. 16). Occasionally, the pollen tube enters through the chalaza or laterally (chalazogamy and mesogamy, respectively).

Fig. 16. Porogamy in cultivated alfalfa

Two sperm cells from the pollen tube enter the embryo sac. One sperm fuses with the egg Cell Nucleus to form a diploid zygote. The second sperm fuses with the polar nuclei, yielding a triploid endosperm nucleus (Fig. 17).

Fig. 17. Mitotic metaphase of the primary endosperm nucleus and zygote in cultivated alfalfa

2.5 Endosperm and Embryo

The endosperm serves as a nutritional source for the developing embryo. Based on the pattern of division of the primary endosperm nucleus, three main types are distinguished: nuclear, cellular, and helobial.

Nuclear endosperm. Nuclear division is not accompanied by Cell wall formation, resulting in a coenocytic phase. Outgrowths known as haustoria may sometimes form.

Cellular endosperm. Nuclear division is accompanied by the formation of cell walls, establishing a cellular phase. Haustoria may also form in this type.

Helobial (intermediate) endosperm. A transverse wall forms initially, dividing the endosperm into micropylar and chalazal chambers. Nuclear division in the micropylar chamber proceeds via the nuclear type, followed by subsequent cell wall formation, whereas the chalazal chamber remains without cell walls (Fig. 18).

Fig. 18. Endosperm development in Echium vulgare: 1, 2 - formation of the partition wall after the First Division of the primary endosperm nucleus, showing lateral and central chambers; 3 - formation of the endosperm haustorium; 4 - increase in nuclear number within the lateral chamber and the endosperm haustorium; 5 - formation of cellular endosperm in the central chamber: цк - central chamber; бк - lateral chamber; эг - endosperm haustorium

Regardless of the mode of development, a fully formed endosperm consists of relatively large cells that accumulate storage reserves in the form of starch, Lipids, aleurone, or hemicellulose.

In certain plants, as the embryo sac develops, the nucellus is not entirely consumed; instead, a portion persists and transforms into a nutritive tissue known as the perisperm, which consists of diploid cells.

Before undergoing division, the zygote passes through a maturation or resting phase. The first division of the zygote produces two cells: an apical cell and a basal cell. The basal cell gives rise to the suspensor, while the apical cell forms a radially symmetrical globular body.

Depending on the number of divisions and the orientation of cell walls, several types of Embryogenesis are distinguished. Let us examine some of them.

1. Paeoniad type. The zygote initially develops as a coenocyte through free nuclear division without the formation of cell walls.

2. Piperad type. The zygote divides by a longitudinal wall.

. The zygote divides by a transverse wall.

a) the apical cell divides longitudinally:

- the basal cell does not contribute to the formation of the embryo — Onagrad type.

- both the apical and basal cells contribute to the formation of the embryo — Asterad type.

b) the apical cell divides transversely.

- the basal cell does not participate in embryo development, differentiating into a suspensor — Caryophyllad type (Fig. 19).

- the basal cell divides to form a suspensor — Solanad type.

- the basal cell participates in the formation of the embryo — Chenopodiad type.

Fig. 19. Embryo development in alfalfa (Caryophyllad type, Medicago variation)

During the Embryonic Stage of development, SHOOT apical Meristems emerge in the apical zone of the globular embryo body, while ROOT and coleorhiza meristems appear in the basal zone. Between them, the tunica and corpus are established.

Later, in dicotyledonous plants, two distinct protuberances—the cotyledons—are formed. In monocotyledonous plants, only a single center of cell division develops, while the second one remains blocked.