CHAPTER 2 MALE AND FEMALE GAMETOPHYTES, FERTILIZATION,

ENDOSPERM DEVELOPMENT AND EMBRYOLOGY

2.1 Preamble
An embryo was originally defined as 'the offspring of an animal before its birth (or emergence
from the egg),' or, as applied to plants, 'the rudimentary plant contained in the seed' (Oxford
Dictionary, 1728). The terms embryo and germ have come to connote the initial developmental
phase in all classes of plants yet germs may develop from spores as well as from fertilized eggs.
The gametophyte is a stage in the life cycle that is found in all plants and certain species of algae.
The fertilization process includes both multicellular diploid generations known as Sporophyte and
a multicellular haploid generation known as Gametophyte. The primary job of gametophyte is the
production of Gametes. The produced gametes are the haploid reproductive cells, such as sperm
and eggs. The word diploid refers to two sets of chromosomes in the cells, and normally written
as ‘2n’whereas haploid to only one set of chromosomes in the cells and written as ‘n’.

Initial embryological studies were essentially morphological in inception and outlook, and indeed
this was necessarily so. The aim was to provide a demonstration or record of development from
the first division of the zygote up to the point where the embryo could maintain itself as a free-
living individual. A great deal of careful and painstaking effort has been necessary to obtain this
information; and it may be said, with due emphasis, that these anatomical studies not only show
us what the embryos of particular species look like: they constitute the basis on which all further
work must rest. Nowadays, however, we recognize that embryology need not, and should not, be
pursued only by the methods of the anatomist: we want to know about the factors which determine
the observed developments. Many new possibilities are opening up. A new outlook and new
investigations have been made possible by recent discoveries in genetics, biochemistry and
physiology and by new manipulative techniques, e.g., surgical treatments, and the methods of
tissue culture. In short, it is the process of embryogenesis, culminating in the organization of the
young plant, that is of paramount interest and importance to the contemporary Student of
embryology; and to this end experimental work should be introduced wherever possible.
2.2 Male and Female Gametophytes
2.2.1 Flower Structure

A typical flower has four main parts, or whorls: the calyx, corolla, androecium, and gynoecium.
The outermost whorl of the flower has green, leafy structures known as sepals, which are
collectively called the calyx, and help to protect the unopened bud. The second whorl is comprised
of petals, usually brightly colored, collectively called the corolla. The number of sepals and petals
varies depending on whether the plant is a monocot or dicot. Together, the calyx and corolla are
known as the perianth. The third whorl contains the male reproductive structures and is known as
the androecium. The androecium has stamens with anthers that contain the microsporangia. The
innermost group of structures in the flower is the gynoecium, or the female reproductive
component(s). The carpel is the individual unit of the gynoecium and has a stigma, style, and
ovary. A flower may have one or multiple carpels.

If both male and female flowers are borne on the same plant (e.g., corn or peas), the species is
called monoecious (meaning “one home”). Species with male and female flowers borne on
separate plants (e.g., C. papaya or Cannabis) are termed dioecious, or “two homes.” The ovary,
which may contain one or multiple ovules, may be placed above other flower parts (referred to as
superior); or it may be placed below the other flower parts (referred to as inferior).

2.2 Alternation of Generations

The lifecycle of angiosperms follows the alternation of generations. In the angiosperm, the haploid
gametophyte alternates with the diploid sporophyte during their sexual reproduction process.
2.3 Male Gametophyte
The male gametophyte develops and reaches maturity in an immature anther. In a plant’s male
reproductive organs, development of pollen takes place in a structure known as the
microsporangium. The microsporangia, usually bi-lobed, are pollen sacs in which the microspores
develop into pollen grains.
The first gametophytic structure is the modification of microspore mother cell into the pollen grain.
The pollen grain grows into the male gametophyte on germination which is initiated before
pollination takes place.
Inside the microsporangium, Pollen mother cell (PMC) undergoes meiosis and results in four
microspores which eventually mature into pollen grains. The inner layer called tapetum nourishes
the developing microspores.
The pollen grains consist of two cells- a vegetative cell and a generative cell. Once the
microsporangium is matured it bursts and releases the pollen from the anther.
The pollen grain consists of two layers, the outer thick layer is called the exine and the inner thin
layer is called intine which protects the pollen from damage.
Male gametophyte development can be classified into two stages: Pre-pollination development
and post-pollination development.
2.3.1 Development of Male Gametophyte
Pollen grains display germination that is initiated in pollen sac which is referred to as precocious
germination wherein a large central vacuole formation causes the nucleus to be pushed to one side
post which the nucleus goes through mitosis that gives rise to two daughter nuclei.
The small cell that is formed is known as the generative cell and the larger cell is known as the
vegetative cell which has cytoplasm in sufficient amounts acting as a food reserve for the male
gametophyte to develop, while the generative cell assembles at the mid-section of the pollen grain.
At this phase, the pollen grains fall on the stigma wherein further development takes place.

Maturing pollen and growth of a pollen tube

The falling of the pollen grains on the stigma causes the nutrient absorption through the germ pore
from the stigma which causes the vegetative cell to enlarge. The enlargement causes the intine to
move out via the germ pore for the formation of the pollen tube.
The nuclei of the vegetative and generative cell travel to the pollen tube. The generative cell at this
stage divides to produce two haploids, the non-motile and unicellular male gametes. For the deeper
insertion of the tube, the size of the developed male gametophyte is diminished and derives the
nutrition from the style’s tissues.

2.4 Female Gametophyte (Embryo Sac)

The overall development of the female gametophyte has two distinct phases. First, in the process
of megasporogenesis, a single cell in the diploid megasporangium undergoes meiosis to produce
four megaspores, only one of which survives. During the second phase, megagametogenesis, the
surviving haploid megaspore undergoes mitosis to produce an eight-nucleate, seven-cell female
gametophyte, also known as the megagametophyte, or embryo sac.
The polar nuclei move to the equator and fuse, forming a single, diploid central cell. This central
cell later fuses with a sperm to form the triploid endosperm following double fertilization. Three
nuclei position themselves on the end of the embryo sac opposite the micropyle and develop into
the antipodal cells, which later degenerate. The nucleus closest to the micropyle becomes the
female gamete, or egg cell, and the two adjacent nuclei develop into synergid cells. The synergids
help guide the pollen tube for successful fertilization, after which they disintegrate. Once
fertilization is complete, the resulting diploid zygote develops into the embryo; the fertilized ovule
forms the other tissues of the seed.

A double-layered integument protects the megasporangium and, later, the embryo sac. The
integument will develop into the seed coat after fertilization, protecting the entire seed. The ovule
wall will become part of the fruit. The integuments, while protecting the megasporangium, do not
enclose it completely, but leave an opening called the micropyle. The micropyle allows the pollen
tube to enter the female gametophyte for fertilization.

Structure of a typical ovule prior to fertilization and fertilization in an angiosperm illustrated.

2.5 Pollen Tube Entry

In the vast majority of angiosperms, the pollen tube enters the ovule porogamously, that is, via the
micropyle. In five dicot families, pollen tube penetration is chalazogamous; it enters the chalazal
end of the ovule and follows a course along the lateral surface of the megagametophyte until it
reaches the micropylar end where it enters the gametophyte itself in the usual fashion. One dicot
family is mesogamous, with the pollen tube initially penetrating the ovule at some side point before
making its way to the micropylar end of the gametophyte. No monocotyledonous genera have been
reported to show either chalazogamy or mesogamy.

2.6 Fertilization

Fertilization in angiosperms, or flowering plants, involves the fusion of male and female gametes,
resulting in the formation of a zygote, which develops into an embryo within the seed. It is a
complex process that ensures the production of viable seeds and the continuation of the plant
species. The process is highly regulated and relies on the successful interaction between the male
and female reproductive structures of the flower, as well as external agents such as pollinators,
which play a crucial role in the transfer of pollen between flowers.
The process of fertilization in angiosperms can be broken down into several key stages:

Pollen Transfer: The male reproductive organs of the flower, known as stamens, produce pollen
grains containing the male gametes. Pollen is transferred from the anthers of the stamen to the
stigma of the female reproductive organ, known as the pistil. This transfer can occur through
various mechanisms, including wind, water, or animal pollinators such as bees, butterflies, or birds.

Pollen Germination: The pollen grains germinate on the where there is usually a liquid secretion,
often containing sugar, but sometimes other substances are also present e.g., malic acid.
Germination results the growing out of the thin inner wall of the pollen grain through one of the
pores in the thick exine. This results in the production of a cylindrical pollen tube into which the
tube nucleus and generative nucleus pass. The pollen tube, tending to grow towards chemical
substances produced by the stigma, towards the moisture and away from oxygen, then enters the
stigma and begins to grow down the style to the ovary, where the ovules are located. The tip of the
tube enters the micropyle of the ovule, possibly directed by a fluid secreted by the synergids. It
thus reaches the nucellus and by penetrating the few overlying layers of this tissue, it comes into
contact with the embryo sac in the vicinity of the egg apparatus. During the downward growth of
the pollen tube the generative cell divides to form two male gametes. These occupy the tip of the
tube, the tube nucleus having disintegrated, fertilization now takes place.

Double Fertilization: Angiosperms are unique in that they undergo double fertilization. The tip of
the pollen tube becomes ruptured and the two male gametes, now spirally coiled, thread-like
bodies, enter the embryo sac. One of the gametes enters the egg and fuses with the egg cell,
resulting in the formation of a diploid zygote and it is from this that the embryo plant develops.
This process is similar to fertilization in other plants. Simultaneously, a second sperm cell fuses
with two polar nuclei in the ovule, resulting in the formation of a triploid cell that develops into
the endosperm, a nutrient-rich tissue that nourishes the developing embryo.

Seed and Fruit Development: Following fertilization, the zygote develops into an embryo, while
the ovule develops into a seed. The ovary, which surrounds the ovules, begins to develop into a
fruit, protecting the developing seeds and aiding in their dispersal.
2.7 Endosperm

Nuclear endosperm development results when division of the primary endosperm nucleus- the
product of fertilization of the polar nuclei- and of subsequent nuclei is not followed by wall
formation leading to a coenocytic condition. The number of free nuclei formed varies from
relatively few to well in the hundreds before wall formation starts, but in most species, though not
all, the endosperm does become cellular—usually completely but sometimes only in part. In direct
contrast to the nuclear is the cellular type of endosperm development in which nuclear divisions
are accompanied by wall formation (most commonly throughout development, but at least during
the early stages) and the endosperm is cellular from the beginning, not just in later stages.

2.8 Embryogenesis

In plants, this is the process by which a multicellular embryo develops from a single fertilized egg
cell, ultimately giving rise to a new plant. This process is critical for the reproduction and growth
of higher plants. Plant embryogenesis is a complex and highly regulated process that ensures the
successful development of new generations of plants. In studying embryogenesis in any plant, our
initial concern is with the growth of a single cell, a zygote or a spore, in its particular environment.
In some, perhaps in all, developing sporophytes we probably ought to begin with the unfertilized
ovum; for some of the orderly changes that take place during the ontogenetic development may
already have been determined by the cytoplasmic organization of the ovum. If we examine the
ovum, whether in an embryo sac or archegonium, there is evidence that it has undergone some
specialized development, e.g., in the archegonium it becomes rounded off and lies free in the
venter, and it is a reasonable assumption that it may be affected differentially by various
biochemical and physical factors in its immediate environment

Here's an overview of the key stages and events in plant embryogenesis:

1. Double Fertilization: In flowering plants (angiosperms), embryogenesis begins with double

fertilization where the two sperm cells from the pollen grain fertilize two different female
gametes within the ovule as outlined above.
2. Zygote Formation: The zygote is the initial cell of the new plant embryo. It is typically a
diploid cell (2n), resulting from the fusion of a haploid egg cell (n) and a haploid sperm
cell (n).
3. Cell Division: The zygote undergoes multiple rounds of cell division through mitosis to
form a multicellular structure called the proembryo. The proembryo consists of a few to
several cells and starts to show polarity with an apical (top) and basal (bottom) region.
4. Apical-Basal Polarity: The proembryo establishes apical-basal polarity, with the apical part
becoming the shoot apex, and the basal part developing into the root apex. In thallophytes,
bryophytes, pteridophytes and seed plants, the earliest perceptible embryogenic
development is the establishment of polarity.

A characteristic, filamentous, axial or spindle-like development is found in the embryogeny of

all classes of plants. The formation of this 'primitive spindle' appears to be a fundamental
feature in the embryogeny of plants and affords evidence of the early establishment of polarity
in the zygote. The action of growth-regulating substances, or their precursors, is closely bound
up with their polarised movement in the plant. Auxins, for example, typically more basipetally,
i.e. from the shoot apex downwards to the root and only to a slight extent in the opposite
direction.

It has been demonstrated that auxins can move with great rapidity from one part of the plant to
other parts. This movement is not, however, equally rapid in all directions, i.e. from the point
of origin, or, in experiments, from the point of application. The move- ment of auxins, which
is considerably more rapid than diffusion, may take place through parenchyma, phloem, or,
more generally, through vascular tissue. It is a reasonable hypothesis that the polarity of the
shoot, or of the organ involved, in some way affects the movement of growth-regulating
substances.

5. Germ Layers: In many plant species, the proembryo develops into a globular or heart-
shaped structure with two main germ layers: the outermost layer, called the protoderm,
which gives rise to the epidermis, and the innermost layer, called the ground meristem,
which develops into the ground tissues.
6. Cotyledon Formation: Depending on the type of plant, one or two cotyledons (seed leaves)
form during embryogenesis. Cotyledons are essential for nutrient storage and may play a
role in photosynthesis in some plants.

7. Suspensor: In some plant species, there is a structure called the suspensor, which provides
support and nutrients to the growing embryo. The suspensor eventually degenerates as the
embryo matures.
 8. Maturation: As the embryo continues to grow and develop, it undergoes further
differentiation and organogenesis. This includes the formation of the shoot and root apical
meristems, which give rise to the future shoot and root systems of the mature plant.

9. Seed Formation: Once the embryo has developed into a mature plantlet, it is surrounded
by protective layers, including the seed coat, which forms from the outer ovule
integuments. The endosperm, which was formed during double fertilization, provides
nutrients to the developing embryo and, in some cases, remains as a storage tissue in the
seed.
10. Seed Maturation: The embryo and its surrounding structures complete their development,
and the seed enters a state of dormancy, which allows it to withstand adverse environmental
conditions until it germinates.

When environmental conditions are favorable, the mature seed undergoes germination, and the
embryonic plant resumes its growth to establish a new plant.