The Sea in the Seed
Sperm of Ginkgo biloba and Reproductive Evolution in Plants

A TokyoCinema Video, color 35min. completed Japanese version January 2000 and English version November 2000, All Rights Reserved
copyright: 2000, TokyoCinema Inc. all rights reserved
41th Science and Technology Film and Video Featival Tokyo, 2000

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The Sea in the Seed

A little more than a century ago an important botanical discovery was reported from Japan.
In 1896 Hirase Sakugoro discovered flagellated sperm in a seed of Ginkgo biloba, then
Ikeno Seiichiro found the same phenomenon in Cycas revoluta. Botanists around the world
were excited by these discoveries.

In the higher plants, like most gymnosperms and angiosperms, male gametes or non-flagellated
sperm cells are sent direct to egg cells through pollen tubes. In lower plants, flagellate sperm
cells to swim towards egg cells in water.

The discoveries made by Hirase and Ikeno indicated that the Ginkgo and cycads are intermediate
forms between higher and lower plants on the evolutionary line. Among the seed plants it is only
the Ginkgo and cycads which have been found to have flagellated sperm cells in their life-cycles.

In the last few years, at the end of the 20th Century, has the detailed process of fertilization of
these higher plants has been recorded successfully in the living state.

It is thought that all life originated in the sea and that, later, certain organisms adapted to life
on land. Land plants are all therefore considered to have evolved from a species of green
fresh-water algae.

The green alga Mesostigma, a flagellate, is thought to be the closest relative to the original
ancestors of land plants.

The green body seen here is a chloroplast where photosynthesis takes place.

The red spot at the centre is the stigma which is photosensitive.

Two flagella are attached to a dimple in the middle. The organism has no cell wall and the outer
body is covered with scales, which is a common feature in primitive green flagellates.

Cell division of Mesostigma is shown here using time-lapse videomicroscopy.
The central part of the organism furrows before dividing into two. This primitive process of
cytokinesis is found in many organisms, including algae and all animal cells.

This multicellular green alga, Coleochaete, lives in fresh water when in the non free-living
state. Cell division enlarges its body size but does not increase its population. For this it produces
zoospores for non-sexual reproduction and eggs and sperm for sexual reproduction.

This transparent sphere is an antheridium containing sperm cells. The large green part at
the tip is the oogonium which contains egg cells.


The fertilized egg in the oogonium is protected by a covering of cells.
This protection is regarded as the first evolutionary step which led ultimately to life on land.

This is a charophyte which is thought to be the closest relative of the first plants that made
the transition from the sea to the land. It has a slender pineapple-shaped oogonium and
a spherical antheridium. Only one egg is held in the oogonium but the antheridium carries many

Here antheridial filaments emerge from the antheridium.

These filaments are divided by cell walls into many compartments, each of which contains
a sperm.

When a sperm matures, it swims out into the water.

Sperm can be seen here swimming in slow motion. In water a sperm can easily swim up to
an egg.

If water is essential to the process of reproduction, this raises the question of how the
first plants made the transition from water to land.

It has long been thought that the first plants to make the transition from freshwater to land
were the mosses and liverworts. This is the liverwort Marchantia polymorpha. The part of
the life-cycle which carries out sexual reproduction is called the gametophyte. The organism
needs to live on a wet surface.

The structures which look like folded umbrellas are female gametophytes or female receptacles.

Many archegonia are found under the umbrellas.

Fertilization takes place when a female receptacle is short and the archegonium is covered
with water on the ground.

This is a male gametophyte. The plate-like male receptacle has many antheridia which produce

When one of these receptacles is covered with water, sperms are released
swim in the film of water from the antheridium to the archegonium to fertilise the egg.

Here there is a white mass on the surface of the male receptacle.

It is a clump of sperm.

Before long they will start to swim.

Bryophyte sperm have two flagella. They swim forward using the flagella at the anterior end.
This is one of the characteristics of plant sperms.

After fertilization in the female receptacle, the egg develops into a sporophyte to produce
spores. Meanwhile the stalk of the female receptacle elevates and opens the umbrella.

The yellow mass seen here is a lump of spores. The spores are released into the air.

The spores are attached to a slender thread-like cell. When the thread shakes, the spores
spring off.

As spores fly off and are carried far afield, the range of mosses and liverworts or bryophytes
spreads and the population enlarges.

Landing on wet ground, a spore immediately germinates.

Dark spots on the back of fern leaves are masses of sporangia which contain many spores.
Ferns as well as bryophtes use spores to increase their population.

Ferns have a specialised organ called a vascular bundle through which water and nutrients
absorbed from roots are transported to every part of the organism. The presence of this
organ is the reason ferns are considered to be higher on the evolutionary line than bryophytes.

The vascular system in ferns allows these plants to grow as high as trees. The height of the
foliage gives a greater chance for the spores from these organisms to be carried further to
extend the range of their habitats.

Lygodium provides another example of fern reproduction. This is asporophyte of Lygodium.

Here many spores are packed in the sporangia which are attached to the tips of the leaves.
When a spore emerges from the sporangium and successfully lands on wet ground, it
germinates into a prothallium -a tiny gametophyte where sexual reproduction takes place.
The gametophyte is extremely small.

Many archegonia and antheridia are formed on the surface of the gametophyte.

These are archegonia.

When an egg matures, the entrance of the archegonium opens to receive the sperms.

These are antheridia producing large numbers of sperm cells.

When the antheridium touches water, the lid opens and the sperms burstb out.

The sperms can swim up to an archegonium, but only if there is a thin layer of water
covering the gametophytes.

In mosses or brophtes and ferns otherwise knows as pteridophytes water essential for
sexual reprodeuction.

However, the supply of water on land is irregular - so how have the multitude of land
plants adapted to this environment?

They have developed seeds!

The Cycads and Ginkgo are now considered to be the most primitive species among
existing seed plants. They have partially followed the same method of reproduction
which the ancestors of green plants did in the water but their solution has been to
produce a similar aqueous environment in a seed.

Ginkgo plants first appeared some two hundred and eighty million years ago and
culminated in the Mesozoic era with the dinosaurs. It is thought that dinosaurs fed
on ginkgo nuts like humans now. Ginkgo biloba is therefore referred to as "a living
fossil", because the form of its leaves and organs of reproduction are not much
different from those of the pre-historic Ginkgos.

The reproduction processes of Ginkgo biloba retain the characteristics of lower land

Ginkgo biloba is a dioeceous plant - meaning that the male and female forms of the
tree exist separately.

Close observation of growth confirms that the Ginkgo's male flowers open several
days earlier than the female flowers.

This time-lapse sequence shows the Ginkgo beginning to bud.

Here a structure which looks something like a bunch of bananas develops in the axil
of the leaves.

These are clusters of anthers which are generally called male flowers .

This is a Ginkgo male tree two weeks after budding.

The male flowers are growing.

When the pollen is mature, anthers rupture and pollen emerges.

These small grains are Ginkgo pollen which is carried to female trees by wind currents.

This is an electron micrograph of Ginkgo pollen. The pollen grain is four-celled. One of
the cells produces sperms. A pollen grain is therefore a "flying gametophyte", though
its appearance resembles a spore.

This is a female tree at the time when pollen is released into the air by male plants.

These small spherical structures seen among leaves are often called female flowers,
but they are actually ovules. In early spring the stalkes bearing ovules extend upwards
the sky.

On the apex of a young ovule there is a pore through which pollen enters.

When it is ready for pollination a mucilaginous droplet is exuded from the inside of
the ovule to catch the pollen.

Exudation and retraction of the droplet happens repeatedly.

Retraction of the pollination droplet brings pollen grains into the pollen chamber.

These are Ginkgo pollen grains.

Pollen flows into the droplet.

Fertilization does not occur immediately after pollination because the egg is not
produced at this time. After pollen enters the pollen chamber, differentiation of
the female gametophyte starts in the ovule.

One month after pollination the young ovules, or female flowers, are shed from
the tree when they can catch no pollen.

The inner structure of a pollinated ovule is shown here.

This is a thin section through an ovule. It shows one of the pollen grains inside.
A female gametophyte is in the process of developing.

Two and a half months after pollination the stalks turn downwards and the ovules
are hanging off.

The longitudinal median section of the ovule, the large green portion at the centre,
shows two young archegonia in which eggs will be produced.

Usually only one of the two fertilised eggs germinates the following year the other
egg degenerates and is eventually absorbed.

Four months after pollination, pollinated ovules have grown large on the female tree.

Inside the archegonia egg cells are maturing.

In the meantime a peculiar column of female gametophyte tissue - known as the
"tent pole" - elevates between two archegonia and extends towards the pollen
chamber to destroy the tissue between the pollen chamber and the archegonia.

The apex of the expanded pollen tube is shown here. Both the pollen chamber and
the space around the tent pole have been united to become a bigger archegonial
chamber. When the two sperm are produced in the pollen tube, this large chamber
is filled with fluid. This fluid could be regarded as "the sea" produced in the seed.

A central cell, which will later differentiate into two sperms, can be seen inside
the stretched pollen tubes. At this stage the archegonial chamber has not yet
filled with liquid."The sea" appears in the archegonial chamber around the time
the sperms are released.

This sequence shows how sperm cells are differentiated from the central cell,
shown here.

The long portion at the centre is a nucleus.

The large globules in the upper and lower parts are lipid bodies.

On the outer part there are small globules called blepharoplasts which are clusters
of sperm cilial bases.

Two doughnut-like nucleoli are found in the nucleus.

At the apex of the pollen tube are the first and second prothallial cells and a stalk cell.

When the central cell becomes elliptical and wide than the stalk cell, cell division
begins to produce sperm.

Chromosomes appear inside the nucleus.

Here nuclear division has finished and the mother nucleus has divided into two.
Cytoplasmic granules begin to accumulate along the furrow and this l eads to cytoplasmic
cleavage, cytokinesis.

Division of the central cell can now be seen.

Look carefully at the line which just appeared. An inwardly invaginated furrow developed
and the cell divided into two. Ginkgo biloba uses the same primitive method of cytoplasmic
division for reproductive cell division which is found in Mesostigma.

Two divided cells can be clearly seen here in longitudinal section. The lines in the middle
are the cell membranes of each cell.

As cytokinesis is completed, a great number of cilia develop on the spiral band from the

In this repeat of the process, watch the cell of left . It shows clearly how the cilial spiral

As if twisted off, the central cell has just divided into two sperm cells.

Now two sperms are produced.

In the spring. pollen grains - or male gametophytes - are carried by wind currents to ovules
- or female gametophytes. The pollen grains are caught and pulled into the pollen chamber
by retraction of pollination droplets.

During the five months following pollination, the pollen grows big enough to form two sperms,
absorbing water and fertilizer from the female tissue - a nucellus. The eggs mature at the
same time.

As the sperms get ready to swim, the female gametophyte prepares "the mother sea" in the
archegonial chamber.

The precise timing of this process is one of the miracles of nature.

The pollen tube ruptures and the sperms swim in "the sea" toward the eggs. As we have
already seen, Ginkgo sperms swim forward with cilia at the front just like the charophytes,
bryophytes and pteridophytes.

The cilia are arranged in a spiral.

Here, in an electron micrograph, can be seen the fine structure of the cilial bases.

The basal bodies of the cilia are aligned in parallel and are attached to a complex multilayered

This multilayered structure of the cilial apparatus is characteristic of sperm and swimming
cells of organisms in the evolutionary line to land plants.

Those algae which have no link with land plants, such as Chlamydomonas, do not have a
multilayered structure.

During fertilization, Ginkgo biloba excretes reproductive liquid from its own tissue to create
"the sea" in the seed. Compared to mosses and ferns, Ginkgo sperm can swim safely in
"this inner sea" and can reach the egg cells more successfully.

The egg cells attract nearby sperms to the entrance of the archegonium. Fertilization occurs
about four months after pollination, between the end of August and the beginning of September.

This is the ovule of Cycas revoluta at the time of fertilization. Fertilization of the cycad resembles
that of Ginkgo biloba. In October sperms are produced in the pollen tube.

These are mature cycad sperms.

The sperms have just been released from the pollen tube.

The sperm of cycads may be the largest in the plant world. They have tens of thousands of cilia.

They also swim in "the sea" toward the egg cells.

Here a sperm enters the archegonium. The dark part at the centre is the entrance of the
archegonium which holds the egg cells. The sperm swims around the entrance, sensing the attracting
chemicals which are probably released by the egg.

The sperm now enters the archegonium. It takes about twenty seconds for a healthy sperm to pass
through and it has to elongate its shape to get through the extremely narrow entrance. After entering
the archegonium, the sperm recovers its shape.

It has just entered. Fertilization and fusion of the sperm and the egg cell then follow.

Conifers are gymnosperms just like Ginkgo and the cycads, but they do not produce ciliated sperms.

They extend pollen tubes in the ovule and send their non-ciliate sperm cells directly to the egg cells
like most other advanced angiosperms.

Here are some examples of male pine flowers. They release pollen which is carried by wind currents to
female flowers.

This is a female pine cone.

The pines need about three years from the development of reproductive organs to the maturation of

Angiosperms are more advanced than conifers.

The pollen of angiosperms is carried not only by wind currents but also by birds and insects.

This is Torenia fournieri which shows the double fertilization characteristic of angiosperms.

At the centre is a pistil and the upper and lower parts are stamens.

The moment pollen grains attach to the tip of the stigma, it encloses them.

Here pollen adheres to the stigma. Pollen tubes extend, absorbing fluid produced from the pistil.

The pollen tubes grow downwards in the style towards eggs in the embryosac.

Here is a section of a style showing many extending pollen tubes.

Each is a long slender cell containing cytoplasm which is actively streaming. The fluid
environment inside the pollen tube and the style is kept stable. It resembles a pipe full of

Here, under a fluorescence microscope, two nuclei can be seen inside the pollen tube.
They are pollen tubal and generative nuclei. At the bottom of the picture they are moving
towards the anterior end of the pollen tube. A generative cell will divide into two sperm cells.
Now the cell division starts. Immediately after nuclear division, cytokinesis produces two
sperm cells. The sperm cells of angiosperms have no flagella or cilia because they do not
swim, though they play the same role as sperms.

The protoplasmic streaming and the movement of the nuclei in the pollen tube seems not
to be random but more of a planned migration targetting the eggs.

Torenia's embryo sac,is seen here protruding from the micropyle. This allows us to look at
the interior and to analyse the fertilization process in detail.In here a part of the embryo sac,
an egg,two synergids and half of the central cell are outside the micropyle.

Synergids attract the approaching pollen tube to the embryo sac with chemicals. Sperm
cells move towards the apex of the pollen tube.

In Torenia a pollen tube will reach an embryo sac approximately nine hours after pollination.
In Cycas revoluta and Ginkgo biloba the time between pollination and fertilization is three
to four months, and in pines - about three years. Compared to gymnosperms, angiosperms
take a shorter time for fertilization. This results in a higher success rate in fertilization.

Here the apex of a pollen tube is firmly inserted in the centre of an embryosac. The pollen
tube bursts open and protoplasm rushes in. Carried along in this flow, two sperm cells also
enter the sac. One of the sperm cells fuses with the egg and the other with the central cell.
This type of fertilization is called "double fertilization"-characteristic of angiosperms. Double
fertilization was first reported almost simultaneously with the discovery of Ginkgo sperm.
A hundred years later the entire process of the double fertilization was video recorded for
the first time. Mosses and ferns discharge sperms into water externally and fertilization
takes place there. Cycads and Ginkgo produce a "sea" inside the seeds where the sperm swim
and fertilize. Angiosperms, on the other hand, send sperm cells direct to eggs through
extended pollen tubes. The process of reproduction is impressive to watch in any organism
because it is the key to life itself.

Plant reproduction processes are directly related to plant evolution. The green organisms
on earth are proof of this and the history of the evolution is carried in this seeds.

Reproduction process in this plant provides a unique key to our understanding of the evolution
of higher plants.

The budding of the further evolution begins with the budding of this tree.

The end.

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