Structure of Plant Cells with Special References to Lower ...

384 Cytologia 21

Structure of Plant Cells with Special References to Lower Plants I. Mitosis in Spirogyra setiformis

Katsumi UedaBotanical Institute, Faculty of Science, Kyoto University, Kyoto, Japan.

Received June 6, 1956

In the morphological study of plant cells, there are two main fields which

have not yet been cleared; that is, the submicroscopic morphology of the cells

and the cytomorphology of the lower organisms.The darkness in the former field is due to a limit of the resolving power

of optical microscopes. Nowadays, however, it may be cleared with an aid

of the electron microscope. In fact, many investigators are throwing light upon this field (Leyon, 1954; Steinmann and Sjostrand, 1955, and others).

Obscureness in the field of the cytomorphology of the lower plants on the other hand, is mainly due to a complicated behaviour of the cell elements

such as nuclei and achromatic figures. This complicated behaviour of the

cell elements has prevented the investigators from obtaining accurate knowledges of the nature and structure of these elements. Among many investi

gators, Belar (1926) contributed considerably in this field, but some of his descriptions are not satisfactory from the view point of the recent cytology.

It is the aim of the present investigation to study the microscopic and submicroscopic structures of nuclei, chloroplasts, chondriosomes and other

elements in plant cells, especially those in the lower organisms.Among many lower organisms, Spirogyra is a frequently studied organism

by many cytologists, but there seems to be no convergent opinion. This divergency in opinions is probably due to facts that the behaviour of the

nucleolus during the nuclear division is peculiar, and that the behaviour of

the nucleolus seems to differ among different species.It is intended in the present paper to report the result of an observation

on the mitosis of Spirogyra setiformis with special reference to the behaviour of the nucleolus in prophase and telophase.

Material and methods

Spirogyra setiformis* used in the present investigation was collected

from the pond of the botanical garden of Kyoto University. After the ma

terial was fixed with Flemming's stronger solution, it was cut in about 10ƒÊ,

then was stained with Heidenhain's iron alum haematoxylin. Alcohol-acetic

acid mixture was also employed for the fixative followed by staining with

methylgreen-pyronin, safranin-light green, Schiff's reagent, or acetocarmine.

* cf . Pascher, A.: Susswasserflora, Zygnemales. 29. (1913).

1956 Mitosis in Spirogyra setiformis 385

Lens system. Obj. Zeiss apo. 2mm ., Oc. Zeiss K. •~15 (•~1500) Figs.

1-8. Fixed with Flemming's stronger solution and stained with Heidenhain's

iron alum haematoxylin. Figs. 9-12 . Fixed with alcohol-acetic acid mixture

and stained with acetocarmine.

Observations

Resting nucleus. In the living cell, the nucleus is found in the cytoplas

mic mass which is hung at the central part of the cell by strings of the

cytoplasm. The nucleus is a shape of disc, and has a spherical nucleolus in

which any structure is hardly visible with an optical microscope. With a

phase contrast microscope, on the other hand, sinuous thick threads are dis

tinctly observed. These threads appeared to move very slowly though it was

very difficult to confirm the movement.

In fixed and stained preparations, the nucleus is round in its polar view,

and is rectangular in its side view, short line being l3ƒÊ and long line being

20ƒÊ (Fig. 1). The nucleolus, 9ƒÊ in diameter, is found within the nucleus.

In acetocarmine preparations, thick threads are found without difficulty within

the nucleolus, while they are not distinct in deeply stained preparations

using haematoxylin.

In the nuclear area outside of the nucleolus, which is called "AuƒÀenkern",

there are many fine sinuous chromonemata. One to three spherical bodies,

most frequently two, 1.5ƒÊ in diameter, are found near the nucleolus. These

granules were called by Czurda (1922) "Nebenkorper". In the living cell

these granules are hardly distinguished.

Prophase. As a result of comparing various figures of the dividing

nucleus in a fixed material, the figure which is slightly different from a resting

one, was assumed to be the figure in the earliest prophase.

In this stage the nucleus is nearly spherical in shape, and the nucleolus

appears to be slightly swollen. Deeply stained threads in the nucleolus are

clearly distinguished from the matrical substance of the nucleolus (Fig. 2).

Besides these threads there are fine threads which are not visible in the

resting stage. These fine threads are found at the peripheral parts of the

nucleolus, and carry several granules within them. The fine threads in

"AuƒÀenkern", which have also several granules within them, are so similar

in appearance to the fine threads in the nucleolus that it is impossible to

identify whether the threads in the most peripheral part of the nucleolus

belong to the nucleolus or the "AuƒÀenkern".

As the prophase proceeds, thick threads in the nucleolus are gradually

unravelled to fine threads, and finally the nucleolus in the resting stage ap

pears to be transformed into a mass of fine threads (Figs. 3 and 14). It

is confirmed by observation of the whole division cycle that the fine threads

both in the nucleolus and in the "AuƒÀenkern" are chromonemata and that

the granules within them are small chromocenters. They are called, therefore,

386 K. Ueda Cytologia 21

"chromonemata" and "chromocenters" respectively in subsequent descriptions . Matrical substance of the nucleolus is hardly visible in this stage.

The chromonemata in the peripheral part of the mass of chromonemata

carry numerous chromocenters (Fig. 14). Then, the chromonemata in the

Figs. 1-6. 1. Resting nucleus. Arrow "A" indicates the "nucleolus"; and arrow "B",

the "Nebenkorper". 2. Early prophase . Nucleus takes a spherical shape. The "nucle

olus" begins to disintegrate. 3. "Nucleolus" is transformed into a mass of chromo-

nemata. Spindle fibers appear. 4. Mass of the chromonemata shifts to equatorial plate . 5. Metaphase. Spindle is the shape of barrel. Arrows indicates the small chromocenters.

6. Early anaphase. Apparent transverse divisions of chromosomes.


"AuBenkern" gradually shift to the mass of ch romonemata. At this stage spindle fibers become visible, each four centers setting on the cytoplasmic

Figs. 7-12. 7. Late anaphase. Several chromosomes indicated by arrows proceed from

the main chromosome-group. 8. Early telophase, A nuclear membrane is indicated by

arrows. 9. Poleward proceeded chromosomes which are unravelling. Arrows indicate

chromocenters. 10-12. From late telophase to interphase. Arrows indicate chromocenters

which are unravelling to fine chromonemata.

strings which hang the nucleus. At the later stage of the prophase, these four centers converge on two mitotic centers (Figs. 3-5).


As the stage proceeds to the late prophase, the nucleus is extended to the direction of mitotic poles which is parallel with the direction of the axis

of cellular filament (Fig. 4). The chromonemata which are now thick and

short gradually shift to equator of the cell taking the direction vertically

against the equator. At the peripheral part of the mass of chromonemata or

chromosomes, many chromocenters are seen. These chromocenters decrease

both their number and size as the stage proceeds and completely disappear in

the early anaphase.

Metaphase. Extention

of the nucleus proceeds,

and two ends of the nucleus

arrive at the mitotic poles.

Spindle fibers undulate, and

connect chromosomes and

mitotic poles. The mitotic

pole at which the spindle

fibers attach is not central

ized to a point but is

spread to some extent so

that the spindle takes the

shape of barrel (Figs. 5

and 16). Even at this

stage many chromosomes

have small chromocenters

which are stained more

deeply than the other part

of the chromosomes. In

most cases, about 20 chro

mocenters was counted.

These chromocenters ap

pear as if they were granu

les, hence they were de

scribed by Geitler and

Godward as chromosomes.

Besides these small chro

mocenters, one or two rod

shaped bodies, 1.5-3ƒÊ in

length, are frequently ob-

served, which are probably

the large chromocenters

(Fig. 16).

Anaphase. Apparently chromosomes are divided transversely, and move

toward the poles. At the beginning of polar separation, chromocenters are

Fig. 13. Earliest prophase.

•~ 1500.

Fig. 14. Mid prophase.

•~1500.

Fig. 15. Late prophase.

•~1500.

Fig. 16. Metaphase.

•~ 1500.


not distinguishable from the other part of the chromosomes, while the chro

mosomes become more distinct than before (Fig. 6). Number of the chro

mosomes was not able to be counted exactly, but the chromosomes appeared

to be more than twenty. In each daughter chromosome-group, two large

chromosomes are frequently observed, projecting their arms from the daughter

chromosome-mass toward the equator . Chromosome-mass in late anaphase

becomes dense and is stained so deeply that individual chromosomes are hardly

distinguishable (Fig. 7). In this stage, about ten chromosomes are pulled

out toward the pole apart from the chromosome-mass or main chromosome

group. Therefore, there are two chromosome-groups in each half spindle:

main chromosome-group and poleward proceeded chromosome-group .

Telophase. In early telophase, the main chromosome group and the

poleward proceeded chromosome-group, are in a membrane. This membrane

becomes distinct as the stage proceeds and at last it develops into a nuclear

membrane in the interphase nucleus (Fig. 8) . Then, the main chromosome

group swells and becomes a less closely packed spherical body. This body

develops into nucleolus at the resting stage . Each chromosome in the pole

ward proceeded group, unravells and becomes a slender thread or a chromo

nema. The unravelling of a chromosome does not take place equally within

the chromosome, that is, it takes place quickly in a part and more slowly

in another. Accordingly, these chromosomes are transformed into granular

chromocenters and fine chromonemata. Although most granular chromo

centers are gradually unravelled to fine chromonemata, two or three of them

remain not unravelled and become "Nebenkorper" (Figs. 9, 10, 11 and 12).

Staining reaction. The nucleus is negative with Feulgen's nucleal reac

tion throughout the whole cycle of the mitosis.

When a resting nucleus is stained with pyronin-methylgreen, the "nucleo

lus" is coloured red with pyronin while the "AuƒÀenkern" is stained with

methygreen. With safranin-lightgreen the "nucleolus" is stained with light

green, while the "AuƒÀenkern" is stained with safranin.

Discussion and conclusion

Mitosis in Spirogyra setiformis, has been observed by Czurda (1922),

Geitler (1930, 1935), and Godward (1950, 1953). But results of the observations reported in the present paper are different to those obtained by these

investigators. The main conclusions derived from the results obtained in the

present study are as follow:In the first place, the granules observed in the prophase and metaphase

nuclei are not the chromosomes itself or the fragments of the nucleolus, but

chromocenters within the chromonemata. What is called "nucleolar substance" of Geitler (1935) and Godward (1953) is nothing but the chromosomes, while

Godward has described that chromosomes are not observed in this stage. The "horns" of Godward which project from the anaphase chromosome-mass


toward equator are, therefore, the arms of large chromosomes.

Secondly, apparent transverse division of chromosomes at early anaphase

might mean that the chromosomes are longitudinally divided at earlier stages

and their mutual attachment at the distal ends are merely broken down at

this stage, but the opening out of the daughter chromosomes along the longi

tudinal split, was not confirmed in the present investigation.

Thirdly, the so-called nucleolus consists mainly of the "main chromo

some-group" stated in the descriptive part of this paper.

The author wishes to express his sincere thanks to Prof. Dr. N. Shinke

for his guidance throughout this work.

Summary

Somatic mitosis of Spirogyra setiformis was studied with special attention

to the behaviour of a "nucleolus".

In the prophase, a "nucleolus" is transformed into fine chromonemata.

Both chromonemata derived from the "nucleolus" and derived from the

"AuƒÀenkern" or the nuclear part outside of the nucleolus, shift together

toward the nuclear plate of the spindle.

In the anaphase, about ten chromosomes are pulled out from the daughter

chromosome-group toward the mitotic pole. A membrane surrounding the

main daughter chromosome-group and the poleward proceeded chromosome

group, develops into nuclear membrane.

The main chromosome-group swells and becomes a spherical body or a

"nucleolus" .

Most of the poleward proceeded chromosomes, are unravelled to fine

chromonemata which exist in the "AuƒÀenkern" at resting stage. Part of

the chromosome which remains without unravelling throughout the resting

stage, is designated as a "Nebenkorper".

Literature

Belay, K. 1926. Der Formwechsel der Protistenkerne. Jena. Czurda, V. 1922. Uber ein bisher wenig beobachtes Gebilde und andere Erscheinungen im

Kerne von Spirogyra (setiformis Kutz.). Arch. f. Protistenk. 45: 163-199. Geitler, L. 1930. Uber die Kernteilung von Spirogyra. Ibid. 71: 79-100.-

1935. Neue Untersuchung uber die Mitose von Spirogyra. Ibid. 85: 10-19. Godward, M. B. E. 1950. On the nucleolus and nucleolar-organizing chromosomes of Spiro

gyra. Ann. Bot. 14: 39-54.- 1953. Geitler's nucleolar substance in Spirogyra. Ibid. 17: 403-416.

Leyon, H. 1954. The structure of chloroplast. IV. The development and structure of the Aspidistra chloroplast. Exp. Cell Res. 7: 265-273.

Steinmann, E. and Sjostrand, F. S. 1955. The ultrastructure of chloroplasts. Ibid. 8: 15-23.

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