UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN

AGRICULTURAL SCIENCES

Vol. 2, No. 8, pp. 243-248, plate 44 September 17, 1924

MICROSPOROGENESIS OF GINKGO BILOBA L.

WITH ESPECIAL REFERENCE TO

THE DISTRIBUTION OF THE

PLASTIDS AND TO CELL

WALL FORMATION

BY

MARGARET CAMPBELL MANN

(Contribution from the Division of Genetics, University of California)

Microsporogenesis in Ginkgo biloba L. is especially interesting
because the plastids are definitely oriented with respect to the division
figures and because they are distributed so that each pollen cell receives
approximately one-fourth of them. The cells are large, and both
plastids and chromosomes can be observed in living cells. The 12
pairs of chromosomes are nicely separated at late prophase, and one
of them is twice as large as the others. This point was previously noted
by Cardiff (1906) and Ishikawa (1910).

Smears stained in aceto-carmine were used for most of this study,
but smears were also fixed in Plemming 's weak and chrom-acetic-urea
and stained in iron-haematoxylin, Flemming's triple, and safranin and
light green. The plastids are easily observed in aceto-carmine since
the large starch grains resist the carmine, remaining a transparent
green while the rest of the cytoplasm stains pink, and the chromosomes
bright red. The starch grains stain a deep blue in iodine, but show no
color in Flemming 's triple or haematoxylin. With the former a layer
of gentian, with the latter a layer of bluish-black cytoplasm, surrounds
each starch grain. Unless one had examined the pollen mother cells
before fixation, he might easily interpret the plastid-bearing area in
fixed material as an unusually coarse cytoplasmic mesh. This is
probably the reason why the phenomena described below have not been
previously observed.

244 University of California Publications in Agricultural Sciences [Vol. 2

The two nuclear divisions precede cell division as in many of the
higher plants. It will be seen from the account which follows that the
reduction division is typical in all respects.

Before the first, or so-called heterotypic division occurs, the plastids
lie between the wall and the nucleus (figs. 1 and 2). As the prophase
progresses they enlarge, and some of them divide. The division is
usually a simple bipartition, but some appearances suggestive of bud-
ding were seen. The cell wall is very thin at first but gradually
thickens during the first division. The plastids remain between the
nucleus and the wall until after the first spindle has disappeared, when
they gradually move, or more probably are moved, between the two
groups of late anaphase chromosomes, there forming a ring which
nearly fills the space between the two nuclei (fig. 4). This position
is retained until after the second division. In the late second ana- or
early telophase a portion of the ring of plastids is drawn between each
of the two sets of daughter nuclei (fig. 7). The ring then breaks in
four places, so that one quarter of it, and consequently about a fourth
of the plastids, come to surround the inner face of each nucleus (figs.
7 and 8). The outer wall now pushes inward between each of the four
nuclei and finally separates the pollen cells (fig. 9) . Before the inpush-
ing of the outer wall two cell plates form at right angles to each other.
The method of cell division in Ginkgo, then, is a combination of cell
plate formation and cytokinesis. The inner portion of the wall (which
lies next to the plastid-bearing area) thickens greatly while the outer
wall remains unchanged. As the inner wall attains its final thickness,
the plastids become generally distributed between it and the nucleus,
and a new wall, staining in gentian-violet, appears about each pollen
cell. The old wall stains in orange or in light green. As the pollen
cells grow they burst the thin outer walls of this case, leaving an empty
shell. The plastids now appear smaller, since the amount of starch in
them is considerably reduced.

Juranyi (1872, pi. 31, fig. 13) figured a light band between the two
daughter nuclei of the pollen mother cells of Ceratozamia, which is
very like the position of the starch grains of Ginkgo at this stage.
Sprecher (1907, p. 155) figured the cell plate formation of the pollen
mother cells of Ginkgo, but neither figures nor mentions plastid or
starch grain distribution. Moore (1903) figured plastids like those of
Ginkgo in the pollen mother cells of Pallavincinia. but does not discuss
their distribution. Smith (1907) shows starch grains in the. pollen

1924] Mann: Microsporogenesis of Ginkgo biloba L. 245

tube of Cycas which greatly resemble those seen in the pollen mother
cells of Ginkgo.

The procedure described above raises a number of interesting
questions. Firstly, the position of the starch grains bears a definite
relation to the formation of the cell walls. They are generally dis-
tributed while the first wall is forming, and it ceases to thicken when
they withdraw. They lie near the forming cell plate, and near the
thick inner wall during its formation. Finally, they become generally
distributed during the formation of the pollen cell wall. They are also
smaller than they were during the formation of the thick inner wall
of the pollen case. It seems possible that they may provide the reserve
material which is utilized in wall formation.

Secondly, the method of cell wall formation differs from that
common to the higher plants. This is of particular interest on account
of the phylogenetic position of Ginkgo.

The changes of position of the starch grains are essentially the same
as those noted by Terni (1914) for the chondriosomes in the spermato-
genesis of Geotriton fuscus, and by Payne (1916) for certain scorpions.
The mitochondrial mass forms the tail sheath in such spermatozoa. It
would be interesting to know the fate of the plastids in spermatozoa
formation of Ginkgo.

The similarity of behavior of the chondriosomes during cell division
to that observed for the starch-filled plastids of Ginkgo indicates that
the distributing mechanism is very similar in each case. It does not
seem necessary to postulate a separate mechanism for this purpose, the
forces already in action being of a type which would, it seems to me,
bring about essentially the observed distribution. Whatever the force
or forces may be by which the chromosomes are distributed during
reduction, the direction of movement of the chromosomes shows the
direction in which these forces act. One would expect that a large
number of movable cytoplasmic structures or inclusions would be
equally distributed between the cell wall and the nucleus, if in- and
out-going currents maintained an equilibrium during the early pro-
phase. After the spindle has formed, and the cell is a bipolar structure,
the forces (which we may think of as currents) move toward the poles,
and presumably back toward the equator. The changes of position
of the plastids at this stage indicate such lines of force. As the nuclei
grow, apparently by taking in fluid, their increase in size would also
tend to force the plastids out of the polar and into the equatorial

246 University of California Publications in Agricultural Sciences [Vol. 2

regions. The barrel-shaped spindle would hold them in ring formation.
When the nuclei have attained their final size, the plastids, now filled
with starch, form two rows with the cell plate between them. With the
formation of the second spindles this line of division is obliterated.
It is possible either that the processes involved in cell plate formation
produce the line of division, or that it results from the action of the
opposing forces concerned in chromosome division. In any case, the
distortion of the ring at late second ana- and early telophase might
result from the pull of the same forces which separated the chromo-
somes. The final division of the plastids into four groups may depend
somewhat upon the invaginations which give rise to the lateral Avails
of the pollen case. In the pollen cell the plastids again revert to the
position which they had at early prophase of the pollen mother cell.

1924] Mann: Microsporogenesis of Ginkgo biloba L. 247

LITERATURE CITED
Cardiff, I. D.

1906. A study of synapsis and reduction. Bull. Torr. Bot. Club, vol. 33,

pp. 271-303.

ISHIKAWA, M.

1910. Uber die Zahl der Chromosomen von Ginkgo biloba L. The Bot. Mag.,
Tokyo, vol. 24, pp. 225-226.

JURANYI, L.

1872. Ubei' den Bau und die Entwickelung des Pollens bei Ceratozamia longi-
folia Miq. Jahrb. f. Wiss. Bot., vol. 8, pp. 382-400, pis. 31-34.
Moore, A. C.

1903. The mitosis in the spore mother cell of Pallavincinia. Bot. Gaz., vol.
36, pp. 384-388.
Payne, F.

1916. Germ cells of Gryllotalpa. Jour. Morph., vol. 28, pp. 287-327.

Smith, F. G.

1907. Morphology of the trunk and development of microsporangium of

Cycas. Bot. Gaz., vol. 43, pp. 187-204, pi. 10.
Sprecher, A.

1907. Le Ginkgo biloba L. (Geneva) 207 pp.

Tern i, T.

1914. Condriosomi, idiozoma e formazioni periidiozomiche nella spermato-
genesi degli Anfibrii. (Ricerche sul Geotriton fuscus.) Arch. f.
Zellf., vol. 12, pp. 1-96, pis. 1-7.
Wilson, E. B.

1916. The distribution of the chondriosomes to the spermatozoa of scorpions.
Science, vol. 43, p. 539.

PLATE 44

The drawings are semi-diagrammatic. They were made with a camera lucida,
using a 4 mm. dry objective and a number 18 Zeiss compensating ocular. The
chromosomes and nuclei are simply outlined, the plastids at the upper focus
are in gray wash with a stippled margin, while the lower ones are left white.
The drawings show the position of the plastids at successive stages in micro-
sporogenesis.

1. Polar view of late first prophase showing 12 pairs of chromosomes, one of
which is about twice the size of the others.

2. Lateral view of same stage showing that the spindle area is largely free
of plastids. Most of the plastids are near the poles.

3. Lateral view of late anaphase showing movement of plastids from poles
toward equator of cell.

1. Late telophase. The plastids are now arranged in a double ring at the
equator.

5. Late second prophase. The plastids are so closely massed that ring
formation cannot be distinguished.

6. Late second anaphase. The ring of plastids is being distorted.

7. Second telophase. The ring is now breaking into four segments, one group
lying about the inner face of each of the four nuclei.

8. Early cell division. The dotted lines show cell plate. The outer wall is
pushing inward between each of the four nuclei.

9. Cell division completed.

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UNIV. CALIF. PUBL. AGRI. SCI. VOL. 2

[ MANN | PLATE 44