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,* Cornell TAnivcrsity . .

DEPARTMENT OF BOTANY

THE DEVELOPMENT OF THE

ARCHEGONIUM AND FERTILIZATION

IN THE HEMLOCK SPRUCE

(Tsuga Canadensis Carr.)

IBy VXiUiam Alpbonso Aurrill

A Thesis submitteci to the University Faculty of
Cornell University for the Degree of Doctor
of Philosophy^ June, (900.

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ITHACA, N. Y.

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WAMurr.ll.del.

MURRILL — TSUGA CANADENSIS.

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VoLXIV, PIXXXI.

IS.

Universityr Press, Orford.

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MURRILL — TSUOA CANADENSIS.

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40.

UnivBrwty Press, Oxford.

; i^ i tll ! ?VWMUi»«W'N iii .

The Development of the Archegonium

and Fertilization in the Hemlock Spruce

(Tsuga canadensis, Carr.).

WILLIAM A. MURRILL*.

With Plate* XXXI and XXXU.

THE material for these studies has been collected with
great regularity for the past three years from a hemlock
standing alone on the bank of a stream in an open pasture
near the University grounds. The tree is well advanced in
years and has fruited heavily every season. The female
cones are terminal on the larger horizontal twigs, the male
cones occurring in great abundance on the smaller ones. At
pollination, there is no change in the position of the female
cones ; they remain slightly pendent, the scales opening
a little and receiving the pollen from below as it floats
upward. Soon after pollination, the pedicels lengthen and
the cones, which are now considerably heavier, hang directly
downward. Pollination on a single tree occupies about three
days, but a week or more elapses before it is completed on
all the trees of this locality. In 1899 it was at its maximum
on May 19, in 1900 on May 2 a. The seasons frequently differ
more than this.

' Read before the Botanical Society of America, at its sixth Annual Meeting in
New York City, June 38, 1900.

[Annala of Botany, Vol. ZIV. No. LVI. December, 1900.]

■I

MWM^SBKvr-

■msms»mmu»i.- — .•—

584 MnniU. — The Development of the Archegonium

Two weeks after pollination the archcgonial rudiments
appear ; a week later the necks are formed ; and two weeks
after this the ventral canal-cell is cut off. Fertilization takes
place five days after the ventral canal-cell ia formed. It
varies for the same and different trees much as pollination
docs, so that stages of fertilization may be obtained for a
week or longer. After one season's experience, it is possible
to determine with tolerable accuracy the date of fertilization,
but, after all, there is an clement of chance that can be
eliminated only by regular and abundant collections. I put
up material from one to three times a day. The hour of
collection seerns to be of little consequence. The central cell
was found in active division in cones collected at 10 a.m. in
bright sunlight, in others collected at 7 p.m., and in still
others on branches taken from the tree at 7 p.m., and kept
in water until 11 p.m. Fertilization stages were abundant in
cones collected at 9 a.m. and at 9 p.m. of the same day.

Methods.

The material was placed in the fixing solution within a few
minutes after it was taken from the tree. Only the middle
portion of the cone was used, as this part contains the best-
developed ovules. In the younger stages, the terminal sterile
portion of the scale was cut away, leaving the two ovules
attached side by side ; in older stages, the ovules were
entirely separated from the scale, and, as the coats became
hardened, they were cut away at the sides, exposing the
endosperm directly to the fixing fluid. The endosperm
should be quite well filled out before this is done, otherwise
it may collapse. After the embryos are well established, it is
well to remove the coats entirely. Many approved fixing
methods have been tried, with variations in strength, time,
and temperature, but for these studies none has been found
equal to Mottier's modification of Flemming's solution, used
fresh and allowed to act for twenty-four hours at about 30° C.
During the preparation and fixing of the ovules the bottles
were repeatedly shaken to ensure equal contact of fresh

^^■« *--«\' (J,^'-*^^

and Fertifizalion in the Hemlock Spruce. 585

golution on all sides. The solution was sometimes changed
at the end of one or two hours. Into each bottle was placed
at the time of fixing a small rectangular piece of linen paper
bearing in pencil the current number. This paper remained
with the material through all of the succeeding chnnges.appear-
ing finally at the bottom of the paraffin block as a permanent
and very convenient label. After fixing, the material was
washed for twelve to twenty-four hours in running water,
dehydrated in grades of alcohol, bleached with a seventy per
cent, alcoholic solution of hydrogen peroxide, and the
dehydration completed in commercial and absolute alcohol
several times changed. It was then brought very gradually
into cedar oil, and transferred with equal care into parailfin
melting at 54", in which it was imbedded. I have found it
expedient to store material in seventy or eighty per cent,
alcohol for a short time after bleaching, and to allow a
number of bottles to accumulate before proceeding farther
with the imbedding. Time and chemicals are thus saved and
more attention is given to the details of the process.

A Mmot-Zimmermann revolving microtome was used in
cutting the sections. In cases where the material was poor
in stages, a number of ovules were imbedded in rectangular
groups and sectioned together. Archegonia were thus
examined by thousands instead of by hundreds. Sections
were cut 6*6 ^l and 1 3-3 (t in thickness. The ribbons were
floated out on water, fixed to the slide with Mayer's albumen
fixative, dried thoroughly, and melted down by placing the
slides in the paraffin bath for an hour or two. The slides
were numbered with figures and letters according to the
system devised by Marks. A mixture of vermilion and
sodium silicate in a little water supplied a convenient and
indelible medium. Preliminary to staining, the slides were
passed through xylol, alcohol, hydrogen peroxide solution
and water. Several staining combinations were used, the
well-known Safranin-Gentian- Violet-Orange G. combination
proving the best ; though Iron- Hematoxylin alone and
combined with acid Fuchsin, and Delafield's Hematoxylin

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586 Murrill. — TJu Development of the Archegoniutn

alone and followed by Orange G. or Bismarck-Brown, were
useful for comparison in some stages of archegonial develop-
ment and oogenesis. Methyl-Green, Fuchsin, and Orange G.
were used in the search for centrospheres and some slides
were covered without staining. After the usual process of
dehydration with absolute alcohol and clearing with oil of
cloves, the sections were treated for several minutes with
Bergamot oil before being covered with balsam. This
removes foreign particles and ensures the permanence of
certain stains that are extracted by the oil of cloves: it
also flows easily and dries quickly.

The Origin and Development of the Archegonia

BEFORE the FORMATION OF THE VENTRAL CANAL-CELL.

, The archegonia of Tsuga arise, as they do in other
Gymnosperms, from superficial cells at the apex of the
prothallium which cease to divide and become conspicuous
for their large size and the abundance of protoplasm which
they contain. Fig. i, Plate XXXI, shows one of these
archegonial rudiments with its large nucleus and its radiating
bands of protoplasm, which, in a later stage (Fig. a) are
confined chiefly to the upper portion of the cell, while the
lower tapering portion is comparatively empty. Between
these rudiments are other cells which continue to divide
(Fig. 3) and later give rise to the archegonial sheath (Fig. 4).
About one week after the differentiation of the archegonial
rudiment, the neck-cell is cut oft" (Figs. a-3). It at first has
the form of a circular disk surmounting the central cell and
equalling it in breadth but not in height. As the central cell
grows, it appears comparatively narrow and more elongated
and also shows considerable variation. In the greater
number of well-developed archegonfa, it divides into two cells
about the time when the ventral canal-cell is formed
(Figs. 4-6). The division-wall may be transverse, oblique,
or longitudinal, but it is most often oblique. In many cases
the neck-cell remains undivided, and, on the other hand, one

■ipi| f ii i|- |I HMrif»» i - . n i «

4».<

and Fertilization in the Hemlock Spruce. 587

frequently finds three or four cells in the neck of a mature
archegonium (Figs. 8-10). Differences of opinion concerning
the number of neck-cells in the archegonia of Tstiga are
probably due to the fact that the division often occurs late.
After examining a large number, I must agree with Hofmeister
that two cells are more frequently present than any other
number. Had Mottier (1892) examined more of the mature
archegonia, he would have probably found two cells even
more frequent than he supposed.

Returning to the condition of the central cell after the
neck-cell was cut off, it will be remembered that it tapers
towards its lower extremity and is almost entirely free from
protoplasmic contents. Though its nucleus remains at the
apex just beneath the neck, rapid changes take place in the
form and contents of the cell. It increases greatly in size,
and a delicate reticulum appears with numerous vacuoles in
its meshes containing cell-sap (Fig. 11). Enveloping the
central cell is a sheath of cells rich in protoplasm which
furnish the central cell with food, the endosperm still growing
vigorously and crowding back the disorganized nucellar
tissue. In place of the delicate network shown in Fig. 1 1, the
central cell soon shows spherical vacuoles filled with granules
and other food-masses (Fig. 12). These appear first at the
periphery near the food-cells and later come to occupy the
whole cavity, with the exception of one or two large vacuoles
at the centre. A transverse section of a prothallium with
five archegonia at this stage is shown in Fig. 16.

I cannot confirm for Tsuga the results of Arnoldi's recent
studies (1900) on the proteid vacuoles of the Abietineae. It
may be that further search on my part will reveal the passage
of the nuclei of the sheath-cells into the central cells, but
very careful examination of numerous archegonia in all stages
of development has thus far failed to show a single un-
doubted example of such passage. I find the nuclei of the
sheath-cells staining diffusely at times, as described by Ikeno
(1898) for Cycas, and I observe collections of granules in the
outer vacuoles of the central cells which very much resemble

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588 Murrill. — The Da^elopmcnt of the Archegonium

the sheath-nuclei, but the sheath-cells are never found without
their nuclei. The sheath remains one-layered, though its
cells often divide as the archegonium grows. At points
where the archegonia come into close contact (Fig. 16), the
sheath is frequently crushed and destroyed, but throughout
most of its extent the cells and their nuclei continue active
during the life of the archegonium.

The Formation of the Ventral Canal-Cell.

Shortly before the division of the central cell, an accumu-
lation of cytoplasm may be observed beneath the nucleus a
little to one side of the longitudinal axis of the archegonium
(Fig. 13). This accumulation is a dense mass of fibres with
small granules of uniform size scattered through it, the whole
being continuous with the cytoplasm around it and taking
the same brownish stain with the Flemming combination.
From the first it is closely pressed against the nuclear mem-
brane, and soon begins to push it inward in the form of blunt,
unequal projections (Fig. 17). This fibrous mass continues to
increase in size and to send out radiations far into the cyto-
plasm, thus forming a support for the free lower pole. These
radiations grow at their free ends from the cyto-reticulum
and extend in all directions, but are of necessity short on the
side next the nucleus.

The spindle-fibres arise within this mass and grow upward
against and press in the nuclear membrane, while they also
draw to a point below and establish the lower pole of the
spindle (Figs. i8-ai). At the pole there is usually greater
density and frequently a rounded granule (Fig. 24), but
nothing could be discovered corresponding to a centrosome,
even with the most favourable methods. Activity at the
upper pole begins late and is always feeble. The cytoplasm
between the nucleus and the neck-rell is all of a density
intermediate between that of the ordinary cytoplasm and that
collected at the lower pole. In a few cases a minute hyaline
lenticular area was observed at the upper pole, resembling
the collections of sap usually seen between the polar caps of

«'.

•ii

and Ferlilization in the Hemlock Spruce. 589

dividing vegetative nuclei and the nuclear membrane (Fig. 15).
In later preparations this area seemed to have broadened and
filled with delicate threads (Fig. 18) which later drew together
above, but I cannot be sure of this K Possibly the collection
of sap is so small that it often disappears in fixing. Careful
search was made for a similar collection at the lower pole,
but without success.

The further development of the spindle is very rapid. The
nuclear membrane disappears below and the spindle-fibres
press into the nuclear cavity and connect with the linen net-
work, already partially arranged in such a way as to continue the
fibres to the chromosomes or through the centre of the cavity.
The fibres soon become homogeneous below, while at their
upper extremities they are still only rows of granules (Fig. 21).
Nothing now remains in the nuclear cavity except the spindle-
threads and the homogeneous chromatin segments. The large
spreading bundle of fibres originating from below traverses
about two-thirds of the nuclear cavity before connecting with
those from the upper pole. The nucleus consequently becomes
pear-shaped at this stage, with the upper end larger. As the
spindle-fibres draw in towards the centre, the chromosomes
are forced from their peripheral position and come to lie along
the central part of the spindle (Fig. 23). At the same time
the upper pole is somewhat elevated and appears as an abrupt
but sharp point rising out of the nuclear cavity. Supporting
fibres run from this pole to the cell-wall and in various
directions through the neighbouring cytoplasm (Fig. 25).

In the plate-stage (Fig. 26), the chromosomes show the
form and arrangement characteristic of that class of divisions
recently denominated 'typical' by Strasburger (1900), and

* It is often impossible to demonstrate tiie existence of an extra-nnclear spindle-
radinient at the upper pole, but the fibres appear to collect in bundles within the
membrane and to unite at one point when the polarity becomes more pronounced.
The narrowing and consequent stretching of the spindle as the metaphase is
approached would elevate the upper pole and give it the appearance of penetrating
the membrane (Fig. 22). In Larix Amerkana there is still less cytoplasm above
the nucleus than in Tsuga, which makes the difference in the two poles yet more
marked. ,

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590 A/urri/L — T/ie Development of the ^rchegonimn

are drawn to the poles as attenuated U's or V's (Fig. 27).
The cell-plate is laid down from the centre outwards. It
begins in the dispirem stage and keeps pace for some time
with the growing daughter-nuclei (Figs. 29, 30). The fibres
thicken at the centre, and the thickened portions fuse together
at the side. The spindle then broadens out and stretches the
young cell-plate until it connects with the wall of the mother-
cell (Fig. 31). The original nuclear membrane does not
entirely disappear until the daughter-nuclei are formed. It
then splits up into slender fibres and fades away into the
surrounding cytoplasm.

The division above described, while it resembles that de-
scribed by Rosen (1895), Hof (1898), Nemec (1899), and
others for the vegetative* cells of various plants, in the extra-
nuclear origin of the spindle and the details of its construction,
nevertheless differs very strikingly from that type by reason
of the fact that the spindle is at first asymmetrical and
originates in a large fibrous mass beneath the nucleus, which
appears early, grows to an immense size, and remains for
some time after the division is completed, being enveloped
during its existence by numerous fibres radiating from it far
into the surrounding cytoplasm.

Various authors (Ikeno, 1898, p. 567; Blackman, 1898;
Chamberlain, 1899, figs, i, a; and others) have described or
figured the nucleus of the central cell as remaining at the
cell-apex until division occurs, and it would be interesting
to know whether more careful study would show such a method
of spindle-formation as I have discovered in Tsuga in these
or similar cases of unequal division.

This collection of cytoplasm is probably due to the need
of a support for the free lower pole, and the numerous
radiations from it doubtless add greatly to its stability. The
origin and growth of the spindle chiefly on the lower side
of the nucleus seem to indicate that the force controlling
division is largely centred there.

1 v

■MMrt*'-^-

and Fcrlilization in the Hemlock Spruce. 591

^•

FuiiTHER Changes in tuE Egg and the Ventral

Canal-cell before the Entrance of the

Sperm-cells.

When the nuclear membrane is deposited about the young
egg-nucleus, the chromatin is in the form of a thick homo-
geneous band which is gradually drawn out until the chromatin
granules appear distinct on the linin threads. At the same
time the threads anastomose to form an ordinary cell-reticulum,
in which several small nucleoli appear (Figs. 29-31). '^^^
nucleus increases rapidly in size and begins to move down
toward the centre of the egg.' The chromatin now occupies
chiefly the upper half, the lower part being comparatively
empty (Fig. 3a). As it begins to move, it usually becomes
elliptical in form (Fig. 33), and remains so until fertilized,
but if the archegonium is very broad it may remain perfectly
spherical. As it increases in size, it takes in considerable
food-material and the network becomes coarser and the
nucleoli larger. When the centre of the egg is reached, it
remains stationary in the resting condition until fertilized.
The granules in the cytoplasm of the egg are arranged in
rows radiating from the nuclear membrane. This arrange-
ment appears with the formation of the egg and disappears
with fertilization.

The nucleoli of the egg-nucleus first appear as minute
spheres hardly distinguishable from collections of chromatin.
They increase rapidly in size, however, and apparently unite
to form larger ones and often a single one of immense size
(Fig. 35). When first formed, these larger ones are very
plastic and easily take the sickle-form under the influence of
fixing agents. Later, the outer shell becomes firm and dense
and takes a deep purple stain with Gentian-Violet, while the
vesicular mass within stains feebly with Orange^ and shows
a delicate reticulum probably consisting of dissolved proteid
substance precipitated by chromic acid. The shell is frequently
found broken in mounted preparations, and the nucleolus then

R r

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MMM

59 2 Murrill. — The Developmenl of the Archegonium

suggests a sperm-nucleus with its membrane breaking away,
and the contents dissolving in the nuclear substance of
the egg.

The large vacuole at the centre of the egg changes its
position as the nucleus moves down, and, passing it on one
side (Fig. 34), comes to lie near the apex of the cell. The
vacuole is apparently somewhat diminished in size as the
egg-nucleus increases, and it is not improbable that some
of the cell-sap from the vacuole passes into the nucleus while
they lie near together. The concentration of cytoplasm and
food materials near the centre, in consequence of the change
in position of the nucleus, leaves more room for the vacuole
near the apical portion of the egg. Another vacuole with
contents very similar to those of the nucleus is regularly
observed * in the mature egg situated just beneath the ventral
canal-cell (Fig. 10). It is quite different from the proteid-
vacuoles in appearance, but seems to originate in one or more
of the latter upon the relief of pressure in the apical region
by the enlargement of their enclosing membranes and the
expansion and distribution of their contents. This ' nuclear '
vacuole sometimes fuses with the 'empty' vacuole as it
approaches the apex, but the latter more often remains to one
side and a little below the fornier. Near these two vacuoles
is the receptive spot of the egg.

The ventral canal-cell is fairly persistent in Tsuga. When
division is completed its nucleus is equal in size and similar
in structure to the nucleus of the egg, and for some time
shows the same stages of development. But when the nucleus
has nearly filled the ventral canal-cell its membrane becomes
wavy in outline, the scanty reticulum occupies only a small
portion of the nuclear cavity, and the nucleoli remain minute
and scattered. Growth having ceased, signs of disorganization
soon appear. The nuclear contents become amorphous and
stain diffusely, while the nucleus and the cell become more

• An archegonium rendered very abnormal by developing fcr down on the «de
of the prothallinm showed this nnclear Tacuole in all respects normal.

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and Fertilization in the Hemlock Spruce, 593

irregular and misshapen in outline. It is at this stage that
the pollen-tube usually enters the neck of the archegonium.

The Entrance of the Contents of the Pollen-
tube INTO THE Egg.

The pollen-tube reaches the egg by penetrating the neck
of the archegonium, the contents of the neck-cells being
pressed to one side or swept away entirely. They are fre-
quently found crowded down near the apex of the egg with
the remains of the ventral canal-cell (Fig. 36). On reaching
the egg the tube spreads out over it and causes its apex to
stain diffusely. Further than this the tube itself does not go,
but the contents of its terminal portion are emptied into the
egg near the empty vacuole, the membrane of the egg usually
closing up again, but sometimes remaining open at the point
of entrance.

The contents of the pollen-tube thus cast into the egg
consist of two sperm-cells, the stalk-cell, the vegetative nucleus,
and some protoplasm and starch from the tube-cavity (Fig.
36). The stalk-cell nucleus is small, perfectly spherical, and
conspicuous by reason of its thick, deeply-stained reticulum
(Fig. 39). The cytoplasm accompanying it is scanty, vacuolate,
and irregular in outline. The vegetative nucleus is larger,
ovoid or irregular in outline, and unaccompanied by cytoplasm.
It contains a delicate network with granules and nucleoli, and
takes a pale, slightly diffuse stain with Gentian- Violet.

Above these smaller nuclei lie the ellipsoidal sperm-cells,
each with dense cytoplasmic contents and a large nucleus. The
sperm-nuclei differ considerably in size and appearance. The
one that was in advance in the pollen-tube, which I shall call
the first sperm-nucleus, is about twice the diameter of its com-
panion, and its contents are so dense that its two small purple-
staining nucleoli are almost hidden from view. The second
sperm-nucleus is less dense and shows two large and prominent
nucleoli that stain a clear red with safranin. It tends to
conform more to the shape of its cell, and also corresponds
more nearly to the resting stage than does the larger nucleus.

R r a

594 Murriil, — The Development of the Archegonium

It is through the first sperm-nucleu:i mat fertilization is
accomph'shed. A short time after its entrance into the egg
it slips from its cell and moves with accelerated velocity
towards the egg-nucleus, the latter remaining stationary and
inactive, but probably exercising chemotactic attraction on
the sperm-nucleus by reason of its rich proteid contents.
There is no depression at the apex of the egg-nucleus, nor
other evidence in it of the approach of the sperm. The
conjugation -path is a straight line from near the point of
entrance. Since this point is ordinarily at the side near the
top of the egg, the sperm-nucleus usually strikes the egg-
nucleus slightly to one side of its apex ; when entrance into
the egg is eflfected at the apex, the sperm-nucleus strikes
the egg-nucleus directly at its apex (Fig. 38). Conjugation
occasionally occurs at the middle of one side of the egg-
nucleus, as shown in Fig. 40. The same figure also shows
the second sperm-nucleus very near the egg-nucleus, but there
is nothing in this or other preparations of mine to indicate
that both sperm-nuclei ever unite with the nucleus of the

egg-
In Conifers, the sperm-cell is very similar to a pure
vegetative cell. In the Pteridophytes, the nucleus is con-
densed and elongated, with reduced cytoplasm and active
cilia. In Phanerogams, the nuclei may be elongated, but
cytoplasm and cilia are absent. The sperm-cells of Ginkgo
and the Cycads diflfer from those of the Conifers in possessing
a cilia-bearing band which propels the cells from the tip of
the pollen-tube to the apex of the egg. In the Conifers, the
pollen-tube penetrates to the egg and the cilia-bearing band
is unnecessary and absent.

After the first sperm-nucleus has moved to the egg-nucleus
the second sperm-nucleus remains for a long time in its
cytoplasm in the upper part of the egg (Fig. 38), and is then
gradually absorbed, usually after the other contents of the
pollen-tube have disappeared. The discovery of a tripolar
spindle (Fig. 46) in the position commonly occupied by the
second sperm-nucleus at first led me to believe that, being

,i '-,-

and Fertilization in the Hemlock Spruce. 595

fed by the egg-cytoplasm, this nucleus had entered upon
mitotic division, just as the functional sperm-nucleus pre-
sumably initiates the division which, with the assistance of
the egg-nucleus, results in the first segmentation. In the
nuclei of unfertilized eggs, also, the process of disintegration
may be accompanied by the fusion of the chromatic reticulum
into rods similar to chromosomes, while the fibres of the
nuclear membrane focus on several points external to the
membrane as the nuclear cavity diminishes, thus forming a
figure resembling a multipolar spindle. Occasionally, before
the nucleus begins to diminish in size, the disintegrating
chromatin contents are found collected near together, with
numerous radiations present in the nuclear cavity, the whole
suggesting a possible stage of fertilization.

Fertilization and the first segmentation.

When the egg-nucleus is reached, the sperm-nucleus flattens
itself against it in the form of a bi-convex lens and soon
comes to lie within its original boundary (Fig. 37). The
surface of separation is at this time quite even and is com-
posed of the two nuclear membranes with some included
cytoplasm. The reticulum in the apex of the egg-nucleus
is pressed down in advance of the sperm-nucleus and furnishes
the first deposit of chromatin at that point.

The difference in the density of the sperm-nucleus and egg-
nucleus is very apparent when they are thus first in contact, but
the former begins almost immediately to lose its density and to
become a perfect resting nucleus like that of the egg. In the
process, no stainable substance is cast out into the cytoplasm,
such as is described by Ikeno (1898) for Cyeas, and Wager
(1899) for some Phycomycetes \ nor can any of its contents
be seen to pass through the membranes into the cavity of
the egg-nucleus ; but increase in the number or in the size
of the nucleoli of the sperm-nucleus is very evident (Figs.
41^ 42). The deeply staining nucleoli occupy the centre of
the nucleus, while its periphery shovyrs a delicate chromatic

. TijiiirtfifimiiiiiwHiin

ZSSEES^

596 Murnll, — The Development of the Archegonium

reticulum, which may have been present before, concealed
by the dense contents of the nucleus, or may arise only after
the contact of the sexual nuclei. Certain granules of this
reticulum now gradually become larger by the addition of
neighbouring granules, and the whole contracts to a coarse,
knotted, slightly anastomosing thread, which, with the assistance
of the nucleoli, passes over into the spirem band (Figs. 43-45)*

While the sperm-nucleus has thus been entering upon the
early stages of division, the chromatin of the egg-nucleus has
been collecting near the centre of the nuclear cavity not far
from the membranes separating the two nuclei. It likewise
presents the appearance of an anastomosing, knotted thread,
in contact with granules and spheres of various sizes apparently
derived from the nucleoli, which latter now become hollow
and stain feebly, and finally disappear.

Changes have also occurred meanwhile where the nuclear
membranes are in contact. Instead of the even surface pre-
sented at the first contact of the nuclei, the membranes are
now separated by numerous spherical granular areas, which
tend to encroach on the cavity of the egg-nucleus and cause
its membrane to show in cross-section a series of crenate
folds. The contents of these spheres stain very slightly, with
the exception of one or two small spherical bodies which are
precisely like nucleoli and take the nucleolar stains. The dis-
appearance of the membranes and the consequent union of
the two nuclear cavities first occurs at points between these
granular spheres, and the latter continue to occupy their
position until the appearance of the spindle-iibres among
them, when all of them disappear except a few upon which
the fibres are centred. Whether they have any direct con-
nexion with the formation of the spindle, or are simply cavities
between the nuclear membranes containing a small amount
of cytoplasm caught between the conjugating nuclei, it is
impossible for mc to say. Their increase in size with the
decrease in density of the sperm-nucleus has suggested to me
the arrangement found in Cycas and Cephalotaxus, where
fusion is accelerated by root-like projections of the sperm-

aMMM

mum

and Fertilization in the Hemlock Spruce. 597

nucleus into the egg-nucleus ; but, even if the analogy were
otherwise perfect, the increase in the amount of nucleolar
substance seems to fully account for all the contents of the
sperm-nucleus not found in its chromatic reticulum.

The breaking down of the membranes separating the two
nuclear cavities occurs before the completion of the spirem
bands, but the chromatin masses remain distinct until the
chromatic segments appear. The achromatic contents of
the cavity, however, undergo a decided change, becoming
denser and more fibrous in appearance with the probable
rearrangement of the linin network under the stimulus causing
division (Fig. 45). The outer membrane also disappears in
places, and some of the cytoplasm presses into the cavity, but
the chief activities of division are intranuclear. With the
union of the nuclear cavities there also occurs a change in
the cytoplasm of the egg. The dense sheath of small granules
and fibres that encircles the unfertilized egg-nucleus partly
disappears, and the larger cytoplasmic granules extend almost
to the nuclear cavity, while the rows of elongated granules,
radiating into the surrounding cytoplasm from the egg-nucleus
since its origin, now lose their radial position and are dis-
tributed without special arrangement. The indications are
that the egg-nucleus has relaxed its hold for a time upon its
cytoplasm to enter upon the changes involved in division.

The fibres originating the spindle of the first segmentation
arise among the segmenting spirems derived from the two
nuclei, and they first draw together at several different points
forming a multipolar spindle-rudiment. On these fibres the
long bent and twisted chromosomes appear, still showing
the chromatin disks distinct on the linin thread (Fig. 47).
As the number of fibres increase, and the spindle becomes
monaxial in form, the chromosomes contract and become
homogeneous, and are mostly bent in the form of a U.
There is no difference to be observed between the chromo-
somes of the sperm and those of the egg, and, at this stage,
they are mingled indiscriminately near the centre of the
common spindle.

598 Murrill, — The Development of the Archegonium

The mature spindle is broad, with many fibres and rather
blunt ends, and, during the metaphase, the twenty-four chromo-
somes occupy all of the equatorial plane (Figs. 50, 51). The
type of this division, so far as the chromatin is concerned, is
the same as that already described for the central cell of the
archegonium. The position of the mature spindle in the nuclear
cavity seems independent of the relative position of the con-
jugating nuclei, and, while the division is usually oblique, it
may be perpendicular to, or parallel with, the longitudinal
axis of the archegonium (F"igs. 5a, 54, 57).

The chromosomes pass to the poles as slender undulated
V's or U's, and fuse to form the network of the daughter-
nuclei (Figs. 53, 54). No cell-plate is formed, but the fibres
fade away into the cytoplasm, the slightly thickened middle
portions being the last to disappear.

The pro-embryo.

The two free nuclei resulting from the first segmentation
increase rapidly in diameter, and soon divide simultaneously
without change of position to form four nuclei of equal size
lying free near the centre of the egg (Figs. 54-58). The type
of the second division is similar to that of the first, but the
spindle is narrower and more pointed at the ends (Fig. 56).
The daughter-nuclei are formed and the spindle-fibres absorbed
as in the first division. When the four nuclei have attained
their full size, they move to the base of the archegonium
(Figs. 59, 60), and by successive divisions in a horizontal plane
give rise to four tiers of nuclei with four nuclei in each tier,
as has been already described for various Conifers.

When the four free nuclei are moving through the egg-
cytoplasm they do not show any special collections of fibres
about their membranes^ but soon after the base is reached
they become enclosed in a dense mass of fibrous substance
(Fig. 60), which supplies the material for the walls that appear
later. Above this fibrous mass is a zone showing a delicate
reticulum almost devoid of stainable substance. Above this

OMMH

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and Fertilization in the Hemlock Spruce. 599

zone the egg shows its normal structure. The cause of this
h^ aline zone is difficult to determine. It appears that the
stored food has been taken from this region in order to
provide for the growing want of supplies for the nuclei below ;
but since the remaining available contents of the egg are
doubtless also transferred to the developing embryo, there is
no apparent reason why this zone should be left. It possibly
represents an intermediate condition of these contents in which
they do not easily take or retain stains.

ated
iter-
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Summary.

The archegonia of Tsuga originate as single superficial
cells, in each of which occurs the usual division cutting off
an outer smaller cell that forms the neck. At maturity, the
neck most commonly consists of two cells, though in very
many cases the neck-cell fails to divide at all. Three and
four cells in the neck are less commonly observed.

In the division of the central cell the spindle-fibres arise
from a large dense fibrous mass beneath the nucleus and grow
into the nuclear cavity, where they are later joined by fibres
from the very small upper pole. The division resembles that
recently described for many vegetative cells, but belongs to
a new and distinct type hitherto undescribed. The mass of
cytoplasm at the lower pole with its extensive radiations
suggests a huge centrosphere.

As the egg-nucleus increases in size and moves to the centre
of the egg, the vacuole moves upward, passing it on its way
and comes to lie near the egg-apex. Just beneath the ventral
canal-cell may always be found another somewhat smaller
vacuole with contents similar to those of the nucleus. These
vacuoles form the receptive spot of the egg.

The contents of the pollen-tube cast into the egg consist
of the vegetative nucleus with some cytoplasm and starch-
grains, the stalk-nucleus surrounded by its own scanty, vacuo-
late cytoplasm, and the two unequal sperm-cells with their
dense cytoplasm and large deeply-stained nuclei. The larger

HlMlillT' "

itfi^iriiii

li 1 nfil iBTJMWfcaiWiW

6oo Murrill. — The Development of the Archegonium

sperm-nucleus slips from the cell and conjugates with the
egg-nucleus, the smaller one being gradually absorbed with
the Ot'ier structures derived from the pollen-tube.

The lunctional sperm-nucleus flattens itself against the
egg-nucleus in the form of a bi-convex lens, and the two
nuclei soon come to lie in the space formerly occupied by the
egg-nucleus alone, their membranes, however, remaining intact
for a long time. In this condition the sperm-nucleus rapidly
loses its density and constructs a delicate peripheral chromatic
reticulum and larger central nucleoli, thus becoming a perfect
resting nucleus similar to that of the egg. The chromatin of
each nucleus collects in the form of a thick knotted thread
near the centre of the separating partition, and the two masses
remain distinct until the spirem-bands begin to segment.

Just before the spirems are formed the separating mem-
branes disappear and the nuclear cavities become united.
The spindle then arises in a multipolar fashion between and
among the two masses, twelve chromosomes being supplied
from the chromatin of the sperm and twelve from that of the
egg, as described by Blackman for Pinus Sylvestris. The
mature spindle is broad with blunt ends, and the manner of
division is typical.

A second division succeeds the first without much delay,
and the four resulting free nuclei soon attain full size and
move to the base of the arch^onium, where the young embryo
becomes established in the manner already so well known
among Conifers.

The investigations leading to the results recorded above were
conducted in the Botanical Laboratory of Cornell University
under the direction of Professor Geoi^e F. Atkinson, at whose
suggestion this work was undertaken, and for whose kindly
sympathy and invaluable aid in its prosecution I aqi deeply
grateful.

wi l Biiii ii ni

.isgi'^

and Fertilization in the Hembck Spruce. 60 1

Bibliography.

Andrews, G. F., '98 : Camera Drawing. Zeitschr. f. wist. Mikr., xiv, 451-45J,

1898.
Aknoldi, W., '00' : Beitrage zur Morphologic der Gymnoipermen : iii. Flora,

Ixxxvii, 46-63, pi. 1-3, 1900.
'OO'' : Beitrage zur Morphologic der Gymnoapermen : iv. Flora,

Ixxxvii, 194-204, pi. 6, 1900.
Blackman, V. H., '98 : On the Cytological Features of Fertilization and Related

Phenomena in Pinus Sylvestris, L. Phil. Trans. Roy. Soc, B., cxc,

395-426, pi. IJ-14, 1898.
Chamberlain, C. J., '99 : Oogenesis in Pinus I.aricio, Poir. Bot. Gaz., xxvii,

268-280, pi. 4-6, 1899.
Dixon, H. H., '99 : The Possible Function of the Nnclcolus in Heredity. Ann.

Bot., xiii, 269-278, 1899.
FULMER, E. L., '98: Cell Division in Pine Seedlings. Bot. Gaz., xxvi, 239-246,

pi. 33, 34, 1898.
GuiGNAKD, L., '91 : Nouvelles Etudes snr la F^condation. Ann. d. so. nat.,

Botaoiqne, 7* s^r., t. xiv, 163-296, pi. 9-18, 1891.
HoF, A. C, '98 : Histologiache Studien an Vegetationspnnkten. Bot. Centralbl.,

Ixxvi, Nos. 3-7, pi. 3-4, 1898.
Ikeno, S., '98: Untersuchnngen iiber die Entwickelnog der Geschlechtsorgane

und den Vorgang der Befmchtnng bei Cycas revoluta. Jahr. f. wiss.

Bot., xxxii, 557-602, pi. 8-10, 1898.
MOTTIER, D. M., '92 : On the Archegoninm and Apical Growth of the Stem in

Tsuga Canadensis and Pinus Sylvestris. Bot. Gaz., xvii, 141-143, pi. 8,

1892.
Nehec, B., '98 : Ueber die Ausbildnng der achromatischen Kemtbeilungsfigur

im vegetativen und Fortpflanzungsgewcbe der hoheren Pflanzen. Bot.

Centralbl., Ixxiv, 1-4, 8 text-figures, 1898.
'99' : Ueber Kern- und Zelltheilnng bei Solanum tubtrosum. Flora,

Ixxxvi, 314-237, pi. 13-14, 1899.

'99* : Ueber die karyokinetische Kemtheilung in der Wnrzelspitze von

Allium Cepa. Jahr. f. wiss. Bot., xxxiii, 313-336, pi. 3, 1899.
Rosen, F., '95 : Kerne und Kemkorperchen in meristematischen und sporogenen

Geweben. Cohn's Beitr. z. Biol. d. Pflanc, vii, 335-313, pi. 3-4, 1895.
Strasburger, £., '84 : Ncne Untersuchnngen iiber die Be&uchtungsvorgange bei

den Phanerogamen als Grundlage fUr eine Theorie der Zeugnng. Jena,

1884.
'88 : Ueber Kern- und Zelltheilung im Pflanzenreiche, nebat

einem Anhang iiber Befruchiung. Histol. Beitr., i, 1888.

'92 : Ueber das Verhalten des Pollens und die Befruchtungi-

vorgiinge bei den Gymnospermen. Histol. Beitr., iv, 1893.

*97' : Kemtheilung und Befruchtung bei Fucus. Jahr. f. wiss.

Bot., XXX, 35'-374. pl- 17-18, 1897.

'97*: Ueber Befruchtung. Jahr. f. wi»s. Bot., xxx, 406-422,

1897.

-A^

6o2 Mtirrill. — The Development of t lie Archegonium

Strasbukuer, K., '00: Ueber Reductionstheilung, Spindel' Mung, Centrosomen,

und Cilienbiklner im Pflanzenreich. Jena, 1900.
Thom, C, '99 : The Process of Fertilization in Aspidium and Adiantum. Trans.

Acad. Sci. St. Lonis, ix, 385-314^ pi. 36-38, 1899.
Wager, H., '99 : The Sexuality of the Fungi. Ann. Bot., xiii, 575-597, 1899.
Wai.deyer, W., '88 : Ueber Karyokinese und ihre Beziehung zu den Befruchtnngs-

vorgiingen. Arch. f. milcr. Anat., xxxii, i -12a. 14 text-figures. 1888.

EXPLANATION OF FIGURES IN PLATES
XXXI AND XXXII.

Illustrating Dr. Murrill's paper on Tsuffi Canadensis.

Most of the figures were first drawn with the aid of a camera lucida from a Zeiss
microscope, using compensation ocular 1 3 and the a"" homogeneous objective, and
then mechanically reduced to the present scale. For some it was necessary to use
a lower magnification in the first instance. In using the camera lucida, I found it
convenient to employ dark shades of paper (Andrews, 1898), usually black or dark
blue, and the outlines were traced upon it in Chinese white with a long pointed
pen. Since most of the preparations are coloured with dark stains, a white medium
is peculiarly suitable for outlining chromatic structures.

PL.VFE XXXI.

Fig. I. ( X 400.) A portion of the apex of the prothallium, showing an arche-
gonial rudiment in the centre, with rudimentary sheath-cells on each side.

Fig. 3. (x 400.) The archegonial rudiment has increased in length, and iti
nucleus is preparing to divide. Almost all the protoplasm is collected at the upper
end of the cell The sheath-cells have increased in number.

Fig. 3- ( X 400.) The archegonial rudiment has divided into an inner central
cell and an outer neck-cell. The outer cell is much smaller than the inner.

Fig. 4. ( X 300.) The neck-cell is in division. The central cell is very much
larger, and is being rapidly filled with protoplasm from the rich layer of sheath-
cells that closely envelop it. Its nucleus remains near the neck-oell and is still in
the resting stage, though the condensation of cytoplasm just below it indicates that
division is not far off.

Fig. 5. ( X 400.) A later stage of the same, showing the anaphase of the
division in the neck-cell.

Fig. 6. ( X 400.) A still later stage, showing the neck-cell divided into two
cells. The central cell is now entering upon division.

Fig. 7. ( X 300.) A mature archegonium, with two cells in the neck. The
division wall is oblique. The ventral canal-cell is also shown.

\um

and Fertilization in the Hemlock Spruce. 60j

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Fig. 8. ( y 300.) A matnre arcbegonium, with three cells in the neck. The
iirst division was transverse, and the upper cell afterwards divided longitudinally.

Fig. 9. ( X aoo.) Four neck-cells are present, formed by one longitudinal
division followed by two oblique ones. The cytoplasm of both cells has divided,
bnt no separating walls could be observed.

Fig. 10. (x aoo.) In this arcbegonium the four neck-cells are in one row,
formed by two successive transverse divisions. Beneath the neck is the dis<
organizing ventral canal-cell, and beneath the ventral canal-cell, at the apex of the
egg, is the nuclear vacuole. The empty vacuole is in another section. At the
centre of the egg is the large resttiig nucleus. About the periphery of the egg,
proteid vacuoles are abundant. The larger granules of cytoplasm are arranged in
rows radiating from the nucleus. The contents of the egg have become so dense
that its reticulum is wellnigh concealed. In many places, also, the reticulum has
been distorted by denser aggregations of nutritive materials.

Fig. II. ( X aoo.) This follows Fig. 3 in the order of development, bnt shows
a marked increase in the size of the central cell, with a well-formed sheath. The
nucleus is at the apex of the cell, where it remains until division. Throughout the
cell-cavity a delicate reticulum has been constructed, which is interrupted at the
centre of the cell by a large vacuole filled with sap.

Fig. I a. (x aoo.) A later stage in the development of the arcbegonium. The
neck-cell has elongated. The central cell has enlarged, and the meshes of its
rnticulum are fast filling with granular food supplies. The first proteid-vacnoles
have been formed.

Fig. 13. ( x 400r) This figure represents the nucleus of the central cell pre-
paring to divide. Its reticulum has become coarser and stams more deeply, and
is balled up in the cedtre of the nuclear cavity in a condition suggesting synapsis.
The preparations showing this condition are fixed as perfectly as one could desire.
Beneath the nucleus is a dense fibrous mass closely pressed against the nuclear
membrane, and sending out radiating fibres into the cytoplasm.

Fig. 14. ( X Soo.') The spindle-fibres are arising in this mass, and growing
upward against the membrane.

Fig. 15. (x 800.) A lenticular hyaline area at the upper pole, seen in only
two or three preparations, but very distinct and apparently perfectly normal.

Fig. 16. (x 150.) A cross-section of a prothallium containing five archegonia
of the stage shown in Fig. la. It will be observed that each archegonium is
enveloped by its own one-layered sheath. The archegonia are, therefore, ordinarily
separated by two rows of sheath-cells, but at points where there is some distance
between the archegonia the cells of the two sheaths have divided to fill the space.
Between the two upper archegonia in the figure, the sheaths have been crushed to
a line. This is quite frequently (he case where the curved surfaces of the arche-
gonia come nearest together.

Fig. 17. (x 800.) Following Fig^ 14, and showing the pressing in of the
nuclear membrane, in the form of blunt protuberances, by the spindle-fibres origi-
nating below. The chromatin thread is now peripheral and almost continuous,
though the disks are still far apart.

Fig. 18. ( X 800.) The spindle-fibres have advanced still farther, and pre-
parations are being made within the nuclear cavity for their continuation.
Segmentation occnn abont this time, and rows of granules and delicate threads
connect the chromatic segmeati with the incoming spindle-fibres. The greater

|]w" i i i l i'

1%-

604 Murrill. — The Development of the Archegonium

part of the chromatin is in the upper half of the nncleui. Activity has now begun
at the upper pole, where delicate fibres are seen growing down a^^nst the nuclear
membrane, but it rarely appears so distinct as m this preparation.

Fig. 19. ( X 800.) This nucleus belongs to a larger archegonium than that
shown in Fig. 18. The spindle is in about the same stage, but the chromosomes
are more advanced than those shown in the preceding figure. Radiations are
present at the upper pole, and possibly a small polar cap, but the latter, if present,
is not distinct enough to figure.

Fig. 20. ( X 800.) This figure represents the same stage as Fig. 19, but the
nucleus remains spherical, and the spindle-fibres seem to have originated at
a gteater distance than usual below the nuclear membrane, and to have advanced
with a more even front. This u exceptional, being observed only a few times in
the examination of a large number of preparations.

Fig. ai. ( X 800.) The nuclear membrane has disappeared opposite the polea,
and the spreading cone^haped bandies of spindle-fibres have grown into the
nuclear cavity, and are uniting somewhat above the centre of the nucleus. The
true pear-shaped form of the nucleus and the inequality of the two poles cannot be
shown in a longitudinal lection Which includes the upper pole, smoe the division is
oblique and much of the lower pole is cut away. When first formed, the spindle
is broad and the chromosomes, which have now become homogeneona, are attached
to its outer threads.

Fig. 23. ( X 800.) The spindle-fibres are now homogeneous throughout, and
the spindle has narrowed and drawn in the completed chromosomes neater to the
centre. In narrowing, it has also elongated, and the upper pole has been elevated.
The section is made as in Fig. ai, and does not show all of the lower pole.

Fig. 23. ( X 800.) Further narrowing of the spindle has taken place, and the
chromosomes are now coming up to the equator, preparing to enter the plate stage.
The equatorial portion of the nucleus approaches very near the wall of the egg,
and above this line the small cap of cytoplasm is dense and full of fibres.

Fig. 24. ( X 400.) The lower pole of the mature spindle is here represented.
The fibres come to a definite point, and, in this case, focus on a small hyaline
granule, which shows none of the properties of a oentrosome.

Fig. 25. ( X 400.) The fibres of the mature spindle also ccnverge to a definite,
though rather abrupt, pomt at the upper pole, from which supporting fibres extend
to the cell-wall. The remainder of the nuclear membrane also appears to function
as a support to the spindle during metakinesis.

Fig. 26. ( X 800.) Separation of the chromosomes has begun at the nuclear
plate, the bundles of nutntle-fibres being attadted on opposite sides of the diamond-
shaped openings in the chromosomes already seen in earlier stages.

Figs. 27, 28. ( X 600.) The daughter-chromosomes pass to the poles as U's or
V's with undulated margins. After they reach the pnles, the central apindle-fibras
appear lax, and the spindle becomes slightly concave in the equatorial region.
This may be due to artificial cause* or to relaxation after removal of the strain.

Fig. 39. (X400.) The daughter-chromosomes have united into a close, dee[dy>
stainhig spirem, but no membrane is yet formed about theiA. The inner spindle*
fibres are beginning to thicken in the equatorial region preparatory to the formation
of the cell-plate.

Fig. 30. (x 400.) The diqiirems have opened ont, bat their loops still
maintain a position parallel with the axis of the spindle. Delicate nuclear

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and Ferltlizalion in the Hemlock Spruce, 605

bianet have been depoiited abont the danghter-nuclei, while the latt tracet of the
membrane of the mother-nucleut have disappeared. The large dente matt of
cytoplaim at the lower pole, however, still remaint, and is present even when the
egg-nncleni begint to past down. The formation of the cell-plate it proceeding
fori passu with the increase in diameter of the daughter-nuclei.

Fig. 31. (x 400.) The danghter-nuclei have reached the resting stage, and
thow a delicate reticnlnm with teveral nucleoli. The egg-nucleus it somewhat
larger than that of the ventral canal-cell. The cell-plate hat been continued to
the wall of the mother-cell.

PiK- 3'- ( >< 400.) The egg-nncleut it now not only larger than the ventral
canaUcell nucleus, but itt reticulum is growing more rapidly. The chromatic
contents of the nucleus are mottly confined to itt upper portion, the lower part
containing chiefly nuclear sap.

P'S- .13- ( '*'■ 4°oO "^^ reticulum of the egg-nncleus has become coarser
and the nucleoli larger. The nucleus has alto changed itt form from ipherical to
elliptoidal, and bat begun to move through the dente polar cytoplatm toward
the centre of the egg. The larger granules of the geneml cytoplasm are
arranged in radial rows abont the nucleus, and the grannies themselvet are radially
elongated.

F'S- 34- ( X >5^) At ^i> *'*8e ^'^ reticulum of the egg nncleui it distributed
throughout the entire nuclear cavity. At the archegonium it nnutually broad, the
nucleut hat retained iu spherical form. Passing it on the left is the large central
vacuole which now takes a position near the egg apex. The nucleus of the ventral
canal cell it irregular in outline, and ita contents thow tignt of disorganization.
Tracet of the divition-tpindle ttill remain above the nucleus.

PLATE XXXII.

Fig. 35- ( X 300.) The nucleus here represented it drawn from an egg into
which the contentt of the pollen-lube have just been diacharged. Situated near
the bate of the nucleus, it the very large nucleolui with its firm, deeply ttaining
outer thell broken at one point. The contenU of the nudeolut appear finely
giannlar and vacuolate in ttained preparations.

Fig- 3<i' (^ ><>°0 "I^ contentt of the pollen-tube have entered the egg
near itt apex on the right hand tide in the figure, and now lie beneath the empty
vacuole with the functional tperm-nndeut in advance, and already free from iU
cytoplatm. Near it on the left it the ttalk-cell, while between it and the tperm-
ocU Uct the vegetative nucleut. The contentt of the firtt tperm-nucleut are very
dense and stahi deeply. The egg-nndeut it apparently unaffected by the near
approach of the tperm. Jutt above the %g are the remaint of the neck-cellt and
the ventral canal-cell puhed atide by the entering pollen-tube.

Fiff- 37' ( ^ '^0 '^'^ ^^ tpeim-nnclent has flattened itielf against the
apex of the egg-nndeus in the form of a biconvex lent. Its contentt have at yet
undergone no change. The tecood iperm-nnclens remaint in itt cytoplatm above,
ihowtaig itt ictkmlam and nucleoli very dittinctly. The tmaller nuclei are in
another tection. The first speim-audcnt hat left slight traces of iU passage to the
cge^nudens ia the intervening cytoplasm. These traces are mwe distinct in

Fig. 38.
Fig. 38. (x 200.) The sperm-nudeus hat lott itt deniity, and it now a true

6o6 MurrilL — The Development of the Archegoniunt

resting nucleus like that of the egg. The membranes of the two nuclei are still
intact The changes in the two nuclei may be better described under later figures.
In this case the stalk-cell lies near the second sperm-nucleus.

Fig- 39- ( X 1600.) The stalk-cell enlarged from the section represented in
Fig. 38 to show its thick nuclear reticulum, and scanty vacuolate cytoplasm.

Fig. 40. ( X aoo.) This figure shows the first sperm-nucleus in contact with
the egg-nucleus, and the second sperm-nucleus almost touching its membrane.
The sperm-cells entered the egg unusually far down on its side, and the bursting of
the pollen-tube was sufficient to force them to this position near the egg-nucleus.
This also accounts for the contact of the first sperm-nucleus at the side instead
of the apex. The remains of the neck-cells could not be found. The egg-
membrane did not close up again after the contents of the pollen-tube entered, as
was the case in the egg represented in Fig. 36.

Fig. 41. (X 400.) This represents a stage succeeding that shown in Fig. 37.
The contents of the sperm-nucleus are losing their density, and numerous small
spheres have appeared in the nuclear cavity. Crenate folds are observed in the
membrane of the egg-nucleus at the surface of contact of the two nuclei.

Fig. 4a. (X 400.) The dense contents of the sperm-nucleus have disappeared
except at the centre, and the nucleoli are larger and fewer in number. About the
periphery of the sperm-nucleus a chromatic reticulum is seen. The chromatic
contents of the egg-nucleus have begun to migrate to a point beneath the sperm-
nucleus.

Fig. 43. ( X 400O The centre of the sperm-nucleus is quite free from chromatic
contents. The peripheral reticulum shows larger collections of chromatin.
Nearly all of the chromatin of the egg-nucleus has collected beneath the sperm-
nucleus. The crenate folds in the nuclear membrane of the egg appear larger,
particularly in the centre.

F'g- 44- C X 400.) The chromatin of the sperm seems to have moved nearer
to that of the egg. The intervening membranes are preparing to break up. The
egg-nncleus has constructed a chromatic reticulum.

Fig. 45- ( X 400.) The membranes have broken up, and the two nuclear
cavities are continuous. Small spheres with granular contents now occupy the
position formerly occupied by the membranes. The thick knotted chromatic
thread* of the two nuclei still remain distinct. All traces of nucleoli have disap-
peared, and the contents of the nuclear cavity have become denser and more
fibrous. At a few points, the outer nuclear membrane is disappearing, and the
cytoplasm is encroaching on the nuclear cavity.

Fig. 46. C X 800.) A tri-polar spindle found near the apex of the egg in the
position commonly occupied by the second sperm-nucleus.

Fig. 47. ( X 800.) Following Fig. 45, and showing the origin of the first
segmentation-spindle, which arises between and among the two groups of
chromatin. The spirems have mostly segmented at this stage, but the segmente
have not yet become homogeneous. The chromatin of the egg does not all appear
in this section. A preparation showing the two groups of chromosomes to. better
advantage was injured so that it could not be satisfactorily figured. The spindle
is at first multipolar, but soon becomes monaxial, the position of the poles
apparently bemg d«tennined by collections of a dense granular substance which
takes a diffuse reddish sUin with the Flemming combination. The contents of the

Viii nil

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and Fertilization in the Hemlock Spruce. 607

nucleus are more fibrous than before, and the nuclear membrane has disappeared,
though the limit of the nuclear cavity remains the same.

Fig. 48. ( X 800.) The spindle is now monaxial though not yet distinctly
bipolar. * The chromosomes are homogeneous, and no longer separated into two
groups. The contents of the cavity remain as shown in Fig. 47.

Fig. 49. (X 800.) This figure apparently represents a later stage than that
shown in Fig. 48, but no trace of a spindle could be found, even in very deeply
stained preparations. It must either be considered as a cross-section in which
the spindle fibres are, for some reason, not easily observed, or the spindle is
unusually late in forming.

Fig. 50. ( X 800.) The spindle of the first segmentation at metakinesis. The
division is typical. No centrosomes are present.

Fig. 51. ( X 800.) Showing a cross-section of the same.

f'g- 5*> (x »ooO Same as represented in Fig. 50, but showing a section
through the entire egg. The division is here oblique, while in Fig. 56 it is
represented as longitudinal, and in Fig. 57 as transverse to the axis of tlic
archegonium.

Fig. S3. ( X 800.) The daughter-chromosomes of the first segmentation ap-
proaching the poles in the form of U's and Vs. By counting them in several
preparations at this stage, their number was found to be twenty-four. The fibres
of the central spindle appear twisted and somewhat thickened at the equator, but
they soon disappear without the formation of a cell-plate.

Fig- 64' (>< '00.) The young daughter-nuclei resulting from the first seg-
mentation.

F'g- 55' (X 'o°0 The same at an older stage with the chromatin in the
'resting condition. The cytoplasm between the nuclei is finely granular. Note the
remains of the ventral canal-cell at the apex of the egg.

Fig. 56. ( X 800.) A spindle of the second segmentation at metakinesis. It is
narrower and more pointed than the first segmentation spindle. The chromatin
divides in the same manner, and there are no centrosomet present.

F'g' S7- (>< '0°*) A fertilized egg containing two nuclei in simultaneous
division. The stage of division is the same as that shown in Fig. 56.

Fig- 58. ( X 100.) The second segmentation is complete, and the four resulting
nuclei are equal in size and without separating walls.

Fig. 59. ( X 100.) The four nuclei are moving to the base of the archegonium.
Their chromatic contents are delicate and scanty. There is no special collection
of fibres about the nuclei. The archegonium is cut obliquely so that the position
and size of the nuclei are not truly represented in the figure.

Fig. 60. ( X 100.) The four nuclei have reached the base and are arranged
horizontally in one plane. A dense fibrous substance surrounds them. Some
distance above the plane of the nuclei is a zone showing a regular reticulum quite
ftee from staining contents. Above this zone the normal contents of the egg are
observed deeply stained.

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