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Title: USDA Bulletin No. 844 - Sweet-Clover Seed
Author: Coe, H. S., Martin, J. N.
Language: English
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Text emphasis is denoted as _Italics_.



                UNITED STATES DEPARTMENT OF AGRICULTURE

                           BULLETIN No. 844

            Contribution from the Bureau of Plant Industry

                         WM. A. TAYLOR, Chief


     Washington, D. C.     PROFESSIONAL PAPER      August 11, 1920


                           SWEET-CLOVER SEED

Part I.--Pollination Studies of Seed Production

Part II.--Structure and Chemical Nature of the Seed Coat and its
Relation to Impermeable Seeds of Sweet Clover

By

H. S. COE, formerly Assistant Agronomist, Office of Forage-Crop
Investigations, and J. N. MARTIN, Professor of Morphology and Cytology,
Iowa State College

CONTENTS

                                                                  Page
  Part I.--Pollination Studies of Seed Production.
    Unsatisfactory yields of sweet-clover seed                       1
    Previous investigations of the pollination of sweet clover       2
    Outline of pollinating experiments                               3
    Structure of the flowers of Melilotus alba                       4
    Development of the floral organs of sweet clover                 5
    Fertilization in Melilotus alba                                  8
    Development of the seed                                          8
    Mature pollen of sweet clover                                    9
    Germination of the pollen                                        9
    Cross-pollination and self-pollination of sweet clover          10
    Artificial manipulation of sweet-clover flowers                 10
    Seed production of Melilotus alba under  ordinary field
        conditions                                                  13
    Efficiency of certain kinds of insects as pollinators of
        sweet clover                                                14
    Relation of the position of the flowers on Melilotus alba
        plants to seed production                                   19
    Influence of the weather at blossoming time upon seed
        production                                                  20
    Insect pollinators of sweet clover                              21
    Effect of moisture upon the production of Melilotus alba seed   22

  Part II.--Structure and Chemical Nature of the Seed Coat
      and its Relation to Impermeable Seeds of Sweet Clover.
    Historical summary                                              26
    Material and methods                                            30
    Structure of the seed coat                                      31
    Microchemistry of the seed coat                                 33
    The seed coat in relation to the absorption of water            34
    A comparison of permeable and impermeable seed coats            34
    The action of sulphuric acid on the coats of impermeable seeds  35

  Literature Cited                                                  36

[Illustration]


WASHINGTON GOVERNMENT PRINTING OFFICE

1920

                UNITED STATES DEPARTMENT OF AGRICULTURE

                           BULLETIN No. 844

            Contribution from the Bureau of Plant Industry

                         WM. A. TAYLOR, Chief


     Washington, D. C.     PROFESSIONAL PAPER      August 11, 1920



                           SWEET-CLOVER SEED

Part I.--Pollination Studies of Seed Production

Part II.--Structure and Chemical Nature of the Seed Coat and its
Relation to Impermeable Seeds of Sweet Clover


By H. S. Coe, _formerly Assistant Agronomist, Office of
Forage-Crop Investigations_, and J. N. Martin, _Professor of
Morphology and Cytology, Iowa State College_.



CONTENTS


                                                                  Page
  Part I.--Pollination Studies of Seed Production.
    Unsatisfactory yields of sweet-clover seed                       1
    Previous investigations of the pollination of sweet clover       2
    Outline of pollinating experiments                               3
    Structure of the flowers of Melilotus alba                       4
    Development of the floral organs of sweet clover                 5
    Fertilization in Melilotus alba                                  8
    Development of the seed                                          8
    Mature pollen of sweet clover                                    9
    Germination of the pollen                                        9
    Cross-pollination and self-pollination of sweet clover          10
    Artificial manipulation of sweet-clover flowers                 10
    Seed production of Melilotus alba under  ordinary field
        conditions                                                  13
    Efficiency of certain kinds of insects as pollinators of
        sweet clover                                                14
    Relation of the position of the flowers on Melilotus alba
        plants to seed production                                   19
    Influence of the weather at blossoming time upon seed
        production                                                  20
    Insect pollinators of sweet clover                              21
    Effect of moisture upon the production of Melilotus alba seed   22

  Part II.--Structure and Chemical Nature of the Seed Coat
      and its Relation to Impermeable Seeds of Sweet Clover.
    Historical summary                                              26
    Material and methods                                            30
    Structure of the seed coat                                      31
    Microchemistry of the seed coat                                 33
    The seed coat in relation to the absorption of water            34
    A comparison of permeable and impermeable seed coats            34
    The action of sulphuric acid on the coats of impermeable seeds  35

  Literature Cited                                                  36



Part I.--POLLINATION STUDIES OF SEED PRODUCTION.


UNSATISFACTORY YIELDS OF SWEET-CLOVER SEED.

In some sections of the country much trouble has been experienced for
a few years past in obtaining satisfactory yields of sweet-clover
seed. This difficulty has been due for the most part to the following
causes: (1) To cutting the plants at an improper stage of development,
(2) to the use of machinery not adapted to the handling of the crop,
(3) to the shedding of immature pods, and (4) possibly to the lack of
pollination. As the first two have been overcome, mainly because of a
better understanding of the requirements for handling this crop, the
subject matter of this bulletin is concerned primarily with the factors
which produce the third and fourth causes.

Where the production of seed was disappointing although the plants
produced an abundance of flowers, it has been observed that many
apparently were not fertilized, or if fertilized the pods aborted. In
order to obtain data in regard to the causes of the failure of sweet
clover to produce a normal seed yield, a study was made of the insects
which were most active in pollinating the flowers, the source of the
pollen necessary to effect fertilization, and the conditions under
which the flowers must be pollinated in order to become fertilized.
The relation of environmental conditions to the shedding of immature
pods was also investigated. In order to overcome local environmental
factors as much as possible, the experiments were conducted on the
Government Experiment Farm at Arlington, Va., and in cooperation with
the botanical department of the Iowa State College at Ames, Iowa.


PREVIOUS INVESTIGATIONS OF THE POLLINATION OF SWEET CLOVER.

Since Darwin (4, p. 360)[1] published the statement that a plant of
_Melilotus officinalis_ protected from insect visitation produced but
a very few seeds, while an unprotected plant produced many, other
scientists have investigated this subject. Knuth (19, v. 1, p. 37),
in giving a list of the best known cases of self-sterility in plants,
mentions _Melilotus officinalis_. The same author (19, v. 2, p. 282)
states that since the stigma projects beyond the anthers, automatic
self-pollination is difficult, and for the same reasons Müller (29, p.
180) believes that self-fertilization is not apt to occur.

[1] The serial numbers in parentheses refer to "Literature cited,"
pages 36-38.

In 1901 Kirchner (18, p. 7) covered a number of _Melilotus alba_
racemes with nets. On one of the plants 12 protected racemes produced
187 seeds and on another plant only one seed was obtained from
10 covered racemes. This experiment was duplicated in 1904, with
the result that 40 netted racemes produced an average of 38 seeds
each. Kirchner concluded from this experiment that spontaneous
self-pollination occurs regularly even though the stigma projects above
the anthers. He (18, p. 8) also performed an experiment with _Melilotus
officinalis_ in 1901. At this time 16 isolated racemes produced a total
of 11 seeds. This experiment was repeated in 1904, with the result
that 16 protected racemes produced an average of 14 seeds each. As
the racemes on one of the plants that was protected in 1904 died,
Kirchner concluded that the flowers of _M. officinalis_ were especially
sensitive to inclosure in nets and that the failures to obtain more
than a very few seeds on protected racemes in Darwin's experiment and
in his first experiment were due to this cause.

According to Kerner (17, v. 2, p. 399) the peas and lentils (Pisum and
Ervum) and the different species of horned clover and stone clover
(Lotus and Melilotus) as well as the numerous species of the genus
Trifolium and also many others produce seeds when insects are excluded
from the plants, and only isolated species of these genera gave poor
yields without insect visitation.


OUTLINE OF POLLINATING EXPERIMENTS.

The yield of sweet-clover seed varies greatly from year to year in many
parts of the United States. It has been assumed that this variation
was due to climatic conditions, as excellent seed crops were seldom
harvested in seasons of excessive rainfall or of prolonged drought just
preceding or during the flowering period. The lack of a sufficient
number of suitable pollinating insects also was thought to be an
important factor in reducing seed production. This was especially true
where the acreage of sweet clover was large and where few, if any,
honeybees were kept.

In order to obtain data upon the factors influencing the yield of
seed, a series of experiments was outlined to determine (1) whether
the flowers are able to set seed without the assistance of outside
agencies, (2) whether cross-pollination is necessary, (3) the different
kinds of insects which are active agents in pollinating sweet clover,
and (4) whether a relation exists between the quantity of moisture in
the soil and the production of seed.

The racemes containing the flowers which were to be pollinated by hand
were covered with tarlatan before any of the flowers opened and were
kept covered except while being pollinated until the seeds were nearly
mature. This cloth has about twice as many meshes to the linear inch as
ordinary mosquito netting and served to exclude all insects that are
able to pollinate the flowers. When entire plants were to be protected
from all outside agencies, cages covered with cheesecloth, glass
frames, or wire netting were used.

A preliminary study of the pollination of _Melilotus alba_ and _M.
officinalis_ showed that both were visited by the same kinds of insects
and that both required the same methods of pollination in order to set
seed. On this account _M. alba_ was used in most of the experiments
reported in this bulletin. Where _M. officinalis_ was employed it is so
stated.


STRUCTURE OF THE FLOWERS OF MELILOTUS ALBA.

[Illustration: Fig. 1.--Different parts of the flower of
_Melilotus alba_: 1, Side view of the flower; 2, side view of the
flower with the carina and alæ slightly depressed; 3, side view of the
flower, showing the carina and alæ depressed sufficiently to expose
the staminal tube and the tenth free stamen; 4, ala; 5, ate and carina
spread apart to show their relative position and shape; 6, flower after
the petals have been removed, showing in detail the calyx and staminal
tube; 7, the staminal tube split open to show the relative size and
position of the pistil, _a_, Alæ; _b_, vexillum; _c_, carina; _d_,
calyx; _c_, stigma; _b_, anthers: _g_, tenth free stamen; _h_, digitate
process of the superior basal angle of an ala; _i_, depressions in the
ala; _j_, staminal tube; _k_, pistil.]

The racemes of _Melilotus alba_ contain from 10 to 120 flowers with
an average of approximately 50 per raceme for all of the racemes of a
plant growing under cultivation in a field containing a good stand.

The flower consists of a green, smooth, or slightly pubescent calyx
with 5-pointed lobes and with an irregular white corolla of five
petals. (Fig. 1.) The claws of the petals are not united nor are they
attached to the staminal tube which is formed by the union of the
filaments of the nine inferior stamens. As the claws of the alæ and
carina are not attacked to the staminal tube; the petals may be bent
downward sufficiently far so that many different kinds of insects may
secure without difficulty the nectar secreted around the base of the
ovary.

The fingerlike processes of the alæ are appressed closely to the
carina, therefore the alæ are bent downward with the carina by insects.
These processes grasp the staminal tube superiorly and tightly when
the carina and alæ are in their natural positions, but when the carina
is pressed downward by insects the fingerlike processes open slightly
but not so far that they do not spring back to their original position
when the pressure is removed. The staminal tube splits superiorly to
admit the tenth free stamen. The filament of this superior stamen lies
along the side of this staminal tube. The filaments of the nine stamens
which compose the staminal tube separate in the hollow of the carina.
All stamens bear fertile anthers. The pistil is in the staminal tube,
the upper part of the style and stigma of which is inclosed with the
anthers in the carina. The stigma is slightly above the stamens.

An insect inserts its head into a sweet-clover flower between the
vexillum and carina, the stigma, therefore, comes into direct contact
with the head of the insect and cross-pollination is effected. At the
same time the anthers brush against the insect, so that its head is
dusted with pollen, to be carried to other flowers.


DEVELOPMENT OF THE FLORAL ORGANS OF SWEET CLOVER.

[Illustration: Fig. 2.--Lengthwise sectional view of a very
young flower of _Melilotus alba_, showing the relative development of
the stamens and pistil. In the upper set of stamens the divisions of
the mother cells are completed, while division is just beginning in
the lower set of stamens. In the ovules the outer integuments are well
started on their development, _a_, Anther; _o_, ovule; _p_, pistil. ×
38.]

The stamens of _Melilotus alba_ and _M. officinalis_ may be divided
into two sets, according to their length and time of development.
(Fig. 2.) The longer set extends about the length of the anthers
above the shorter set, and the pollen mother cells in the longer set
divide to form pollen grains at least two days earlier than those
in the shorter set. At the time the pollen mother cells divide, the
longer set of stamens is approximately three-eighths of a millimeter
in length and the pistil about half a millimeter long. The stigma and
a portion of the style project beyond the stamens, and this relative
position is maintained to maturity. The pollen mother cells undergo the
reduction division while the megaspore mother cells are just being
differentiated and while the outer integuments are barely prominent at
the base of the nucellus. The pollen grains are formed while the embryo
sac is beginning to develop. The division of the megaspore mother cell
does not occur until a number of days later, and the embryo sac is
not mature until the flower is nearly ready to open. Thus, the pollen
grains are formed a week to 10 days before the embryo sac is ready for
fertilization. The pollen grains increase in size and undergo internal
changes after their formation. These changes, which are not completed
until the flower is one-half or more of its mature length, may be
regarded as the ripening processes, and they are undoubtedly necessary
before the pollen is capable of functioning. For this reason it is
probable that the pollen grains are not able to function much before
the embryo sac is mature.

[Illustration: Fig. 3.--Stigma at the time of pollination,
showing its papillate character and the position of the pollen in
reference to the papillæ in pollination. × 175.]

The pistils of _Melilotus alba_ and _M. officinalis_ are straight for
the greater part of their length, but curve rather abruptly toward
the keel just below the capitate stigma. The surface of the stigma is
papillate. (Fig. 3.) In their reaction with Sudan III, alkanin, and
safranin the Walls of the papillæ of the stigma show that some fatlike
substances are present. Aside from water, the contents of the papillæ
consist chiefly of a fine emulsion of oil.


DEVELOPMENT OF THE OVULES.

The number of ovules in the ovary of _Melilotus alba_ varies from
two to five; however, most commonly, three or four ovules occur. In
_Melilotus officinalis_ the number in each ovary ranges from three to
six. In both species the ovules are campylotropous at maturity with the
micropylar end turned toward the base of the ovary.

Mature ovules contain two integuments, but the inner one does not close
entirely around the end of the nucellus. The outer integument develops
considerably ahead of the inner one. The outer integument is much
thickened at the micropylar end, the seed coat is formed from it, and
the inner integument is used as nourishment by the endosperm and embryo.

The number of megaspore mother cells in an ovule varies from one to
many. Two or more embryo sacs often start to develop in the same ovule,
but seldom more than one matures. (Pl. I, figs. 1, 2, and 3.) In
general, the development of the embryo sac proceeds in the ordinary
way, as described by Young (44, p. 133), with the inner megaspore
functioning. (Text fig. 4 and Pl. II, fig. 1.) In its development
the nucellus is destroyed rapidly, the destruction being most rapid
first at the micropylar end proceeding backward. The nucellus is
completely destroyed at the micropylar end by the time the embryo sac
is mature, and consequently the embryo sac comes in contact with the
outer integument in this region. (Pl. II, fig. 1.) As the destruction
of the nucellus extends toward the chalazal end the embryo sac becomes
much elongated and tubelike. The antipodals disappear early, so that
a mature embryo sac consists of the egg, the synergids, and the two
polars. The two polars lie in contact in the micropylar end of the sac
near the egg until fertilization.


STERILITY OF THE OVULES.

In _Melilotus alba_ and _M. officinalis_ there is very little tendency
toward sterility of ovules. In an extended study of ovules developing
under normal and under excessive moisture conditions only an occasional
one was found in which no reproductive cells were differentiated, and
no ovaries were found in which all of the ovules were sterile.

[Illustration: Fig. 4.--Median section through an ovule,
showing the embryo sac with four nuclei and the position of the
integuments. × 150.]


DEVELOPMENT OF THE POLLEN.

The pollen mother cells do not separate, but previous to the reduction
division the protoplasm shrinks from the walls, thus forming a dense
globular mass which often occupies less than half the lumen of the
mother cell. (Pl. I, fig. 4.) Nuclear division occurs while they are
in this contracted condition, and four nuclei are formed from two
successive divisions. The cytoplasm is equally distributed around each
nucleus. The four masses of protoplasm separate, and as they enlarge
a number of times and develop into mature pollen grains they become
binucleate, and a wall is gradually formed around each. (Pl. I, figs. 5
and 6.) At first the cytoplasm is quite dense and contains some starch
but no fatty oils. However, the cytoplasm of mature pollen grains is
vacuolate, and it contains a fatty oil in the form of an emulsion.
Soon after the pollen grains are formed, the walls of the mother cells
disappear, thus permitting the pollen grains to lie loose in the anther.


FERTILIZATION IN MELILOTUS ALBA.

The time intervening between pollination and fertilization was
investigated with both self-pollinated and cross-pollinated flowers.
In cross-pollination the parents were separate plants. This point was
investigated with plants out of doors during the summer of 1916 and
with plants in the greenhouse during the following winter. The time
elapsing between pollination and fertilization ranged from 50 to 55
hours and was not longer in the case of self-pollinated than with
cross-pollinated flowers. Furthermore, the rate of the development of
the embryo in each kind of pollination was studied and was found to
be as rapid in self-pollination as in cross-pollination. Therefore,
self-pollination is apparently as effective as cross-pollination in
_Melilotus alba_ so far as the vigor of pollen tubes and the rate at
which embryos develop are concerned. _Melilotus officinalis_ was not
studied in reference to this point.

Considerable difference often exists in the size of the young embryos
in the ovules of the same pod. This is due in part to a difference in
the time of fertilization, although some of it is due to a difference
in nourishment. It was observed that the ovule first fertilized might
be an upper one, lower one, or any one between these. Occasionally one
or more ovules are not fertilized.


DEVELOPMENT OF THE SEED.

A proembryo with a rather long suspensor is developed from the
fertilized egg. (Pl. II, fig. 2). The endosperm, which quite early
forms a peripheral layer around the entire embryo sac, develops most
rapidly about the embryo, which soon becomes thoroughly embedded in it.
(Pl. III, figs. 1 and 2.) After the embryo has used up the endosperm in
the micropylar end and has enlarged so much as to occupy nearly all of
the space in this region, the development of the endosperm becomes more
active in the chalazal end, and when the embryo is mature there is very
little endosperm left.

The seed coat begins to form about the time of fertilization,
although it apparently does not depend upon it, for in ovules where
fertilization is prevented the outer integument undergoes the early
modifications in the development of the seed coat before the ovule
breaks down. The development of the seed coat is apparent first at
the micropylar and chalazal ends, where the outer cells of the outer
integument become much elongated and their outer walls thicken very
soon after fertilization. The modifications in the development of
the seed coat extend around the ovule from these points, involving
at first only the outer or epidermal layer of cells which form the
malpighian layer. Later, the cells just beneath the malpighian layer
form the osteosclerid layer. Accompanying or closely following the
formation of the osteosclerid cells, the remaining cell layers of the
outer integument become modified into the nutritive and aleurone layer,
and the seed coat is fully formed. Meantime the inner integument is
practically all used as food.

Plate I.

[Illustration]

Development of the Ovules and Pollen in Sweet Clover.

Fig. 1.--Section through the nucellus of an ovule of
_Melilotus alba_, showing two megaspore mother cells. × 360. Fig.
2.--Median section through an ovule of _Melilotus alba_, showing the
two cells resulting from the first division of the megaspore mother
cell, and the relative development of the different parts of the ovule.
× 300. Fig. 3.--Section through the nucellus of an ovule of _Melilotus
alba_, showing two embryo sacs, one being more advanced than the other.
× 360. Fig. 4.--Protoplasm of the pollen mother cell of _Melilotus
alba_ contracted and ready to undergo division. × 560. Fig. 5.--Pollen
grains of _Melilotus alba_ just formed, showing their dense cytoplasm
and the presence of the mother-cell wall. × 560. Fig. 6.--_a_, Mature
pollen grain of _Melilotus alba_, showing the binucleate condition at
the time of shedding; _b_, surface view. × 560.


Plate II.

[Illustration]

Fig. 1.--Median Section through an Ovule of Melilotus alba.

The embryo sac is shown ready for fertilization. The egg and synergids
are in contact with the outer integument at the micropylar end. The
remains of the antipodals may be seen at the chalazal end. × 180.

[Illustration]

Fig. 2.--Section through an Ovule of Melilotus alba, about Three
Days After Fertilization.

The proembryo, the endosperm, and modifications of the integuments are
shown. At this stage the suspensor prominent part of the proembryo, and
the endosperm is most abundant around the embryo. The inner integument
is being rapidly destroyed, and the outer integument is beginning to
form the seed coat, as is indicated by the modifications in the outer
layer of its cells, which are elongating and thickening their outer
walls. × 33.


Plate III.

[Illustration]

Fig. 1.--Section of an Ovule of Melilotus alba after
Fertilization.

The stage of development is a little later than that shown in Plate II,
figure 2. The embryo is embedded deeply in endosperm tissue. × 45.

[Illustration]

Fig. 2.--Section through an Ovule of Melilotus alba after the
Embryo is Nearly Half Mature.

But little endosperm remains except in the chalazal end, and very
little remains of either the nucellus or inner integument. The
modifications which transform the outer integument into a seed coat are
well under way. Not only the outer layer of cells which becomes the
Malpighian layer is quite well modified, but also the layer beneath is
being transformed into the osteosclerid layer. × 30.


Plate IV.

[Illustration]

Stubble of Melilotus alba.

These plants, which were cut 12 inches above the ground during rainy
weather, had made a 40 to 42 inch growth. The stubble became infected
at the top and the light-colored portions of them were killed by
disease, thus checking the water supply to the growing branches above
the infection.


MATURE POLLEN OF SWEET CLOVER.

The pollen grains of _Melilotus alba_ and of _M. officinalis_ are quite
similar. Each grain contains three germ pores, and when viewed so
that the pores are visible they present a slightly angled appearance.
The average dimensions of the pollen of _Melilotus alba_ and of _M.
officinalis_ are 26 by 32 microns and 24 by 30 microns, respectively,
when measured in the positions shown in _b_ in Plate I, figure 6.

The walls of the pollen grains have cutin deposited in them, as shown
by their reactions with Sudan III, alkanin, safranin, and chloriodid of
zinc. The contents of the pollen grains give a distinct reaction when
tested for fat, and Millon's reagent shows that also some protein is
present. Tests for sugars and starch showed that these substances are
not present in perceptible quantities in mature pollen grains, although
some starch is present in immature pollen.


GERMINATION OF THE POLLEN.

The germination of the pollen of _Melilotus alba_ permits considerable
variation in moisture, as is illustrated in Table I.

Table I.--_Germination of the pollen of Melilotus alba in water and in
solutions of cane sugar of different strengths._

  ---------------+--------+---------------------------------------
                 |        | Cane sugar in solution (per cent).
                 | Pure   +----+----+----+----+----+----+----+----
  Melilotus alba.| water. |  8 | 12 | 18 | 24 | 30 | 35 | 45 | 55
  ---------------+--------+----+----+----+----+----+----+----+----
  Germination    |        |    |    |    |    |    |    |    |
    of pollen    |   33   | 23 | 64 | 46 | 60 | 46 | 31 | 22 |  0
      per cent   |        |    |    |    |    |    |    |    |
  ---------------+--------+----+----+----+----+----+----+----+----

The results given in Table I represent the average of 12 tests. Some
of the pollen grains burst in pure water and in the weak cane sugar
solutions, the percentage of bursting being greatest in pure water and
decreasing as the percentage of sugar in the solution was increased.
There was considerable variation in the percentages of germination in
both water and in the solutions of different strengths, and at times
there was very little bursting which was not accompanied by a high
percentage of germination. The pollen tubes grew as rapidly in water as
in any of the sugar solutions, some reaching a length of 100 microns
in six hours. As the pollen tubes made no more growth in the solutions
of sugar than in water, it is evident that the sugar is not used as
food, but helps in germination by reducing the rate at which water is
absorbed.

To judge from Table I, the pollen of sweet clover can be effective not
only under ordinary conditions but also when the flowers are wet with
rain or dew or when the stigma is so dry that in order to obtain water
from the papillæ the pollen must overcome a high resistance offered by
the sap of the papillæ, a resistance that may be equal to the osmotic
pressure of a 45 per cent solution of cane sugar. This is in accord
with results obtained under field conditions; as flowers that were
pollinated while rain was falling set seed satisfactorily, indicating
that a high percentage of humidity in the atmosphere does not check the
germination of the pollen sufficiently to interfere with fertilization.
Neither was the setting of seed affected when the soil about the roots
of plants was kept saturated with water, showing that the excessive
quantity of water in the stigmas resulting from an abundance of water
in the soil did not interfere with the fertilization of the flowers.

No definite counts were made of the germination of the pollen of
_Melilotus officinalis_ in the solutions of cane sugar of different
strengths, but observations show that the moisture requirement of the
pollen of this species is approximately the same as that of _Melilotus
alba_.


CROSS-POLLINATION AND SELF-POLLINATION OF SWEET CLOVER.

Results published by previous investigators on the cross-pollination
and self-pollination of sweet clover do not agree. The experiments of
Darwin (4) show that the flowers are self-pollinated to only a small
extent. On the other hand, Kirchner (18) and Kerner (17) find that
self-pollination occurs generally and that cross-pollination is not
necessary for the production of seed. However, all investigators agree
that many different kinds of insects are able to pollinate sweet clover.

Because of the diverse opinions as to the pollination of sweet clover,
a number of experiments were conducted to determine (1) whether insect
visitation was necessary to pollinate the flowers, (2) if necessary,
whether the flowers must be cross-pollinated, and (3) what insects are
active agents as pollinators of sweet clover.

ARTIFICIAL MANIPULATION OF SWEET-CLOVER FLOWERS.[2]

[2] The writers wish to acknowledge their indebtedness to Mr. Carl
Kurtzweil for assistance in conducting part of the field experiments at
Ames.

Experiments were conducted to determine, if possible, the effect of
various types of artificial manipulation of sweet-clover flowers when
in full bloom on the production of seed. Only healthy, vigorous plants
growing on well-drained soil were selected for these experiments.
Before any of the flowers were open, the individual racemes were
covered with tarlatan and labeled. (Fig. 5.) As soon as part of the
flowers opened, the racemes were uncovered and after removing all
flowers that were not open the open flowers were pollinated and the
racemes re-covered. If the flowers of sweet clover are not fertilized
they will remain open for two to three days, then wither, and in a
short time drop. But after being fertilized the ovules enlarge very
rapidly, and the corollas usually drop in about seven or eight days.
Therefore, all fertilized flowers can be distinguished a few days after
fertilization has taken place. Counts were made of the number of pods
which formed in 10 to 12 days after pollination. An outline of the
experiments is given in Table II.

[Illustration: Fig. 5.--Individual racemes of white sweet
clover covered with cheesecloth to protect them from insect visitation.]

Table II.--_Treatment of sweet-clover flowers in the
artificial-manipulation experiments._

  ------------+---------------------------------------------------------
  Experiment. |          Method of pollinating the flowers.
  ------------+---------------------------------------------------------
    A         | Check--covered.
              |
    B         | Check--open to insect visitation at all times.
              |
    C         | A separate toothpick was used to spring the keel of each
              |   flower on the raceme.
              |
    D         | One toothpick was used to spring the keels of all the
              |   flowers on a raceme.
              |
    E         | Cross-pollinated.
              |
    F         | Raceme rolled several times between thumb and finger.
  ------------+---------------------------------------------------------

As insects, and especially honeybees, usually visit all recently
opened flowers on a raceme, experiments C and D were conducted to
determine whether more seed would be produced when pollen from other
flowers on the same raceme was placed on the stigmas of the flowers
than when only the pollen produced by each flower was placed on its
own stigma. The effect of pollination when only the pollen produced by
an individual flower was placed on its own stigmas was also obtained
in experiment F, as by this method of pollination no pollen was
transferred from one flower to another. It can not be stated definitely
that the seed produced by the cross-pollinated flowers was the result
of fertilization with foreign pollen, as the anthers were not removed
from the flowers pollinated because it would be necessary to remove
the anthers when the flowers were not more than two-thirds mature, and
in doing this the flowers would be so mutilated that only occasionally
would pollination at this time or at a later date be effective. The
flowers listed in experiment E were pollinated a short time before they
opened, and in each case pollen taken from flowers of other plants
was placed on the stigmas. The petals of the cross-pollinated flowers
were not mutilated, and in each case they returned to their original
positions soon after pollination. The results obtained in experiment B,
where the racemes were simply labeled and left open to the action of
insects at all times, serve for comparison with other experiments where
the flowers were protected from insect visitation and were artificially
manipulated.

Martin (25) found the setting of alfalfa seed and Westgate (40)
found the setting of red-clover seed to be affected by an excessive
quantity of moisture in the soil or atmosphere. In order to overcome
the possible effect of this or of other detrimental factors, in each
experiment only the flowers on a certain number of racemes were
pollinated at one time. All of the experiments were repeated a number
of times during the months of July and August, 1916, and the results
given in Table III show the total number of flowers pollinated and the
number of pods that formed during the two months.

The results presented in Table III show that flowers fertilized with
pollen transferred from another plant produced a higher percentage
of pods than any of the other treatments. The results obtained in
experiment D, where the same toothpick was used to spring the keels
of all the flowers on a raceme, show that this method of pollination
produced an average of 7.24 pods per raceme more than the racemes in
experiment C. where a separate toothpick was used for each flower.
These results indicate that pollen transferred from one flower to
another on the same raceme is more effective than when the pollen
produced by an individual flower is used to fertilize its own stigma.
However, the results of experiment C prove that self-pollination
is effective in _Melilotus alba_. In experiment B. which was the
open check, 4.3 per cent more flowers set seed than on the racemes
where the same toothpick was used to spring all the keels, but 11.57
per cent more seed was obtained than in experiment C. Spontaneous
self-pollination occurs to only a very small extent, as will be seen
from the results of experiment A, in which an average of only 2.9 per
cent of the flowers set seed.

Table III.--_Effect of different types of artificial
manipulation on the seed production of sweet clover at Arlington, Va.,
and at Ames, Iowa, in 1916._

  ----------+--------+---------------------------+-------------------
            |        |    Total number of--      | Flowers that set
            |        |                           |  seed (per cent).
            | Experi-+--------+--------+---------+---------+---------
  Location. |  ment. |Racemes.|Flowers.|Pods set.| At each |
            |        |        |        |         | station.| Average.
  ----------+--------+--------+--------+---------+---------+---------
            |        |        |        |         |         |
  Arlington |    A   |    49  | 3,510  |    144  |     4.1 |}     2.9
  Ames      |    A   |    84  | 4,536  |     92  |     2.0 |}
            |        |        |        |         |         |
  Arlington |    B   |   100  | 5,599  |  3,973  |   70.95 |}   66.51
  Ames      |    B   |   196  | 1,276  |    600  |   47.02 |}
            |        |        |        |         |         |
  Arlington |    C   |    50  | 1,229  |    701  |   57.03 |}   54.94
  Ames      |    C   |    75  |   289  |    133  |   46.02 |}
            |        |        |        |         |         |
  Arlington |    D   |    50  | 1,480  |    936  |   63.24 |}   62.18
  Ames      |    D   |    88  |   575  |    342  |   59.47 |}
            |        |        |        |         |         |
  Arlington |    E   |    31  |   377  |    307  |   81.43 |}   70.10
  Ames      |    E   |    48  |   175  |     80  |   45.71 |}
            |        |        |        |         |         |
  Arlington |    F   |    30  |   933  |    524  |   56.16 |.........
  ----------+--------+--------+--------+---------+---------+---------


SEED PRODUCTION OF MELILOTUS ALBA UNDER ORDINARY FIELD CONDITIONS.

The production of seed of _Melilotus alba_ under ordinary field
conditions varies considerably, not only in different parts of the
country but also on different fields in the same region. A number
of factors contribute to this variation, one of the most important
of which appears to be the inability of the plant to supply all the
developing seed with sufficient moisture, causing some of them to
abort. As pointed out on page 22 this condition was very marked in
certain parts of the country in 1916. However, poor seed production
is not always correlated with lack of moisture, for the seed crop
was a failure in 1915, where cloudy and rainy weather prevailed much
of the time the plants were in bloom. It is believed that the lack
of pollination by insects was the principal cause for the failure of
seed to set, as very few insects visit sweet-clover flowers when such
conditions prevail. As sweet-clover pollen will germinate in pure
water and as plants which have their roots submerged in water set seed
abundantly when pollinated, the failure of the seed crop in 1915 was
not due to excessive moisture.

As a rule, thin stands of sweet clover produce more seed to the acre
than thick stands and isolated plants more seed than those growing
in either a thick or thin stand. The correlation of seed production
with the thickness of stand is probably due to the shading and partial
prevention of insect visitation to part of the racemes on the lower
branches. Most of the flowers upon the lower branches of isolated
plants are directly exposed to sunlight and to insect visits: therefore
the racemes on these branches produce as large a percentage of seed as
the racemes on the upper branches. In a thick stand, little seed is
produced by racemes on the lower branches.

A plant approximately 3 feet high growing close to the center of
a field at Arlington. Va., in which was an average stand of four
sweet-clover plants to the square foot was selected in order to
determine the number of racemes produced and the average number of
seeds to the raceme. This plant produced 196 racemes, which contained
an average of 20.4 pods each. The racemes varied from 2 to 10 cm. in
length, and the number of pods to the raceme ranged from to 75. The
racemes on the upper and most exposed portions of the plants were
larger and the flowers produced a much higher percentage of pods than
the racemes close to the bases of the larger branches. Many of the
small racemes on the lower branches produced less than five pods each.

The data obtained from the two plants at Arlington that were protected
from night-flying insects may also be cited here, as the results of
that experiment show that night-flying insects are not an important
factor in the production of sweet-clover seed, and, further. because
they were growing under the same conditions, in the same plat, and were
approximately of the same size. These two plants produced a total of
544 racemes, with an average of 20.9 pods each. The number of pods to
the raceme varied from to 86.


EFFICIENCY OF CERTAIN KINDS OF INSECTS AS POLLINATORS OF SWEET CLOVER.

In order further to test the self-sterility of sweet clover and to
determine the relative efficiency of night-flying and of different
kinds of day-flying insects as pollinators of the flowers, a number
of cages covered with cheesecloth, glass, or wire screen having 14
meshes to the linear inch were placed over plants at Arlington. Va.,
and at Ames. Iowa, in July and August. 1916. The bases of the cages
were buried several inches in the ground, so that insects could not
pass under them. Cheesecloth was used to cover most of the cages and
was made into sacks of such a size that they could be put on or removed
from the frames of the cages without difficulty. It was stretched
tightly over the frames and fastened to their bases with laths.

A cage having two sides and the top of glass but with ends covered with
cheesecloth to permit ventilation was used at Ames to protect a number
of plants from insect visitation at all times. The purpose of this
cage was to determine whether the partial shading of the plants in the
cages covered with cheesecloth would have any effect upon the setting
of seed.

The cage covered with wire netting having 14 meshes to the linear inch
was used to determine the efficiency as pollinators of sweet clover of
insects so small that they could pass through openings of this size.

The plants used in the experiments at Arlington were growing close to
the center of a field of sweet clover. Volunteer plants in a field
that contained only a scattering stand were used at Ames. The cages
were placed over the plants in all of these experiments before any of
the flowers opened, and the work was continued until they were through
blooming.

PLANTS SUBJECT TO INSECT VISITATION AT ALL TIMES.

A plant subject to insect visits at all times and growing in the same
plat as those inclosed in the cages at Arlington was selected as a
check to those inclosed in the cages during their entire flowering
period or for only a portion of it. This plant, which was in bloom at
the same time as those inclosed in the cages, produced 196 racemes with
an average of 20.4 pods each. As all of the racemes were collected and
as those on the lower portions of the plant were smaller than those
on the upper branches, the average number of seeds per raceme is much
lower than it would have been if only the larger racemes had been
collected.

An isolated plant that was subject to insect visits at all times was
selected for a check to the cage work conducted at Ames. This was
necessary in order to get results that would be comparable with those
obtained from the plants inclosed in the cages, as the cage experiments
at Ames were conducted with isolated plants. The plant produced 239
racemes, with an average of 41.6 pods.

PLANTS PROTECTED FROM INSECT VISITATION DURING THEIR ENTIRE FLOWERING
PERIOD.

On July 3, 1916, a cage 3 feet square and 3½ feet high, covered with
cheesecloth, was placed over three sweet-clover plants at Arlington.
(Fig. 6.) This cage was not opened until August 3, when practically
all of the racemes had passed the flowering stage and the few seeds
that formed on some of them were practically mature. The three plants
inclosed in the cage produced 904 racemes, with an average of 0.63 pod
each. No pods were produced on 594 racemes, while 150 produced but one
each. None of the racemes produced more than five pods.

This experiment was duplicated at Ames in August, 1916, with the result
that the three protected plants produced a total of 776 racemes, with
an average of 0.19 pod each.

[Illustration: Fig. 6.--Cage covered with cheesecloth to
protect plants from insect visitation.]

The plants inclosed at Arlington produced 0.44 pod to the raceme more
than the plants inclosed at Ames, and the average for the six plants
at Arlington and at Ames is only 0.42 pod to the raceme. Results given
below for nine plants inclosed in the glass-covered cage show that the
pods produced per raceme by different plants varied from 0.1 to 0.45,
which is slightly less than the variation in the two cages covered with
cheese-cloth.

In order to determine whether the shading of the plants in the
cheesecloth-covered cages had caused the production of seed to be
reduced, a cage 4 feet wide, 4 feet high, and 10 feet long, having
glass sides and top, but with ends covered with cheesecloth to permit
ventilation, was placed over nine plants at Ames in August, 1916. The
results obtained in this experiment are presented in Table IV.

Table IV.--_Production of sweet-clover seed by plants
protected from insect visitation during their entire flowering period
at Ames, Iowa, in 1916._



            |  Racemes  | Pods produced   | Average number of
  Plant.    | per plant.| by all racemes. | pods to the raceme.
  ----------+-----------+-----------------+-------------------
            |           |                 |
  No. 1     |      84   |         17      |       0.20
  No. 2     |     130   |         58      |        .44
  No. 3     |     166   |         30      |        .18
  No. 4     |     199   |         88      |        .44
  No. 5     |     243   |         35      |        .27
  No. 6     |     131   |         36      |        .27
  No. 7     |     119   |         13      |        .10
  No. 8     |     182   |         83      |        .45
  No. 9     |     340   |        142      |        .41
            +-----------+-----------------+-------------------
    Total   |   1,594   |        592      |      .......
  Average   |           |                 |        .31
  ----------+-----------+-----------------+-------------------


The results given in Table IV show that an average of 0.31 of a pod
to the raceme was obtained from 1,594 racemes and that the variation
in seed production of the different plants was from 0.1 to 0.45 to
the raceme. The average seed production for the nine plants is 0.11
seed to the raceme less than the average results obtained from the six
plants that were covered with cheesecloth. As this difference is well
within the limit of variation for individual plants, it may be stated
that the shading of the plants in the cheesecloth-covered cages did not
reduce the production of seed. The results of this experiment show that
spontaneous self-pollination does not occur regularly, as stated by
Kirchner.

FLOWERS POLLINATED ONLY BY NIGHT-FLYING INSECTS.

In order to determine the importance of night-flying insects as
pollinators, two cheesecloth-covered cages 3 feet square and 3½
feet high were placed over sweet-clover plants at Arlington on July
10, 1916. The covers of the cages were removed each evening at 7:30
and replaced each morning at 4:30 o'clock. Practically all the flowers
on these plants had bloomed by August 2, and the seed produced was
nearly mature. The few racemes that contained opened flowers or buds
were discarded. The three plants in one cage produced 723 racemes,
with an average of 3.76 pods each, while the one plant in the other
cage produced 227 racemes, with an average of 3.58 pods to the raceme.
The four plants, therefore, produced a total of 950 racemes, with an
average of 3.71 pods each. The only night-flying insect found working
on sweet clover while these plants were in bloom was _Diacrisia
virginica_ Fabr.

This experiment was duplicated at Ames in August, 1916, with the result
that one plant subject to visitation only by night-flying insects
produced 486 racemes, with an average of 16.5 pods each.

The results obtained in these experiments show that night-flying
insects were much more active in pollinating sweet clover at Ames than
at Arlington. However, as the results obtained from the plants subject
to visitation by day-flying insects only were practically the same as
those obtained from plants which were subject to insect visitation at
all times, it is concluded that night-flying insects were not a factor
in the pollination of sweet clover at Arlington or at Ames in 1916.

FLOWERS POLLINATED ONLY BY DAY-FLYING INSECTS.

A cheesecloth-covered cage, 3 feet square and 3½ feet high, was
placed on July 7, 1916, over two sweet-clover plants at Arlington,
before any of the flowers opened. As the cover of this cage was
removed at 7.30 a. m. and replaced at 4.30 p. m. each day during the
experiment, the plants were subject to visitation by day-flying insects
only. As soon as all of the flowers on most of the racemes had bloomed,
and before any mature pods shattered, the racemes were removed from the
plants and the pods produced by each raceme counted. The two plants
produced a total of 544 racemes, with an average of 20.9 pods each.

This experiment was also conducted at Ames. One plant was protected
from insect visitation at night in August, 1916, with the result that
it produced 418 racemes, with an average of 41.11 pods each.

PLANTS PROTECTED FROM ALL INSECTS THAT COULD NOT PASS THROUGH A WIRE
SCREEN HAVING 14 MESHES TO THE LINEAR INCH.

It is well known that many small insects, and especially those
belonging to the family Syrphidæ and to the genus Halictus, frequent
sweet-clover flowers, but no records have been noted that show how
important these insects are as pollinators of this plant. In order to
obtain data on this subject a cage 12 feet square and 6½ feet high,
made of wire screen having 14 meshes to the linear inch, was placed
over a few plants at Ames, in July, 1916, before they began to bloom.
The base of the cage was buried several inches in the soil, so that
no insects could get into it. As these plants were growing in a field
where there was a sufficient supply of moisture at all times, they
made a growth of 5 to 6 feet. For this reason all the racemes were
collected from only a portion of one of the plants instead of from
the entire plant, as was done with the smaller ones inclosed in the
cheesecloth-covered cages. The branches selected contained 224 racemes,
with an average of 24.53 pods each. Many insects that were able to pass
through the wire netting were observed working on the flowers of the
inclosed plants.

A check plant, subject to visitation by all insects and growing within
a few yards of the cage, contained 264 racemes, with an average of
28.23 pods each.

This experiment shows that small insects are efficient pollinators
of sweet clover and that the plant to which all insects had access
produced an average of only 3.7 pods to the raceme more than the
one inclosed in the cage. As these plants were growing close to a
strip of timber and some distance from a field of sweet clover, it
is probable that more small insects worked on the flowers than would
have been the case if the cage had been located in the center of a
field of sweet clover. Though these results show that small insects
are able to pollinate sweet-clover flowers freely, it is very doubtful
whether insects of this kind would be numerous enough to pollinate
sufficient flowers in a large field of sweet clover for profitable
seed production. The honeybee is the most efficient pollinator of this
plant, and it is believed that in many sections it is responsible for
the pollination of more than half of the flowers.

SUMMARY OF INSECT-POLLINATION STUDIES.

The data secured in the different experiments where sweet-clover
flowers were subject to insect visitation at one time or another are
presented in detail in Table V.

Table V.--_Summary of the insect pollination studies conducted
at Arlington, Va., and Ames, Iowa, in 1916._

  ----------+-------+-------------------------+----------------------------
            |       |                         |        Number of--
            |Number |                         +--------+---------+---------
  Location. |  of   |  Method of treatment.   |Racemes.|  Pods   |Pods per
            |plants.|                         |        |produced.| raceme,
            |       |                         |        |         | average.
  ----------+-------+-------------------------+--------+---------+---------
  Arlington.|   1   |Check--subject to insect |   196  |  4,013  |  20.47
            |       | visitation at all times.|        |         |
  Ames.     |   1   |     do.                 |   239  |  9,943  |  41.60
  Arlington.|   3   |Protected from all       |   904  |    577  |    .63
            |       | insects.                |        |         |
  Ames.     |  12   |     do.                 | 2,370  |    653  |    .27
  Arlington.|   3   |Visited by night-flying  |   723  |  2,720  |   3.76
            |       | insects only (cage 1).  |        |         |
     Do.    |   1   |Visited by night-flying  |   227  |    152  |    .67
            |       | insects only (cage 2).  |        |         |
  Ames.     |   1   |Visited by night-flying  |   486  |  8,024  |  16.51
            |       | insects only.           |        |         |
  Arlington.|   2   |Visited by day-flying    |   544  | 11,397  |  20.95
            |       | insects only.           |        |         |
  Ames.     |   1   |     do.                 |   418  | 17,186  |  41.11
     Do.    |   9   |Protected from all       | 1,594  |    502  |    .31
            |       | insects.                |        |         |
  ----------+-------+-------------------------+--------+---------+---------

The results in Table V show that an average of 0.37 pod to the raceme
was obtained from the plants protected from visitation by all insects
during the flowering period. As the racemes of _Melilotus alba_ will
average approximately 50 flowers each, less than 1 per cent of them
set seed without being pollinated by insects. The results obtained in
the cages in which only night-flying insects had access to the flowers
show that these insects pollinate sweet clover to a slight extent,
but that the number of pods produced by them is so few that it may be
assumed that these flowers would have been pollinated by day-flying
insects. This assumption is borne out by the results obtained in the
cages where only day-flying insects had access to the flowers, as the
results obtained in these cages at Arlington and Ames, respectively,
are approximately the same as those obtained on the plants subject
to insect visitation at all times. It will be noted that the yield
of seed on the plants visited by insects at Ames is much higher than
that of the plants subjected to insect visits during the same period
at Arlington. This difference in seed yield may be attributed to the
fact that isolated plants were used in the experiments at Ames, and
at Arlington the experiments were conducted with plants growing under
field conditions.


RELATION OF THE POSITION OF THE FLOWERS ON MELILOTUS ALBA PLANTS TO
SEED PRODUCTION.

Observations of sweet-clover plants grown under cultivation, and
especially when the stands were thick, showed that the flowers of the
racemes on the upper and exposed branches produced a larger percentage
of seed than those on the lower branches which were less exposed. It is
thought by some that the failure of the flowers on the lower racemes
to be fertilized is due to shading; but the results obtained in the
cheesecloth and glass covered cages do not warrant this belief, as it
is doubtful whether the shading of the flowers on the lower racemes is
more than that caused by the cheesecloth. It is probably the lack of
pollination that causes this decrease in seed production on the lower
branches of plants growing close together, as a vast number of flowers
open each day on portions of the plants which are exposed directly to
visitation by insects and are therefore more accessible to them.

In order to obtain information upon the number of flowers that produce
seed on the upper and lower portions, respectively, of sweet-clover
plants when grown under field conditions and where the stand contained
four to five plants to the square foot, a number of racemes were
labeled on different portions of the plants at Ames in 1915 and 1916.
When the pods were partly mature, records were made of the number of
flowers that produced pods. The results obtained are given in Table VI.

Table VI.--_Relation of the position of sweet-clover flowers
on the plants to seed production, at Ames, Iowa, in 1915 and 1916._

  -----+------------------------+---------+------------------------------
       |                        |         |       Pods formed.
       |                        |Number of+--------+------------+--------
  Year.|Position of the flowers.| flowers.| Number.| Percentage.|  Average.
  -----+------------------------+---------+--------+------------+--------
       |                        |         |        |            |
  1915 | Upper half of plants   |   812   |   357  |    43.9 }  |
  1916 |   do                   |   261   |   101  |    38.7 }  |  42.6
       |                        |         |        |            |
  1915 | Lower half of plants   |   344   |    44  |    12.7 }  |
  1916 |   do                   |   216   |    59  |    27.3 }  |  18.3
  -----+------------------------+---------+--------+------------+--------

The flowers on the upper racemes of the plants produced 31.2 per cent
more pods than those on the lower racemes in 1915. and 11.4 per cent
more in 1916. These results prove that insects more frequently visit
the flowers that are directly exposed and are therefore more accessible.

INFLUENCE OF THE WEATHER AT BLOSSOMING TIME UPON SEED PRODUCTION.

The seed production of sweet clover is seldom satisfactory when rainy
or muggy weather prevails during the flowering period. In order to
obtain data as to the relation existing between the visits of insects
and the prevailing weather conditions, a record of insect visits and
of the number of flowers that opened each day was kept for a period of
nine days at Ames in August, 1915.

In this experiment the racemes were marked early each morning just
above the last flowers which had opened the previous day, and early
the following morning the number of flowers which had opened the
previous day was noted. The number of flowers that were pollinated was
determined by the number of pods that formed. Table VII gives in detail
the results obtained.

Table VII.--_Influence of the weather at blossoming time upon
the yield of sweet -clover seed, at Ames. Iowa, in 1915._

  -------+-------------------------+---------+-------+-------+-----------
         |                         |         |Number |       |
         |                         |         |  of   |       | Percentage
  Date,  |   Weather conditions.   | Insect  |flowers| Pods  | of flowers
  1915.  |                         |visitors.| that  |formed.|   that
         |                         |         |opened.|       |  matured.
  -------+-------------------------+---------+-------+-------+-----------
  Aug. 16|Cloudy and showery       | Very few|  102  |   18  |    17.6
  Aug. 17|Rain all day             | None    |   69  |    4  |     5.7
  Aug. 18|Cloudy most of the day   | Very few|   60  |   20  |    33.3
  Aug. 19|Clear and cool           | Numerous|   94  |   53  |    56.3
  Aug. 20|Mostly clear and warm    |    do   |   61  |   38  |    62.2
  Aug. 21|Clear and warm           |    do   |   81  |   44  |    54.3
  Aug. 22|Partly cloudy and warm   |}        |       |       |
  Aug. 23|    do                   |}   do   |  181  |  100  |    55.2
  Aug. 24|Cloudy till mid-afternoon| Few     |   37  |   12  |    32.4
  -------+-------------------------+---------+-------+-------+-----------

The data given in Table VII show that the percentage of effective
pollination is much higher in clear weather, when insects are active,
than in cloudy or rainy weather, when but few insects visit the flowers.


INSECT POLLINATORS OF SWEET CLOVER.

On account of the ease with which the heavy flow of nectar of
sweet-clover flowers may be obtained many insects visit the flowers,
thereby pollinating them. While the useful insect visitors of flowers
of red clover are limited to a few species of Hymenoptera, those
pollinating sweet-clover blossoms are many and belong to such orders as
Coleoptera, Lepidoptera, and Diptera, as well as to the Hymenoptera.
However, in the United States the honeybee is the most important
pollinator of sweet clover. In many parts of the country the different
species of Halictus, commonly known as sweat bees, rank next in
importance. The margined soldier beetles (_Chauliognathus marginatus_
Fabr.) were very active pollinators at Arlington, Va., in the latter
part of June and first part of July, 1916, but the woolly bear
(_Diacrisia virginica_ Fabr.) was the only night-flying insect found
working on sweet clover at Arlington.

Insects belonging to the genera Halictus, Syritta, and Paragus were
very active pollinators at Ames, Iowa, in 1916, and ranked next in
importance to the honeybee. In fact, the results obtained in the
cage where the plants were protected from visitation by insects that
could not pass through a screen having 14 meshes to the linear inch
showed that these small insects were able under the conditions of that
experiment to pollinate practically as many flowers as larger insects.

The insects listed below were collected while visiting _Melilotus alba_
and _M. officinalis_ flowers in 1916.


AT ARLINGTON, VA.

 _Neuroptera._--_Perithemis domitia_ Dru., _Enallagma_ sp.

 _Hemiptera._--_Adelphocoris rapidus_ Say, _Lygus pratensis_ Linn,
 (tarnished plant bug).

 _Coleoptera._--_Chauliognathus marginatus_ Fabr. (margined soldier
 beetle), _Diabrotica 12-punctata_ Oliv. (southern corn rootworm).

 _Lepidoptera._--_Pieris protodice_ Bd. (imported cabbage butterfly),
 _Heodes hypophleas_ Bd., _Lycaena comyntas_ Gdt., _Hylephila
 campestris_ Bd., _Scepsis fulvicollis_ Hubn., _Ancyloxypha numitor_
 Fabr., _Pholisora catullus_ Fabr., _Pyraustid_ sp., _Loxostege
 similalis_ Gn. (garden webworm), _Thecla melinus_ Hubn., _Colias
 philodice_ Gdt. (the common sulphur butterfly), _Tarachidia
 caudefactor_ Hubn., _Pyrameis atalanta_ Linn., Drasteria (2 species),
 _Diacrisia virginica_ Fabr. (the woolly bear).

 _Hymenoptera._--_Halictus lerouxi_ Lep., _H. provancheri_ (sweat
 bee), _H. pectoralis_ Sm. (sweat bee), Halictus (3 unidentified
 species), _H. legatus_ Say, _Bombus affinis_ Cr., _B. impatiens_
 Harris (bumblebee), _Melissodes bimaculata_ Lep., _Polistes pallipes_
 Lep. (paper wasp), _Megachile_ sp. (leaf-cutter bee), _Coelioxys
 octodentata_ Say, _Xylocopa virginica_ Drury (common carpenter bee),
 _Pompiloides_ sp., _Apis mellifica_ Linn, (honeybee), _Philanthus
 punctatus_ Say, _Sphex nigricans_ Dahlb. (caterpillar hawk), _S.
 pictipennis_ Walsh (caterpillar hawk).

 _Diptera._--_Archytas analis_ Fabr., _Chrysomyia macellaria_ Fabr.
 (screw-worm fly),. _Pollenia rudis_ Fabr. (cluster fly), _Ocyptera
 carolinae_ Desv., _Trichophora ruficauda_ V. D. W., _Eristalis
 arbustorum_ Linn., _Physocephala tibialis_ Say.


AT AMES, IOWA.

 _Hemiptera._--_Lygus pratensis_ Linn., _Adelphocoris rapidus_ Say,

 _Coleoptera._--_Coccinella transversoguttata_ Fabr.

 _Lepidoptera._--_Eurymus eurytheme_ Bdv., _Chrysophanus_ sp., Lycaena
 (2 species),. _Libythea bachmani_ Kirtland, _Pieris rapae_ Linn.

 _Hymenoptera._--_Angochlora_ sp., _Apis mellifica_ Linn., _Colletes_
 sp., _Halictus lerouxi_ Lep., _H. provancheri_ D. J., _Halictus_ sp.,
 _Elis_ sp., _Calliopsis andreniformis_ Smith, _Polistes_ sp., _Sphex_
 sp., _Eumenes fraterna_ Say, _Sceliphron_ sp., _Isodontia harrisi_,
 Fern., _Cerceris_ sp., _Oxybelus_ sp.

 _Diptera._--_Syritta_ sp., _Paragus_ sp., _Chrysomyia macellaria_
 Desv., Syrphidæ (2 unidentified specimens).


EFFECT OF MOISTURE UPON THE PRODUCTION OF MELILOTUS ALBA SEED.

Careful inspection of a number of sweet-clover fields in Iowa and
Illinois in the autumn of 1916 indicated that many plants were unable
to obtain sufficient moisture for the proper development of their
flowers. Examination of flowers that aborted shortly after reaching
their mature size showed that the anther sacs had not burst, even
though the pollen grains were mature. Apparently for the same reason
many immature pods aborted. The precipitation for July, 1916, in
Livingston County, Ill., where the sweet-clover seed crop suffered
materially for lack of moisture, was 3.2 inches less than normal, while
the temperature was 4.5° F. above normal. In August the precipitation
was 0.96 of an inch below normal and the temperature 4.2° F. above
normal. At Ames, Iowa, the precipitation was 3.54 inches below
normal and the temperature 5.4° F. above normal in July. Both the
precipitation and temperature were about normal at Ames in August, but
most of the precipitation fell before the experiments were commenced.

In north-central Illinois the seed production of sweet clover was
very irregular. Some fields produced an abundance of seed, while a
large percentage of the pods on the plants in other fields near by,
where the thickness of the stand, size of the plants, and conditions
in general were approximately the same, aborted. It was evident that
all stands producing a good seed crop were growing on well-drained
soil and that those which were not yielding satisfactorily were on
poorly drained land. It is well known that sweet clover will produce
deep taproots only when the plants are growing in well-drained soil
and that a much-branched surface root system will be formed on poorly
drained land, and especially when there is an excess of moisture or a
high water table during the first season's growth. During this droughty
period in 1916 the upper layer of soil became so depleted of moisture
that the plants with surface root systems were unable to obtain
sufficient water to mature their seed. On the other hand, the lack of
precipitation and the high temperatures did not affect the moisture
content of the subsoil sufficiently to interfere with the normal seed
production of deep-rooted plants. According to Lutts (22, p. 47) this
same condition was found to be true in Ohio in 1916.

As a rule, under droughty conditions the second crop of sweet clover
will produce a higher yield of seed than the first crop, as the second
growth of the plants is seldom more than half as much as the first,
thereby requiring less moisture. However, if showery hot weather
prevails when the first crop is cut, the end of each stub is very apt
to become infected, usually with a species of Fusarium, which kills all
the cortex as far back as the upper bud or young shoot and that part
of it on the opposite side of this bud to the bud below. If the second
bud from the top of a stub is not directly opposite the upper one the
decay may extend nearly to the ground. (Pl. IV.) The destruction of
half to two-thirds of the cortex from 2 to 4 inches below the upper bud
materially reduces the quantity of water that can be conveyed to the
branch above the base of the dead area. Plants thus infected obtain
sufficient moisture for seed production only under the most favorable
conditions. When the first crop is cut during warm dry weather, and
especially when the first crop has not been permitted to make more than
a 30 to 32 inch growth, the stubble seldom decays, and in no instance
have the plants been observed to decay as far back as the upper buds.

An experiment was conducted at Ames in the latter part of August and
first part of September, 1916, to determine the effect of watering
plants that were aborting a large percentage of their flowers and
immature pods. For this purpose several volunteer plants growing in
a meadow were selected. A hole 12 inches square and 14 inches deep
was dug 8 inches from the crown of one plant, and each evening during
the experiment 2 gallons of water were poured into the hole. The top
of the hole was kept covered, so as to check evaporation from it as
much as possible. Another plant of the same size and growing about 15
yards from the watered plant served as a check. On both plants many
of the flowers and immature buds were aborting at the beginning of
the experiment. The soil in this field was so depleted of moisture
that the leaves of the plants wilted during the hottest part of the
days preceding the experiment. The foliage on the check plant wilted
each day for the first five days of the experiment. On the sixth day
0.96 of an inch of rain fell and four days later 0.23 of an inch
more. The dropping of the flowers was temporarily checked by these
precipitations, but owing to the dry, compact condition of the soil
the rain was not sufficient to check entirely the fall of flowers and
immature pods. At the beginning of the experiment the racemes on both
plants were divided into three classes, according to the development of
the flowers, and labeled. They were collected and the seeds counted as
soon as the pods at the bases of the racemes commenced to turn brown.
Table VIII presents the results obtained.

Table VIII.--_Effect of water upon the seed production of
sweet clover when growing under droughty conditions at Ames, Iowa, in
1916._

  ------------------------+-------------------------------------+---------
                          |Plant not watered.|  Plant watered.  |
                          +--------+---------+--------+---------+
                          |        | Average |        | Average |
  Stage of development    | Number |number of| Number |number of|Increase
     when labeled.        |   of   |pods per |   of   |pods per |  from
                          |racemes | raceme  |racemes | raceme  |watering.
                          |labeled.|  that   |labeled.|  that   |
                          |        | matured.|        | matured.|
  ------------------------+--------+---------+--------+---------+---------
  Flowers at the base of  |        |         |        |         |
    the racemes just ready|   49   |  27.39  |   110  |  55.63  |  28.24
    to open.              |        |         |        |         |
                          |        |         |        |         |
  Pods 3 to 6 days old    |   30   |  21.13  |   112  |  39.81  |  18.68
                          |        |         |        |         |
  Pods 9 to 12 days old   |   35   |  15.23  |    50  |  29.86  |  14.63
                          |        |         |        |         |
  ------------------------+--------+---------+--------+---------+---------


The effect of the water was noticeable soon after the first
application, as the leaves and flowers on this plant became turgid and
the anther sacs burst at the proper stage of their development. Very
few flowers fell after the second day. The water decidedly checked
the aborting of immature pods, as is shown by the results obtained on
the racemes which were labeled after the pods had formed. The racemes
which contained pods 3 to 6 days old when labeled matured 9.95 pods to
the raceme more than those which contained older pods at the beginning
of the experiment, but this was expected, as most of the aborting
which caused this difference had taken place before the racemes were
labeled. As very few pods aborted before they were 3 to 6 days old, the
difference of 9.95 pods to the raceme in favor of the ones labeled
when the flowers at their bases were just ready to open was largely
due to the dropping of the flowers on the older racemes before the
experiment was begun.

It will be seen that the production of mature pods on the plant not
watered was much greater on the racemes that were labeled before
the flowers opened than on the older racemes. This difference is
undoubtedly due to the precipitation which fell on the sixth and tenth
days of the experiment. It is believed that the yield of 15.23 pods
to the raceme on the ones labeled when the pods were 9 to 12 days old
is representative of the production of pods per raceme previous to
the precipitation and that the other racemes on this plant would have
yielded proportionately if conditions had remained the same.

In the early spring of 1916, _Melilotus alba_ was planted in several
large pots in the greenhouse of the Department of Agriculture at
Washington, D. C. These pots were placed outside the greenhouse in
the late spring, where they remained until the following January,
when they were taken into the greenhouse. The plants grew rapidly and
began to flower during the latter part of April, 1917. At this time
two pots were placed in a large cage made of screen having 20 meshes
to the linear inch. One pot was submerged in a tub of water, so that
the soil was saturated at all times, while the plant in the other pot
was given only sufficient water to keep it from wilting. The pods on a
few racemes were self-pollinated and the results obtained are given in
Table IX.

Table IX.--_Effect of moisture on the seed production of
Melilotus alba at Washington, D. C, in 1917._

  -------------------------+------------------+---------+--------+--------
                           |Total number of-- | Flowers that matured
                           |                  |      (per cent).
                           +--------+---------+---------+--------+--------
  Soil treatment.          |        |         |  Pods   |        |
                           |Racemes.| Flowers.| formed. | Total. |Increase.
  -------------------------+--------+---------+---------+--------+--------
                           |        |         |         |        |
  Soil given only a limited|   12   |   227   |    65   | 28.63  | ......
    quantity of water.     |        |         |         |        |
  Soil saturated.          |   17   |   425   |   235   | 55.03  |  26.22
  -------------------------+--------+---------+---------+--------+--------

The results of this experiment compare favorably with those obtained
under field conditions at Ames in 1916.



Part II.--STRUCTURE AND CHEMICAL NATURE OF THE SEED COAT AND ITS
RELATION TO IMPERMEABLE SEEDS OF SWEET CLOVER.[3]

[3] The writers wish to acknowledge the service rendered by Mr. H.
S. Doty, Instructor in Botany, Iowa State College, Ames, Iowa, in
assisting in the preparation of this article.


HISTORICAL SUMMARY.

When agriculturists first began to cultivate wild legumes they observed
that many seeds would not germinate within a comparatively short time
after planting. Thus the problem of impermeable seeds began to demand
attention many years ago. However, impermeable seeds are not confined
to the Leguminosæ, as they occur also in the Malvaceæ, Chenopodiaceæ,
Convolvulaceæ, Cannaceæ, and other families.

Since the first account of the structure of legume seed coats by
Malpighi (23 v. 1) in 1687, many investigators have contributed to our
knowledge of the structure of the coats of seeds belonging to this
family.

Pammel (31) made an extensive study of legume seeds, including all the
genera in the sixth edition of Gray's Manual, as well as genera not
included in that publication. He found that the seed coat uniformly
consisted of three layers, namely, the outer layer of Malpighian cells,
the osteosclerid layer, and the inner layer of nutrient cells. Pammel's
work included a study of the seed coats of _Melilotus alba_ and _M.
officinalis_, and the descriptions and illustrations in his publication
agree for the most part with the results obtained in the investigations
reported in this article. However, more variation was noticed in the
different layers of the seed coat than he describes.

The cause of impermeability in seeds has been investigated by many.
It has been found to be due to the embryos in some seeds, such as the
hawthorns, but in most cases to the structure of the seed coat, and
especially so in the Leguminosæ. Crocker (3) states that, exactly
opposite to the common view, the cause of delayed germination generally
lies in the seed coats rather than in the embryos. Nobbe (29) thought
that the hardness of leguminous seeds was due to the Malpighian layer,
and in a later publication Nobbe and Haenlein (30, p. 81) state that
the absorbent power of many seeds is inhibited or entirely arrested
by the cones of the Malpighian cells and the shields built up between
them, which consist principally of cutin. Huss (15) agrees with Nobbe
and Haenlein. Verschaffelt (39) found that the impermeability of the
seeds of Cæsalpiniaceæ and Mimosaceæ investigated was due to, the
inability of water to pass through the canals of the seed coat. By
soaking the seeds in alcohol or other substances which change the
capillarity of the pores, the seed coats were made readily permeable
to water. Gola (6) states that the cause of the impermeability of seeds
is the peculiar character of the Malpighian cells, which prevents their
infiltration and consequent increase in volume, while Bergtheil and Day
(2) found that the hardness of the seeds of _Indigofera arrecta_ was
due to their possession of a very thin outer covering of a substance
resistant to water. Ewart (5, p. 185) believes that in most impermeable
seeds the cuticle prohibits the absorption of water, but gives as an
exception _Adansonia digitata_, in which the whole integument seems to
be permeable to water with difficulty. The following is quoted from
White (42, p. 205):

 As a general rule in small and medium-sized seeds the cuticle is well
 developed and represents the impermeable part of the seed coat, while
 in the cases of large seeds, such as those of _Adansonia gregorii_,
 _Mucuna gigantea_, _Wistaria maideniana_, and _Guilandina bonducella_,
 the cuticle is relatively unimportant and inconspicuous. In these
 seeds the extreme resistance which they exhibit appears to be located
 in the palisade cells.

In discussing the seed coat of _Melilotus alba_, Rees (33, p. 404)
states that the outer layer consists of palisade cells covered,
externally by a structureless membrane, which, however, did not
appear to be cuticle but hemicellulose, as it stained magenta with
chloriodid of zinc. The greater part of the walls of the palisade
cells also appears to be composed of hemicellulose and the outer ends
only were cuticularized. In order to find whether the outer membrane
was in itself impermeable to water, this author treated seeds for
short intervals in sulphuric acid to dissolve the outside covering
without directly affecting the palisade cells. Seeds treated in this
manner swelled in water and microscopic examination showed that the
ends of the palisade cells were quite intact, but had separated from
each other. From this it was concluded that the outer membrane is
instrumental in conferring impermeability on the seed, although not
directly responsible for it, as is the case with a true cuticle. It
is further believed that it probably served as a cement substance
by means of which the cuticularized ends of the cells were held
together closely, thus forming a barrier through which water could not
penetrate, but that as soon as this barrier was removed the ends of the
palisade cells separated and water passed in between them.

More than 20 years ago machines were devised by Kuntze, Michalowski
(27, p. 86), Huss (15), and later by Hughes (14), to scarify
impermeable seeds. Other methods have been recommended and employed to
some extent for hastening the germination of seeds. Hiltner (13, p.
44) treated seeds of red clover, white clover, and alfalfa 10, 30, and
60 minutes with concentrated sulphuric acid and found that the best
germination resulted from the 60-minute treatment. Love and Leighty
(21) also treated the seeds of various legumes with concentrated
sulphuric acid and obtained a better germination in all cases. In
their investigations with _Melilotus alba_ it was found that a 2-hour
treatment resulted in some injury to the seed, but that a treatment
varying from 25 minutes to 1 hour gave good results. In most cases in
our investigations the seed coats of sweet clover became permeable
to water after a treatment of 15 minutes in concentrated sulphuric
acid, and within 5 minutes all of the Malpighian cells were destroyed
down to the light line. Harrington (10) found that the soil, season,
climate, color, or size of red-clover seeds had no influence upon
the percentage of impermeable seeds and that the good germination
ordinarily obtained with red clover was due to the scarifying of the
seed coats by the rasps of hulling machines. Harrington (11) also
studied the agricultural value of impermeable seeds and found that
alternations of temperature cause the softening and germinating of
many impermeable clover seeds when a temperature of 10° C. or cooler
is used in alternation with a temperature of 20° C. or warmer and that
the effect of such an alternation of temperature is greatly increased
by previously exposing the seeds to germinating conditions at a
temperature of 10° C. or cooler and is decreased by previously exposing
the seeds to germinating conditions at a temperature of 30° C. It is a
well-known fact that impermeable seeds which remain in the field over
winter germinate readily the following spring.

The light line is the most important and interesting feature of
the Malpighian cell, at least so far as _Melilotus alba_ and _M.
officinalis_ are concerned. But one light line occurs in the
Malpighian cells in most Leguminosæ, although Pammel (32) reports two
well-developed light lines in _Gymnocladus canadensis_, Junowicz (16)
found three in _Lupinus varius_, and Sempolowski (36) two in _Lupinus
angustifolius_.

Many investigators have studied the light line, and different theories
have been advanced as to its function, physical properties, and
chemical nature. Schleiden and Vogel (35, p. 26) in describing the
mature testa of _Schizolobium excelsum_ in 1838 undoubtedly referred to
the light line when they stated that the walls of the Malpighian cells
were not equally thickened. Mettenius (26), in 1846, was probably the
first definitely to describe the light line. This author believed it
was composed of pore canals, all appearing at the same height in the
cells, but he was unable to prove this by cross sections. Lohde (20)
studied the light line in seeds of _Hibiscus trionum_ and found it
cutinized. Hanstein (8) states that the Malpighian cells are composed
of two cell layers and the light line is produced by the adjoining
walls of the ends of the cells. Later, this same author (9), according
to Harz (12), refers to the light line as a perforated disk composed of
tissue of strong refracting power.

Russow (34) concludes that the light line is produced by neither
chemical nor mechanical changes but is caused by a modified molecular
structure containing less water than the remainder of the cell wall.
Hiltner (13) agrees with Russow's explanation. Harz (12, p. 561) also
agrees with Russow and adds that he has observed that the light line
disappeared in a number of cases after applications of nitric acid.
Wigand and Dennert (43) suggested that the light line is due to a
series of erect fissures, while Tietz (37, p. 32) believes it is due
to a chemical modification and that the phenomenon results from the
exceptionally extreme density of parts of the cellulose membrane.
Junowicz (16) found evidence of cellulose material. The cell wall
at this point was strongly refractive and had a different molecular
structure. After studying _Phaseolus vulgaris_, Haberlandt (7, p.
38) agrees with the Russow explanation. In the seed of this plant
the light line colored blue after being treated with chloriodid of
zinc. Sempolowski (36), who investigated the light line in _Lupinus
angustifolius_, states that there is not only a difference in the
molecular structure but also a chemical modification of the cell wall
at this point, since with iodin and sulphuric acid the cell wall
colored blue, whereas the light line colored yellow. Wettstein (41),
who studied seeds of Nelumbo, agrees with Russow (34) and Sempolowski
(36) that chemical and physical modifications occur. He found that
iodin and sulphuric acid colored the Malpighian cells intensely blue,
the light line at first yellowish, and then later it gradually became
blue. This reaction may be accelerated by heat. Iodin produced the same
effect, and the light line colored blue more rapidly. When treated with
a water-withdrawing medium the light line was not altered for some
time, but finally disappeared with continued application. Cooking for a
long time in caustic potash or standing in cold caustic potash caused
the cells to swell, while the light line remained uninjured at first
but finally disappeared. He also believed that the absence of pore
canals in the region of the light line caused it to be more dense.

Nobbe and Haenlein (30) treated sections of seed coats of _Trifolium
pratense_ with iodin and sulphuric acid and found that the light line
colored blue as readily as the thickened ridges that radiate inward
from it, but that the outer processes of the palisade cells projecting
from the light line toward the cuticle stained dark brown. They also
state that various causes work to produce such unusual lusters in
the light line, the principle one of which is the thickened ridges
which radiate inward, reach their greatest development at this point,
and coalesce in the lumen of the cell. The result is that the light
line falls upon a continuously homogeneous medium, while in the inner
portions of the ridges the light passes through media of varying
opacity, such as cellulose, water, and protoplasm, whereby it is
progressively subdued in varying degrees by partial reflection. Pammel
(31, p. 147) studied the light line in _Melilotus alba_ and found that
it consisted of a narrow but distinct refractive zone below the conical
layer. The refractive zone colored blue with chloriodid of zinc. The
whole upper part was, however, more or less refractive, while the
remainder of the cell wall contained pigment and colored blue with
chloriodid of zinc. Small canals project into the walls, in some cases
extending beyond the light line.

Beck (1) found that the light-refracting power of the light line was
much greater than that of the undifferentiated membrane and stated that
there may be in addition to this a chemical difference which can not be
detected with the present microchemical methods. He does not believe
that it is cuticularized or that it contains less water than the rest
of the cell.

Marlière (24, p. 11) gives a physical explanation and states that
the true cause of the light line lies in the peculiar structure
of the secondary membrane of the Malpighian cell. Tunmann (38, p.
559) observed that it did not hydrolize in weak acids and therefore
decided that it was not hemicellulose. He found that it dissolved in
concentrated sulphuric acid more readily than the regions surrounding
it and that it was composed of pectin or callose. In our investigations
the main portion of the light line of _Melilotus alba_ and _M.
officinalis_ was very resistant to concentrated sulphuric acid, only
the narrow outer portion being attacked. It showed evidence of callose.


MATERIAL AND METHODS.

Permeable and impermeable seeds[4] of _Melilotus alba_ and _M.
officinalis_ were obtained from commercial samples and also from
samples collected in the field. Those selected for sectioning were
allowed to dry after being removed from the germinator and then
embedded on the ends of pine blocks in glycerin gum, which was made by
dissolving 10 grams of powdered gum arabic in 10 c. c. of water and
adding 40 drops of glycerin. After the glycerin gum had dried for 24
hours, the seeds were easily sectioned. This method of embedding causes
no change in the seed coat. It is more satisfactory than the paraffin
method for holding the seeds firmly. The glycerin gum dissolved readily
when the sections were mounted in water.

[4] The term "permeable" is used in this paper to designate seeds whose
coats are permeable to water in two weeks or less at temperatures
favorable for germination, while the term "impermeable" is used to
designate seeds whose seed coats are impermeable to water for this
length of time when temperatures are favorable for germination.
Impermeable seeds are commonly referred to as "hard seeds," and they
may become permeable in time.

In the microchemical studies Sudan III, alcanin, chlorophyll solution,
and phosphoric acid iodin were used to test for cutin or suberin;
sulphuric acid and iodin, chloriodid of zinc, and chloriodid of
calcium for cellulose; phloroglucin and hydrochloric acid for lignin;
ruthenium red for pectic substances; and sulphuric acid, Congo red, and
aniline blue for callose.

Where very thin sections were necessary for detailed study of the
structure of the seed coat, pods in various stages of development were
collected, and after the usual preliminary treatment they were embedded
in paraffin and sectioned with the microtome. Microchemical tests were
made with these sections by using various specific stains. Safranin was
used to test for cutin, suberin, and lignin; haematoxylin and methyl
blue for cellulose ; methylene blue, methyl violet B, mauvein, and
ruthenium red for pectic substances; and aniline blue and Congo red for
callose. In studying some points with reference to the pore system of
the seed coat, it was necessary to use free-hand sections of fresh pods.

In studying the seed coat in relation to the absorption of water,
both permeable and impermeable seeds were soaked in water solutions
of safranin, gentian violet, eosin, and haematoxylin, then dried and
embedded in glycerin gum for sectioning. Seeds were soaked in stains
dissolved in 95 per cent alcohol to test the penetration of alcohol. It
was evident that the seed coats did not act as a filter, as the stains
passed through them with the water or alcohol.


STRUCTURE OF THE SEED COAT.

There is very little endosperm present in mature seeds of _Melilotus
alba_ or _M. officinalis_. That which is present is quite permeable to
water and therefore bears no relation to the impermeable seeds of these
plants.

The outer layer of the seed coat, which is the modified epidermal layer
of the ovule, is known as the Malpighian layer. (Pl. V, figs. 1 and
2.) The cells constituting this layer, commonly called palisade cells,
are the most highly modified cells of the seed coat. They are very
much elongated, their length varying in the different regions of the
coat, and their outer tangential walls and the outer portions of their
radial walls are so much thickened that their lumina are confined to
the inner portion of the cells, sometimes occupying less than half the
length of the cells. The inner tangential walls and inner portions of
the radial walls are thickened just previous to the death of the cells,
the thickening sometimes being only slight and sometimes so much as to
leave only very narrow lumina.

There is a very thin layer on the outer surface of the Malpighian
cells which has been called cuticle by previous investigators, but
the chemical composition of this layer and its perviousness to water
indicate that there is very little cutin present. This layer is
probably the primary epidermal cell wall rather than a deposit on the
outer surface of the wall. To determine this a study of the development
of the Malpighian cells is necessary.

Beneath the so-called cuticle there is the much thickened outer portion
of the Malpighian cells in which there are two rather distinct regions,
one constituting the conelike structures and the other forming a
continuous layer over the conelike structures, separating them from
the cuticle and filling in between them. These two regions separate
easily, and in cutting sections the outer region, called by some the
cuticularized portion, often breaks away, leaving the entire surface of
the cones exposed.

The term "cuticularized layer" will be used to designate all of the
thickening covering the cones, including that around the cones as well
as the portion between the cones and the cuticle. This term is not
entirely appropriate, for the region is practically free from cutin,
but for the want of a better term it will be used. There are canals
in the cuticularized layer and cones, which are easily seen when the
sections are treated with chloriodid of zinc or sulphuric acid. A
surface view of a section showing the cones and cuticularized layer
when mounted in glycerin shows the canals as dark lines due to the
air inclosed. The canals are most abundant along the lines where the
lateral walls of the cells join, but many are within the cones and in
the cuticularized substance between the cones. (Pl. V, fig. 5.)

The well-developed light line in _Melilotus alba_ and _M. officinalis_
is found just below the bases of the cones. In some seed coats only
a few and in others none of the canals which are common in the cones
and cuticularized region cross the light line. A very distinct line of
small canals filled with air and thus forming a dark band is present
just above the fight line, thus making the light line more conspicuous.
(Pl. V, fig. 3.) When the lumina of the cells extend across the light
line, they are exceedingly small. The light line is the most compact
region of the Malpighian layer and is conspicuous because it refracts
the light much more than the regions above and below it.

Just below the Malpighian is a layer of cells variously modified and
known as the osteosclerid. The cells of this layer are often referred
to as the hourglass cells on account of their shape. In some regions
of the seed coat they are expanded at both ends and their walls are
much thickened, the thickenings forming ridges on the radial walls,
while in other regions only the upper tangential wall and a portion of
the radial walls are thickened and the cells are expanded only at the
inner end, thus having the shape of the frustum of a cone. Beneath the
osteosclerid layer is the nutrient layer.

The nutrient layer contains chloroplasts. It varies not only in the
number of layers of cells composing it, but also in the modifications
of these cells. This layer ranges from four to seven cells in thickness
in the different parts of the seed coat.


PLATE V.

[Illustration]

Structure of the Seed Coat of Sweet Clover.

Fig. 1.--Section of the seed coat of _Melilotus officinalis_. × 450.
Fig. 2.--Another section of the seed coat of _Melilotus officinalis_,
showing the variation in size and modifications that occur in the
three layers. × 450. Fig. 3.--Section of the Malpighian layer of a
_Melilotus alba_ seed, showing a line of canals just above the light
zone. × 450. Fig. 4.--Section of the Malpighian layer of a permeable
_Melilotus alba_ seed. × 450. Fig. 5.--Tangential section of the
Malpighian cells cut between the cuticle and tops of the cones, showing
pores. × 530. Fig. 6.--Section through the Malpighian layer of an
impermeable _Melilotus alba_ seed. × 450. Fig. 7.--Section through
the Malpighian layer of an impermeable _Melilotus alba_ seed, showing
the region through which water and stains readily passed. × 450. Fig.
8.--Cross section of a Malpighian cell of a permeable _Melilotus alba_
seed through the region of the light zone, showing the lumen not
entirely closed. × 530. Fig. 9.--Section through the Malpighian layer
of a _Melilotus alba_ seed shaded to show the portions which react to
the cellulose and pectose tests. × 450. Fig. 10.--Section through the
Malpighian layer of a _Melilotus alba_ seed which shows the condition
of the seed coat after 60 minutes' treatment of concentrated sulphuric
acid. That portion above the light zone was destroyed, and the lumina
as small pores through which much of the stain now passed were seen
extending across the light line. The lines between the cells were much
more distinct, appearing as small intercellular spaces through which
some stain passed. × 450. _a_, Cuticle; _b_, cuticularized layer; _c_,
conelike portion of the thickening of the Malpighian cells; _d_, light
line; _e_, region of a hard seed coat through which water and stains
readily passed; _l_, lumen; _M_, Malpighian cells; _N_, nutrient cells;
_O_, osteosclerid cells; _p_, canals just above light zone.


MICROCHEMISTRY OF THE SEED COAT.

Tests for cutin showed that there was very little present in the seed
coat. Slight reactions for cutin were observed in the cuticle, in the
outer margin of the cuticularized layer, and in the basal portion of
the cones. These reactions were so slight as to be almost negligible.
It is evident that the cuticle and cuticularized layer are not well
named in _Melilotus alba_ and _M. officinalis_. Tests for cellulose
showed that it was present in the cuticle, cuticularized layer, cones,
the walls of the Malpighian cells below the light line, and the walls
of the cells of the osteosclerid and nutrient layers. (Pl. V, fig. 9.)
The reaction for cellulose in the Malpighian cells was quite distinct
in the walls below the light line, less distinct in the cones and
cuticle, and least distinct in the cuticularized layer.

Tests for lignin occasionally showed slight traces in the Malpighian
cells below the light line. When treated with reagents for pectic
substances, the cuticle, cuticularized layer, cones, and all cell walls
below the light line gave a definite reaction. The reaction of the
cones and cuticle was more pronounced than the cuticularized layer.
Tests for callose gave no reaction except in the upper part of the
light line. This part of the light line stained slightly blue with
aniline blue and was easily dissolved with sulphuric acid. In cutting
free-hand sections of fresh material the Malpighian layer sometimes
broke along this line. The greater part of the light line reacted to
none of the tests, and its chemical nature was not determined.

When microtome sections of seeds in different stages of development
were treated with various stains, the results were in accord with those
obtained with free-hand sections. Thus with safranin the periphery and
cones of the Malpighian cells were slightly stained, while haematoxylin
and methyl blue stained all the seed coat except the light line. The
cones and cuticle stained more readily than the cuticularized layer,
but neither stained as deeply as the cell walls below the light line.
Methylene blue, methyl violet B, and mauvein stained all above the
light line, indicating the presence of pectic substances; however, the
staining was more prominent in the cones and cuticle.

The difference in reaction of the cones and cuticularized layer to
the cellulose and pectose tests probably indicates a difference in
density rather than a difference in chemical composition. Since the
cuticularized layer separates readily from the cones, there may be a
difference in physical properties.

With Congo red the upper part of the light line was only very slightly
stained, but aniline blue had a more pronounced effect.

The microchemical tests applied to the seed coat show that in the
region above the light line there is only a slight trace of cutin
or suberin, but a considerable amount of cellulose and pectose. All
cell walls below the light line are mainly cellulose but contain some
pectose. The upper portion of the light line contains callose, but the
remainder of the light line appears to be chemically different from all
other parts of the seed coat or else so dense as to resist the attack
of the reagents.


THE SEED COAT IN RELATION TO THE ABSORPTION OF WATER.

A study of permeable seeds soaked in water containing stains showed
that there were no local regions through which the water passed.
The stains passed through all regions of the seed coat. Coating the
micropylar region with vaseline retarded germination, but had no
effect upon the percentage of germination at the end of three days. In
seed coats through which the stain had passed, the light line was not
stained. Some stain was found in the canals which crossed the light
line, and much more in the cell cavities. There was no evidence that
the stain had permeated the substance of the light line. It was able to
cross the light line only when pores were present.

In impermeable seeds the stains passed readily to the light line.
(Pl. V, fig. 7.) It was evident that the absorption of water was
not prevented by either the cuticularized layer or the cone-shaped
structures of the Malpighian layer, but by the light line. The region
outside of the light line became stained in a few hours, but there
was no trace of the stain within the light line after the seeds had
remained a week in the stains. Alcohol did not penetrate the seed coat
more readily than water.


A COMPARISON OF PERMEABLE AND IMPERMEABLE SEED COATS.

No difference in chemical structure was found between the coats of
permeable and impermeable seeds. The principal differences were in the
character and amount of thickening of the cell walls.

In many of the permeable seeds some of the canals were found to extend
across the light line, but this was not true for all permeable seeds.
(Pl. V, fig. 8.) Oblique sections of permeable seed coats showed that
the cell cavities, although reduced to mere pores by the thickening of
their radial walls, extended across the light line into the base of the
cones, thus forming a passageway through which the stains passed to the
larger portions of the cell cavities below the light line. (Pl. V, fig.
4.)

In the coats of the impermeable seeds the light line was usually
broader, the Malpighian cells thickened more below the light line,
and the main cavities of the Malpighian cells were more reduced and
farther below the light line than in the coats of permeable seeds. (Pl.
V, fig. 6.) No canals except occasionally a few very small ones were
seen crossing the light line in impermeable seeds. Cross and oblique
sections showed that the lumina of the Malpighian cells were closed
in the region of the light line. Thus it was found that permeable and
impermeable seeds differ mainly in the amount of thickening which
occurs in the walls of the Malpighian cells. In the impermeable seeds
the thickening which begins at the outer tangential wall of the
Malpighian cell extends farther toward the inner tangential wall,
leaving the cell lumina smaller and farther below the light line than
in permeable seeds. The thickening is also more complete in impermeable
seeds, leaving fewer and smaller canals across the light line as well
as closing the cell lumina in the region of the light line.


THE ACTION OF SULPHURIC ACID ON THE COATS OF IMPERMEABLE SEEDS.

Impermeable seeds were soaked in concentrated sulphuric acid (sp. gr.
1.84) for 15, 30, and 60 minutes; then washed and put in the staining
solutions. After they had swollen, they were removed from the staining
solutions, dried, and embedded in glycerin gum. A study of these seeds
showed that the acid had eaten away all of the material outside of the
light line and that the stain had passed through all regions of the
seed coat. (Pl. V, fig. 10.) When observed under the microscope, it was
seen that the action of the acid was rapid, destroying the cuticle,
cuticularized layer, and cones in about 5 minutes. After 15 minutes
treatment with acid the light line, aside from the presence of canals
and pores not previously visible, seemed to be very little affected.
The division lines along which the lateral walls of the Malpighian
cells were joined now became much more distinct across the light line,
thus indicating that there was some swelling in this region. When a
close examination of the path of the stain was made the cell lumina,
and occasionally very small pores, were found to extend across the
light line. The presence of the stain in the pores indicated that
they were paths of the stain across the light line. Some of the stain
passed along the lines between cells and through the occasional canals
crossing the light line, but judging from the intensity of the stain in
the lumina the canals appeared to be the principal passageways.

The action of the acid in opening the cell cavities across the light
line was not determined. It may be due to the swelling of the light
line or to the removal of substances closing the pores.

No seeds were exposed to the acid for longer than an hour, but at the
end of this period the light line was still intact. As compared with
other portions of the Malpighian layer, it is extremely resistant to
concentrated sulphuric acid. Since all cell walls below the light line
are mainly cellulose, the resistance of the light line prevents the
acid from destroying the entire seed coat and reaching the embryo.



LITERATURE CITED.


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(42) White, Jean.

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Transcriber Note


Minor typos may have been corrected. Illustrations were moved to
prevent splitting of paragraphs. Content produced from files generously
provided by the USDA through The Internet Archive and all resultant
files are placed in the Public Domain.





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