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diff --git a/old/62998-0.txt b/old/62998-0.txt deleted file mode 100644 index 9cb6626..0000000 --- a/old/62998-0.txt +++ /dev/null @@ -1,2638 +0,0 @@ -Project Gutenberg's USDA Bulletin No. 844, by H. S. Coe and J. N. Martin - -This eBook is for the use of anyone anywhere in the United States and -most other parts of the world at no cost and with almost no restrictions -whatsoever. You may copy it, give it away or re-use it under the terms -of the Project Gutenberg License included with this eBook or online at -www.gutenberg.org. If you are not located in the United States, you'll -have to check the laws of the country where you are located before using -this ebook. - - - -Title: USDA Bulletin No. 844 - Sweet-Clover Seed - -Author: H. S. Coe - J. N. Martin - -Release Date: August 21, 2020 [EBook #62998] - -Language: English - -Character set encoding: UTF-8 - -*** START OF THIS PROJECT GUTENBERG EBOOK USDA BULLETIN NO. 844 *** - - - - -Produced by Tom Cosmas from files generously provided by -the USDA through The Internet Archive and placed in the -Public Domain. - - - - - - - - - - -Transcriber Note - -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. - - -(1) Beck, Gunther. - - - 1878. Vergleichende Anatomie der Samen von Vicia und Ervum. _In_ - Sitzber. K. Akad. Wiss. [Vienna], Math. Naturw. Kl., Bd. 77, - Abt. 1, p. 545-579, 2 pl. - -(2) Bergtheil, C, and Day, D. L. - - 1907. On the cause of "hardness" in the seeds of Indigofera - arrecta. _In_ Ann. Bot., v. 21, no. 81, p. 57-60, pl. 7. - -(3) Crocker, William. - - 1906. Role of seed coats in delayed germination. _In_ Bot. Gaz., v. - 42, no. 4, p. 265-291, 4 fig. Literature cited, p. 290-291. - -(4) Darwin, Charles. - - 1885. The effects of cross and self fertilisation in the vegetable - kingdom. 482 p. New York. - -(5) Ewart, Alfred J. - - 1908. On the longevity of seeds. _In_ Proc. Roy. Soc. Victoria, v. - 21, pt. 1, p. 1-203. Literature, p. 3-4. - -(6) Gola, Guiseppe. - - 1905. Ricerche sulla biologia e sulla fisiologia dei semi a - tegumento impermeabile. _In_ Mem. R. Accad. Sci. Torino, s. 2, - t. 55, p. 237-270, 1 pl. - -(7) Haberlandt, G. - - 1877. Ueber die Entwickelungsgeschichte und den Bau der Samenschale - bei der Gattung Phaseolus. _In_ Sitzber. K. Akad. Wiss. - [Vienna], Math. Naturw. Kl., Bd. 75, Abt. 1, p. 33-47, 2 pl. - - Hanstein, [Johannes]. - -(8) 1863. Erläuterung des Nardoo genannten Nahrungsmittels der - Urbewohner Australiens, einer Marsilea-Frucht, nebst Bemerkungen - zur Entwicklung dieser Gattung. _In_ Monatsber. K. Preuss. Akad. - Wiss. Berlin, 1862, p. 103-119, 1 pl. - -(9) 1866. Pilulariae globuliferae generatio cum Marsilia comparata. - 16 p. Bonnae. Dissertation. - - Harrington, George T. - -(10) 1915. Hard clover seed and its treatment in hulling. U. S. - Dept. Agr., Farmers' Bul. 676, 8 p. - -(11) 1916. Agricultural value of impermeable seeds. _In_ Jour. Agr. - Research, v. 6, no. 20, p. 761-796, 6 fig., pl. 106. Literature - cited, p. 796. - -(12) Harz, C. D. - - 1885. Landwirtschaftliche Samenkunde. 1362 p., 201 fig. Berlin. - -(13) Hiltner, L. - - 1902. Die Keimungsverhältnisse der Leguminosensamen und - ihre Beeinflussung durch Organismenwirkung. _In_ Arb. K. - Gesundheitsamte, Biol. Abt., Bd. 3, Heft 1, p. 1-102, 4 fig. - -(14) Hughes, H. D. - - 1915. Making legumes grow. _In_ Farm and Fireside, v. 38, no. 19, - p. 7, illus. - -(15) Huss, Mathias. - - 1890. Über Quellungsunfähigkeit von Leguminosensamen und Mittel zu - deren Abhilfe. 73 p. Halle. Dissertation. - -(16) Junowicz, R. - - 1878. Die Lichtlinie in den Prismenzellen der Samenschalen. _In_ - Sitzber. K. Akad. Wiss. [Vienna], Math. Naturw. Kl., Bd. 76, - Abt. 1, p. 335-352, 2 pl. - -(17) Kerner von Marilaun, Anton. - - 1891. Pflanzenleben. 2 Bd. Leipzig. - -(18) Kirchner, O. - - 1905. Über die Wirkung der Selbstbestäubung bei den Papilionaceen. - _In_ Naturw. Ztschr. Land-u. Forstw., Jahrg. 3, Heft 1, p. 1-16; - Heft 2, p. 49-64; Heft 3, p. 97-111. Literatur-verzeichnis, p. - 110-111. - -(19) Knuth, Paul. - - 1906-8. Handbook of flower pollination, based on Hermann Müller's - work upon "The fertilisation of flowers by insects," transl. by - J. R. Ainsworth Davis. 3 v., illus. Oxford. Bibliography, v. 1, - p. 212-380. - -(20) Lohde, Georg. - - 1874. Ueber die Entwicklungsgeschichte und den Bau einiger - Samenschalen. 42 p., 1 pl. Naumburg. Dissertation. - -(21) Love, Harry H., and Leighty, Clyde E. - - 1912. Germination of seed as affected by sulphuric acid treatment. - N. Y. Cornell Agr. Exp. Sta. Bul. 312, p. 293-336, fig. 78-85. - -(22) Lutts, F. M. - - 1917. Sweet clover, advantages of the crop for soil improvement. - _In_ Ohio Agr. Exp. Sta. Mo. Bul., v. 2, no. 2, p. 45-47, illus. - -(23) Malpighi, Marcello. - - 1687. Opera omnia, seu thesaurus locupletissimus - botanico-medico-anatomicus ... New ed. 2 v., pl. Lugduni - Batavorum. - -(24) Marlière, H. - - 1897. Sur la graine et spécialement sur l'endosperme du Ceratonia - siliqua. _In_ Cellule, t. 13, fasc. 1, p. 5-60, 2 pl. - -(25) Martin, J. N. - - 1915. Relation of moisture to seed production in alfalfa. Iowa Agr. - Exp. Sta. Research Bul. 23, p. 301-324, 2 fig. - -(26) Mettenius, Georg. - - 1846. Beitraege zur Kenntniss der Rhizocarpeen. 65 p., 3 pl. - Frankfurt am Main. - -(27) Die Hohenheimer Samenritzmachine. - - 1894. _In_ Braunschweig. Landw. Ztg., Jahrg. 62, Nr. 19, p. 86. - illus. - -(28) Müller, Hermann. - - 1883. The fertilisation of flowers, transl. by D'Arcy W. Thompson. - 669 p., 186 fig. London. Bibliography, p. 599-630. - -(29) Nobbe, Friedrich. - - 1876. Handbuch der Samenkunde. 631 p., 339 fig., 1 pl. Berlin. - -(30) --------, and Haenlein, H. - - 1877. Ueber die Resistenz von Samen gegen die äusseren Factoren der - Keimung. _In_ Landw. Versuchs Sta., v. 20, p. 71-96, 13 fig. - - Pammel, L. H. - -(31) 1899. Anatomical characters of the seeds of Leguminosae, - chiefly genera of Gray's Manual. _In_ Trans. Acad. Sci. St. - Louis, v. 9, p. 91-275, pl. 7-35. Bibliography, p. 224-257. - -(32) 1886. On the structure of the testa of several leguminous - seeds. _In_ Bul. Torrey Bot. Club, v. 13, no. 2, p. 17-24, pl. - 52-53. - -(33) Rees, Bertha. - - 1911. Longevity of seeds and structure and nature of seed coat. - _In_ Proc. Roy. Soc. Victoria, n. s., V. 23, pt. 2, p. 293-414, - pl. 79-81. - -(34) Russow, Edmund. - - 1872. Vergleichende Untersuchungen betreffend die Histiologie - (Histiographie und Histiogenie) der vegetativen und - sporenbildenden Organe und die Entwickelung der Sporen der - Leitbündel-Kryptogamen, mit Berücksichtigung der Histiologie der - Phanerogamen, ausgehend von der Betrachtung der Marsiliaceen. - _In_ Mém. Acad. Sci. St. Petersb., s. 7, t. 19, no. 1, 207 p., - 11 pl. - -(35) Schleiden, M. J., and Vogel, J. R. Th. - - 1842. Über das Albumen, inbesondere der Leguminosen. _In_ Nova - Acta. Acad. Caes. Leop. Carol. Nat. Cur., v. 19, pt. [2], p. - 51-96, pl. 40-45. Reprinted. - -(36) Sempolowski, Anton. - - 1874. Beitraege zur Kenntniss des Baues der Samenschale. 57 p., 3 - pl. Leipzig. Dissertation. - -(37) Tietz, A. O. Q. - - 1876. Über die Keimung einiger Coniferen und Laubhölzer bei - verschiedenen aber constanten Temperaturen. 44 p., 8 pl. - Reudnitz-Leipzig. Dissertation. - -(38) Tunmann, O. - - 1913. Pflanzenmikrochemie. 631 p., 137 fig. Berlin. - -(39) Verschaffelt, E. - - 1912. Le traitement chimique des graines à imbibition tardive. _In_ - Rec. Trav. Bot. Néerl., v. 9, livr. 4, p. 401-435. - -(40) Westgate, J. M., and Coe, H. S. - - 1915. Red clover seed production: Pollination studies. U. S. Dept. - Agr. Bul. 289, 31 p., 7 fig. Literature cited, p. 29-31. - -(41) Wettstein, Richard von. - - 1888. Beobachtungen über den Bau and die Keimung des Samens von - Nelumbo nucifera Gärtn. _In_ Verhandl. K. K. Zool.-Bot. Gesell. - Wien, Bd. 38, p. 41-48, pl. 1. - -(42) White, Jean. - - 1908. The occurrence of an impermeable cuticle on the exterior of - certain seeds. _In_ Proc. Roy. Soc. Victoria, v. 21, n. s. pt. - 1, p. 203-210, 2 pl. - -(43) Wigand, Albert, and Dennert, E. - - 1888. Nelumbium speciosum W. Eine monographische Studie. 68 p., 6 - pl. Cassel. (Bibliotheca Botanica, Heft 11.) - -(44) Young, W. J. - - 1906. The embryology of Melilotus alba. _In_ Proc. Ind. Acad. Sci. - 1905, p. 133-141, 50 fig. Bibliography, p. 136. - - - ================================ - - ADDITIONAL COPIES - - OF THIS PUBLICATION MAY BE PROCURED FROM - - THE SUPERINTENDENT OF DOCUMENTS - - GOVERNMENT PRINTING OFFICE - - WASHINGTON, D. C. - - AT - - 15 CENTS PER COPY - - - - * * * * * - - -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. - - - - - -End of the Project Gutenberg EBook of USDA Bulletin No. 844, by -H. S. Coe and J. N. 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