Sweet-Clover Seed

HISTORICAL SUMMARY.

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):

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.

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.

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.

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.

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.

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.