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.