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.