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.])