Some of the ferns are sure to be available in almost any locality, and all stages in the lifehistory are easily secured, except early stages in the Ophioglossaceae.
Vegetative Structure - From a technical standpoint, the vegetative structures of Filicales present a wide range of conditions, some being so soft that the greatest care must be taken to get them into paraffin, while others are so hard that it is almost impossible to cut them at all.
The stem - Growing points, even of the largest ferns, can be cut in paraffin. If the growing point is covered with dense hairs or ramentum, either remove the covering entirely or, in case of rather fleshy ramentum, remove only the scales which are beginning to turn brownish. The white scales will fix and cut. Use chromo-acetic acid (1 g. chromic acid and 1 c.c. acetic acid to 100 c.c. water). Unless mitotic figures are particularly desirable, it is just as well not to add any osmic acid. For illustrating the development of the stem from the apical cell, sections 10, 15, or even 20( are not too thick.
Older portions of the stem, or rhizome, in most ferns are easily cut while fresh, the sections being transferred to 95 per cent alcohol after cutting. But even fairly well-developed rhizomes, after the xylem has become lignified sufficiently to stain sharply in safranin, can be cut in paraffin, and much finer sections can be obtained than by cutting without imbedding (Fig. 81). In digging up rhizomes, do not merely dig down until the rhizome can be grasped and then pull it up, for such material is sure to show the pericycle of the bundles torn away from the parenchyma. Dig carefully around the rhizome and then with a very sharp knife cut off pieces which are perfectly free. The pieces can be wrapped in wet paper and taken to the laboratory. Then, if they are to be cut without imbedding, cut into pieces about 3 cm. long; but material which is to be imbedded should be in pieces not longer than 1 cm.
Pteris aquilina is a good form to practice with. For freehand sections, cut as thin as possible, allow 15 to 30 minutes in 95 per cent alcohol, transfer to 50 per cent alcohol and stain over night in safranin. Rinse in 50 per cent alcohol and stain in light green. Paraffin sections 2 or 3 cm. back of the apex are very instructive, for only the protoxylem will be sufficiently lignified to stain red with safranin, the metaxylem still being thin walled and staining with the light green. This rhizome affords an excellent illustration of a mesarch polystele.
Dicksonia punctilobula has a small rhizome, often on the surface of the soil or rock, so that it is easy to get good clean pieces. About 2 or 3 cm. back of the growing point, the xylem is well lignified, but the material still cuts well in paraffin. One could hardly find a better illustration of a mesarch amphiphloic siphonostele.
Botrychium is widely distributed but individual plants are not abundant. The stem is erect, subterranean, and has an endarch siphonostele with secondary wood. Trim away the roots, which arc very thick and fleshy in B. obliquum, fix in formalin-alcohol-acetic acid, and imbed in paraffin. Even the older parts of old stems car be cut in paraffin if you are sufficiently careful. Transverse sections from the base of the bud down to the secondary wood will give a beautiful series in the development of the stele.
The bud at the top of this rhizome is an interesting object. The leaf is in its fourth year when it appears above ground, and, consequently, the bud contains young leaves of three successive seasons. Two of the three show a differentiation into sterile and fertile portions.
In Osmunda, and in many other ferns of similar habit, the rhizome is surrounded by the very hard leaf bases. Good sections of the central cylinder can be secured only by dissecting away these hard leaf bases and any hard portions of the cortex before attempting to cut sections. A short distance back of the growing point will be found a region which will show practically all the structures of the mature stem, which will be easy to cut. Even in this region the leaf bases should be dissected away. From the apical cell back to the region where the sclerenchyma is beginning to turn brown, the material is easily cut in paraffin. Older portions should be cut freehand. Osmunda affords an excellent illustration of the mesarch siphonostele.
The rhizome of Adiantum affords a good illustration of leaf gap and leaf trace. The vascular cylinder is a mesarch siphonostele; but there are few sections like Figure 81, because the clyinder is so interrupted by leaf gaps. This rhizome cuts well without imbedding. The petiole of Botrychium, in transverse sections below the fertile spike, shows the interesting leaf-trace situation which proves that the fertile spike consists of a pair of pinnae fused together.
The ferns of the Gray's Manual range afford no very satisfactory material for illustrating the protostele, although protosteles occur in Lygodium and Trichomanes. The most satisfactory material is Gleichenia, a very common and very beautiful fern in tropical and subtropical regions, but almost never seen in greenhouses nor even in botanical gardens. Formalin alcohol material is easily cut without imbedding and is easy to stain.
The stems of tree ferns require special treatment. With the large leaf bases partly cut away with a sharp razor, transverse sections are easily cut for a considerable distance below the apex. Material fixed in formalin alcohol cuts very well. If fresh material is to be cut, the softer portions should be flooded with alcohol after each section. Farther down, there will be a region where sections can be cut without any flooding, and still farther down, it will be difficult or impossible to cut sections across the whole stem. Sections 1 or 2 cm. thick, cut smooth on the ends, may be kept in 95 per cent alcohol or in glycerin in large glass dishes of the Petri dish pattern. Better still, clear such sections in xylol and preserve in cedar oil.
The root - The roots of Filicales develop from a strong apical cell. For mitotic figures and the development of the root from the apical cell, fix the tip in chromo-acetic acid with a little osmic acid. If the development of the root is the principal object, stain in safranin and light green, or in the safranin, gentian-violet, orange combination; if mitotic figures are to be studied, stain in iron-haematoxylin with a very light counter-stain in orange. The comparatively large roottips of Botrychium are excellent for the apical cell and its segments. Dicksonia punctilobula can also be recommended; but even the very small root-tips of most of our ferns will yield good preparations.
Roots of tree ferns are sometimes available in greenhouses. In some species the stem is covered by a dense felt of small roots, some of which will be white and soft at the tip. These roots are likely to have about the diameter of onion root-tips, and the beauty of preparations made from them could hardly be excelled. In the tropics, where the plants are often in the spray of cataracts and the lower part of the trunk is often washed by mountain streams, a thousand tips might be secured from a single specimen.
The older roots of Botrychium, especially the large fleshy roots of B, obliquum, cut very easily and show a simple exarch protostele with 4 or 5 protoxylem points.
The roots of Angiopteris and Marattia, which become as large as a lead pencil, maybe secured in some greenhouses. They cut easily after fixing in formalin alcohol and furnish a fine example of the exarch protostele, common to all roots.
The structure of the leaf will appear in sections cut to show the sporangia.
The Sporangia - To illustrate the character of the annulus, select sporangia which are just beginning to turn brown. Fix in formalin alcohol and dehydrate as if for paraffin sections; after the absolute alcohol, transfer to 10 per cent Venetian turpentine. Staining is neither necessary nor desirable.
The various relations of sorus and indusium are best illustrated by rather thick sections (10 to 20() of material in which the oldest sporangia have barely reached the spore stage. Fix in formalin alcohol and stain in safranin and anilin blue.
For the development of sporangia use hot corrosive sublimate- acetic acid-alcohol, or chromo-acetic acid (1 g. chromic acid and 2 c.c. acetic acid to 100 c.c. of water). A larger proportion of figures will be secured by adding 5 or 6 drops of 1 per cent osmic acid to this solution. Sections should not be more than 10( in thickness and, for mitotic figures, 5( is thick enough.
For the reduction of chromosomes, the sections should not be thicker than 5(. Osmunda is particularly good for this purpose because the number of chromosomes is comparatively small. The young sporangia of Osmunda cinnamomea and 0. Claytoniana show the mother-cell stage in the autumn, but the division into spores does not occur until the following spring, in the vicinity of Chicago, the mitotic figures being found during the latter part of April, regalis does not reach the mother-cell stage in the autumn. Material for mitosis should be collected during the first two weeks in May.
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