Various species of Pteris are common in greenhouses and are very good for development of sporangia. Any fern of the Aspidium type will yield a good series, and some, like Cyrtomium, may show a fine series in d single sorus. Marattia, which is likely to be found in botanical gardens, will illustrate the "synangium" type; Angiopteris has a sporangium which forms an easy transition to that of the Cycadales.
For the development of a typical sporangium of the eusporangiate type, nothing is better than Botrychium. Buds of B. virginianum taken in September or October show sporangia with well-marked sporogenous tissue. For a study of the development of sporangia, cut off the fertile portion and fix it separately, using chromo-acetic acid with 5 or 6 drops of 1 per cent osmic acid to 100 c.c. of the solution. Stain in iron-alum haematoxylin. The reduction divisions in the spore mother-cell take place after the leaf arrives above the surface.
The Prothallia - Prothallia can usually be found on the pots in the ferneries of greenhouses. Ripe spores of some fern or other can be obtained at any greenhouse at any time in the year, and spores of most of our native ferns germinate well and produce good prothallia, even if the sowing is not made for several months after the spores have been gathered.
Fine prothallia of Pteris aquilina have been grown two years after the spores were gathered. Some, however, must be sown at once, or they will not germinate at all. Spores which are large and contain enough chlorophyll to make them appear greenish should be sown at once. The spores of the common Osmunda regalis, and of the other members of the genus, must be sown as soon as ripe, or they fail to germinate. The prothallia of 0. regalis, if carefully covered with glass, may be kept for a long time, and they become quite large. Prothallia of this fern in the writer's laboratory produced ribbon-like outgrowths 5 mm. wide and more than 5 cm. in length. These prothallia continued to produce archegonia, anther- idia, and ribbon-like outgrowths for more than a year, when they suddenly "damped off." Lang watered prothallia with a weak solution of permanganate of potash, which kills the fungi but does not injure the prothallia. He does not state the strength of the solution, but 4 or 5 crystals to 1 liter of water seems to be effective.
The prothallia of most ferns will grow for a long time under such conditions. Pteris aquilina and many other ferns often furnish a good supply of antheridia 3 weeks after sowing, and the archegonia appear soon after, but it is well to make sowings 6 weeks before material is needed for use. In P. aquilina and in many others, if the spores are sown too thickly only antheridial plants will be obtained. If prothallia are to produce archegonia, they must have sufficient room and nutrition.
While there should always be a study from living material, it is worth while to make permanent mounts, even for habit study. For such study, the prothallia should be mounted whole. Fix in the special chromoacetic-osmic-acid solution. If the material shows any tendency to break up, use 2 c.c. of acetic acid instead of 3 c.c. In cities where water is treated with copper or other substances, the difficulty may sometimes be due to the water rather than to any excess of acetic acid. Formalin-acetic acid (10 c.c. formalin, 5 c.c. acetic acid to 100 c.c. water) is good for material which is to be mounted whole. Stain some in iron-alum haematoxylin, and some in Magdala red and anilin blue. Mount in Venetian turpentine, using material from each stain for each mount. Select stages so that each preparation will show the filamentous stage, the apical cell stage, the group of initials stage, and also antheridia and archegonia.
For sections, the chromic series is better than formalin or corrosive sublimate. If the gradual processes of dehydrating, clearing, and infiltrating have been carefully observed, about 15 or 20 minutes in the bath should be sufficient.
For the development of the antheridium and sperm, and especially for the blepharoplasts and their transformation, cut about 3 ( in thickness and use iron-haematoxylin; for the development of archegonia, cut at 5( and stain in the safranin, gentian-violet, orange combination.
The gametophyte of Botrychium is subterranean and tuberous. It sometimes reaches a length of 7 to 12 mm. and a thickness of 4 to 5 mm. Usually, it is not more than 5 or 6 mm. long and 2 or 3 mm. thick. Gametophytes showing the development of antheridia and archegonia are not likely to be more than 2 or 3 mm. long and 1 or 2 mm. thick. Near large plants, look for small sporelings, not more than 1 or 2 cm. in height. Dig very carefully and you may find the gametophytes attached. The soil should be examined for smaller specimens. Most of the gametophytes will be found at a depth of 1 to 3 cm. Fix in chromo-acetic acid.
No one has yet succeeded in raising the prothallia from the spores. The prothallia always contain an endophytic fungus, but even when this is present no one has succeeded in raising prothallia. The prothallium of Ophioglossum is harder to find or, perhaps it would be better to say, harder to recognize, for it is also subterranean and tuberous and, besides, looks so much like the root that you may not recognize it, even when you have it in your hand.
The Embryo - Instructive mounts of the whole embryo, with the prothallium still attached can be made by the Venetian turpentine method. Iron-alum haematoxylin, with the stain not too deep, is good; Magdala red and anilin blue are more transparent and will show the structure of the root, if the two stains are well balanced.
For sections, cut longitudinally, perpendicular to the prothallium. Pteris longifolia may show the young embryo within 3 or 4 weeks; Osmunda, somewhat later.
Nephrodium is generally parthenogenetic. The embryos, developed while the prothallia are still very small, come from tissue cells of the prothallium. Pteris cretica is frequently parthenogenetic.
The Heterosporous Filicales - The four genera, Pilularia, Marsilia, Salvinia, and Azolla are aquatic, the first two growing rooted but more or less submerged, and the other two floating freely on the water. Marsilia is the most available and convenient laboratory type of this group. It is easily grown in a pond or in an aquarium in the greenhouse. In setting it out in a pond, select a place with a gently sloping bank, so that part of the material may be under water and part may creep up the bank. In the greenhouse, a rectangular aquarium may be tilled to secure the same conditions. The portions which are not under water will continue to fruit during the summer and autumn. The whole sporocarp cuts easily in paraffin during the development of sporangia, the division of the spore mother-cells, and even during the earlier stages in the formation of spores. Except in the case of the youngest sporocarps, it is better to cut off a small portion at the top and at the bottom to facilitate fixing and infiltration. The mother-cell stage and the young spores will be found in sporocarps which are just beginning to turn brown. In nature, no further nuclear divisions take place within the sporangium until the next spring, but the wall of the sporocarp becomes extremely hard. Sporocarps for germinating should not be collected until they are so hard that it is impossible to crush them between the thumb and finger. They can be kept in a box until needed for use. When you find them in good condition, make a big collection, for they retain their power of germination almost indefinitely, sporocarps from poisoned herbarium specimens 50 years old germinating readily. Sporocarps which have been kept in 95 per cent alcohol for years germinate almost as quickly as those which have been kept in a dry box.
To germinate sporocarps, cut away a portion of the hard wall along the front edge and place the sporocarp in a dish of water. The gelatinous ring with its sori will sometimes come out in a few minutes. In less than 24 hours, sometimes within 10 or 12 hours, microspores, starting from the one-cell stage, will produce the mature sperms; and the development of the female gametophyte is equally rapid. Starting with the uninucleate megaspore, the stage found when the gelatinous ring comes out, the archegonium may be developed and fertilization may occur within 12 hours. At the end of a week, there may be green sporophyte more than a centimeter in length.
It is obvious that material should be fixed at frequent intervals if one is to secure a series of stages in the development of the gametophytes and embryo.
For sections showing the development of the antheridium and sperms, it is better to remove the megaspores from the sorus, since they occasion considerable difficulty in cutting. Fix in the special chromo-acetic-osmic-acid solution, cut 5 ( thick, and stain in ironalum haematoxylin.
The older megaspores, with the archegonium or embryos at the apex, are very hard to cut. Fix in hot alcoholic corrosive sublimate- acetic, or in alcohol-formalin-acetic acid. It will facilitate infiltration and cutting it you prick each megaspore with a sharp needle before fixing. Since the archegonium is at the apex of the megaspore, the pricking need do no damage. If sections come loose from the slide, use Land's fixative. Cut 5 to 10( thick and stain in safranin, gentian-violet, orange.
The sperm, which in Marsilia has an unusually large number of turns in the spiral, is easily mounted whole. When the sperms have become numerous, put several megaspores upon a slide and heat gently until dry. Then wet the preparation in any alcohol and stain sharply in acid fuchsin. Dehydrate in absolute alcohol for at least 10 minutes, clear in clove oil, and mount in balsam. Such a preparation will often show a score of sperms in the gelatinous funnel leading down to the neck of the archegonium.
Azolla is not difficult to obtain, and it is easy to get a series of stages in the development of the micro- and megasporangia; but it is not at all easy to find the gametophytes, since the spores germinate only after they have been set free by the decay of the plant. Azolla does not fix well in any of the chromic-acid series, because it catches so much air that it will not sink. Alcohol-formalin-acetic, or hot alcoholic corrosive sublimate-acetic acid, formalin (4 g. corrosive sublimate, 5 c.c. formalin; 5 c.c. acetic acid, 100 c.c. of 50 per cent alcohol) can be recommended for both Azolla and Salvinia. Both of these forms grow well in the greenhouse, floating on the water in tanks or aquaria; but Azolla seldom fruits under such conditions. Salvinia sometimes produces microsporangia in the greenhouse, but megasporangia are comparatively rare.
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