Sporophytes: Study and cultivation

Sporophytes in early stages of development often yield good preparations without very much trouble, but in later stages they are frequently difficult to cut on account of the secondary thickening of the capsule wall and the stubborn exine of the mature spores. Great care must be taken to get Riccia natans into paraffin without shrinking, and the same thing may be said-of other forms which have such loose tissue with large air cavities. The gradual processes already described have obviated the difficulty, so that the student should be able to get thin paraffin sections as free from distortion as were the old celloidin sections. But even with well-fixed material great care must be taken not to let the paraffin get too hot. Remember that in most paraffin ovens the temperature is different in different parts of the oven. Do not let the temperature of the paraffin go above 53 C., and, preferably, not above 52 C. In Riccia natans it is even more difficult to get median longitudinal sections of the sporophyte than of the archegonium. Sections perpendicular to the groove, whether longitudinal or transverse, are almost- sure not to give median longitudinal sections of the sporophyte, and these are the sections the beginner is sure to cut. Examine the material and note very exactly the orientation of the sporophyte; then, for fixing, cut out sections about 2 mm. thick, taking these sections in such a plane that paraffin sections parallel to the thick section will give the desired median longitudinal sections of the sporophyte.

In forms like Pellia and Aneura, it is desirable to show the sporophyte still enclosed in the calyptra. For such sections, we should recommend fixing in formalin alcohol. Aqueous fixing agents are likely to cause trouble on account of air bubbles. For cytological studies, the calyptra must be removed and a thin slab should be cut from opposite sides of the capsule to facilitate fixing and infiltration. Chromo-acetic acid, with the addition of a little osmic acid, is best for fixing. In Pellia and Conocephalus the spores are very large and have a rather thin wall. Both these genera show a peculiar intrasporal development of the gametophyte, i.e., the gametophyte develops to a considerable extent before it ruptures the spore wall and before it is shed from the capsule. Mitotic figures during the first three divisions in these spores are exceptionally beautiful and are very easy to stain with the safranin, gentian-violet, orange combination, the chromosomes taking a very brilliant red, while the asters take the violet. Achromatic structures are very prominent during these three divisions, but become less and less conspicuous as division progresses and before the intrasporal stage is over, the radiations are scarcely demonstrable.

For the older sporophytes of Marchantia, it is better not to cut the whole receptacle. Remove the radiating branches. The sporophytes are in radiating rows, alternating with the branches. A piece 2 mm. wide can be cut so as to include two of the radiating rows, one on each side of the stalk, and such a piece will include early stages in other rows. By taking such care, you can get median longitudinal sections of nearly all the sporophytes. For class work, 5 to 10( is a good thickness, but for figures, especially the reduction mitoses in the spore mother-cells, the sections should not be thicker than 2 or 3(.

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