298 METHODS IN PLANT HISTOLOGY 



The gametophytes. — In most cases the spores germinate while still 

 within the sporangium and, in some cases, like Selaginella apus, the 

 female gametophyte develops up to the archegonium initial stage be- 

 fore shedding. If strobili of this species at the stage shown in Figure 93 

 be broken off and laid down on moist ground so as to keep the sporan- 

 gium moist, dehiscence may not be vigorous enough to discharge the 

 megaspores; but development of archegonia and antheridia will con- 

 tinue, fertilization will take place, and an embryo will be developed 

 while the megaspore is still inclosed within the sporangium. Such a 

 structure satisfies the definition of a seed. Ordinarily, to get the later 

 stages, shake the spores out from the older strobih into a Petri dish 

 with the bottom covered by several thicknesses of wet filter paper. 

 There is enough nutritive material in the spores to carry them up to 

 the sperm and egg stage. The female gametophytes within the old 

 spore coats generally orient themselves in the paraffin, the base of the 

 spore being down and the archegonium end of the gametophyte being 

 up. A little of some nutrient solution, added to the water, will carry 

 the development up to the cotyledon stage. All stages after the mega- 

 spore has fallen out from the sporangium may be fixed in the Chicago 

 chromo-acetic-osmic solution. After washing in water, the very hard 

 outer spore coat may be softened somewhat by treating with 5 per 

 cent hydrofluoric acid for 2 days. The gametophyte is so delicate at 

 this stage that a stronger acid will injure the tissues. Wash for 24 

 hours to remove all traces of hydrofluoric acid. 



The young embryo, with its two cotyledons, its root, and the mega- 

 spore still attached, makes an instructive preparation when mounted 

 whole in Venetian turpentine. 



The various stages of the female gametophyte and embryo are not 

 hard to stain; but the walls throughout the development of the male 

 gametophyte are very thin and extremely hard to stain. Safranin and 

 light green is a good combination. Light green in clove oil is more 

 satisfactory than the alcoholic solution. 



Isoetes. — This peculiar genus is widely distributed, and 16 of its 

 64 species occur within the United States. It looks so much like a 

 sedge that it is easily overlooked, even when rather abundant. As a 

 genus, it is hydrophytic, growing in wet places or even under water. 

 A monograph by Dr. Norma Pfeiffer not only gives an ecological, 

 morphological, and taxonomic account (with keys in English) but 

 gives hundreds of stations, a feature which will enable many to find 

 material. 



