SPERMATOPHYTES— GYMNOSPERMS 333 



this, in a thin layer of protoplasm surrounding a large central vacuole 

 are the most difficult structures to fix well that the botanist encoun- 

 ters. They occur in various groups of plants. 



For sections of the entire ovule, use safranin, gentian violet, 

 orange; for free nuclear stages in both gametophyte and sporophyte, 

 use iron-haematoxylin with a touch of orange; for the megaspore 

 membrane, safranin seems to be the best stain. 



The entire ovule, even at a late embryo stage, makes an effective 

 demonstration when cleared whole in equal parts of xylol and carbon 

 disulphide. If living material is available, it would be worth while to 

 try Gourley's basic fuchsin method. 



GYMNOSPERMS— CONIFERALES 



Since Pinus is an available laboratory type, we shall describe meth- 

 ods for demonstrating various phases in the life-history of this genus, 

 hoping that the directions will enable the student to experiment in- 

 telligently with similar forms. The dates are for Pinus laricio in the 

 vicinity of Chicago, but dates will be different for different species and 

 even for the same species in different regions; P. laricio, at Chicago, 

 sheds pollen about the middle of June, but P. maritima at Auckland, 

 New Zealand, sheds its pollen about the first of October. After a year's 

 collecting in any region, there should be no difficulty, since the dates 

 do not vary much from year to year. 



The vegetative structures. — The stem, root, and leaf will be treat- 

 ed separately. 



The stem. — The vascular cylinder is an endarch siphonostele, a type 

 which, with few exceptions, is found throughout the living gymno- 

 sperms. 



The young stem in its first year's growth is green and soft and is 

 easily cut in paraffin. The best time to collect material is soon after 

 the young shoot has emerged from the bud scales in the spring. 

 Resinous material, like these young stems, do not fix well in aqueous 

 media. With a thin safety razor blade, cut the stem transversely into 

 pieces about 5 mm. in length; fix in formalin (10 c.c), acetic acid 

 (5 c.c), 70 per cent alcohol (85 c.c), for several days, or until needed 

 , for use. Imbed in paraffin, and stain in safranin and light green, or in 

 safranin and Delafield's haematoxylin. 



Longitudinal sections of the buds in winter or early spring condition 

 are instructive for comparison with longitudinal sections of the ovu- 



