1906] CHAMBERLAIN—OVULE OF DIOON 327 
sion layer at the base of the petiole of the sporophyll, so that the 
whole sporophyll drops easily when the seeds are ripe. A second 
abscission layer, which separates the ovules from the sporophyll, 
does not mature until a much later period. 
Il. MATERIAL AND METHODS. 
In March the ovulate cone has almost reached its full size, and 
the staminate cone of the same season has decayed. The first 
staminate cones were sent from the field on June 30, 1904. These 
showed the four microspores still held together by the wall of the 
spore mother cell. From this point to the discharge of the motile 
sperms a complete series in the development of the male gameto- 
phyte was secured. In the female gametophyte the series is com- 
plete from the appearance of the archegonium initial to the germi- 
nation of the seed. The series showing the development of the 
embryo is very complete. 
Nearly all the material was fixed in chrom-acetic acid in various 
Proportions, with or without the addition of osmic acid. The fol- 
lowing formula is excellent for the pollen tube structures and for 
young ovules: chromic acid, 18"; glacial acetic acid, 4°°; 1 per cent 
osmic acid, 2°¢; water, roo°*. This fluid will not penetrate the 
microsporangia, and with older ovules it not only causes some plas- 
molysis but makes the endosperm very hard to cut. After the 
endosperm has become starchy, better results were secured by using 
a slight modification of a formula suggested by Dr. Lynps JONEs: 
5° per cent. alcohol, r00°°; commercial formalin, 6°°. This reagent 
penetrates well and fixes rapidly. Iron-alum haematoxylin stains 
brilliantly after it, but the safranin gentian-violet orange combina- 
tion does not give as bright a stain as with material fixed in chromic 
solutions. After this reagent, the starchy endosperm is not so hard 
to cut. After the stony coat of the ovule has become hard, it is 
almost impossible to cut it with any knife without injuring the arche- 
gonia. For such stages the ovules were sawed in two transversely 
with a fine wire-like fret saw. The upper part of the endosperm 
with its archegonia was then trimmed into suitable shape for cutting. 
The extremely thin blade of the Gillette safety razor, soldered to any 
suitable handle, is particularly adapted for such trimming, since it 
