2 So Ishikawa.—Studies on the Embryo Sac 
which was reported by Goldschmidt ( 23 ) as having occurred in those plants. 
Lately Werner ( 73 ) published a paper on the embryo sacs of Oe. biennis , 
Oe. Lamarckiana, and some others, and she discusses in the same paper 
some ecological relations existing between the gametophyte and ovular 
tissues. Besides, there are a considerable number of papers dealing with 
the reduction phenomena in Oenothera , which lie beyond the scope of the 
present paper. 
Although the nuclear phenomena of the gametophyte and fertilization 
of Oenothera seem to have been fully described in the papers referred to 
above, there still remain not a few points which require a careful investiga¬ 
tion. The present work was therefore undertaken in order to get an 
accurate idea about the fertilization, especially with special reference to the 
behaviour of the synergids at the time of the attack of the pollen-tube. In 
the course of the present investigation, the materials selected were found 
very suitable for the purposes. 
Several fixing reagents were tried, but Bouin’s solution as re¬ 
commended by Gates ( 19 ) was found to give the best result. Some good 
results were also obtained by using Gilson’s fluid, recommended by Renner 
( 55 ), while Flemming’s fluids gave bad results, and Juel’s fluid proved still 
less satisfactory. Sections were cut 8-12 /z, sometimes 20 y in thickness. 
Flemming’s triple stain was applied, but very good preparations were 
obtained by staining with safranin followed by light green, saturated in 
clove oil. The latter method, which was recommended by Blackman and 
Welsford ( 4 ), was used to the greatest advantage for any developmental 
stage of the embryo sac, especially for the structures of the egg apparatus, 
before and after the fertilization, on account of its transparency and clear¬ 
ness, good differentiation of the cell-wall, and the capacity of showing a fine 
display of the minute plasmic structure. Heidenhain’s iron-alum-haemato- 
xylin, Congo red, and orange G were also used for certain special 
purposes. 
Before commencing the study of the embryo sac, the number of the 
chromosomes in each species and hybrid were counted in the pollen mother- 
cells just in the reduction division as well as in the dividing cells in the 
young ovular tissue ; 7 and 14, as expected for ^ and ix number, were 
respectively found in them. The pollen and ovular structures were found 
to be also quite identical in one species with any other of these plants 
under consideration. 
Embryo Sac and its Development. 
The meiotic phenomena of the embryo sac mother-cell of Oenothera 
have been repeatedly studied by several investigators, as for instance Geerts 
( 21 ) in Oe. Lamarckiana and Davis ( 15 ) in Oe. biennis. The investigation 
of those interesting phenomena is, however, excluded in the present paper. 
