O’ NEAL: EMBRYO SAC DEVELOPMENT IN OENOTHERA 137 
became more or less crowded they were set out in small pots 
and, after several transferals, they were transplanted about May 
1 in the university garden, where they were staked to prevent 
breakage by the wind and given the usual cultivation. TExtT FIG. 
1 shows three of our young plants with their usually shaped 
leaves. 
Treatment of material—The ovaries in various stages of 
development were partly stripped of their outer wall and dropped 
into the chromo-osmo-acetic solution. After two or three hours, 
they were transferred to a solution lacking the osmic acid, as 
recommended by Davis (5) for Oenothera material. For the 
younger stages of the development of the ovule, this treatment 
gives excellent results, but for the older stages, after the nucellus 
has become extensively developed, it is not so satisfactory. At 
this time the sporogenous cells are so deeply imbedded in the nu- 
cellar tissue that fixation is very difficult. For this part of the 
study a number of killing and fixing agents were tried out. The 
chromo-osmo-acetic mentioned above, Carnoy’s fluid, corrosive 
sublimate acetic solution, and Bouin’s fluid. Bouin’s fluid, 
recommended by Gates (10) and used by Ishikawa (17), was 
found to give the best results and was used in securing all 
stages after the megaspore tetrad was formed. 
After the usual method of dehydration and imbedding, 
sections 4-20 uw in thickness, depending upon the stage of 
development, were made and stained with anilin-safranin and 
gentian violet followed by orange G in clove oil. In all, 204 
ovaries were cut and examined in sections. Considerable con- 
fidence is felt in the statements of development and attending 
conditions that are given below. 
The ovule-—The ovule of Oenothera rubrinervis is of the 
usual anatropous type and is borne on the end of a short funi- 
culus. One ovary was found in which the ovule-stalks were 
forked and bore two ovules. This abnormality may be added to 
the long list already noted by Gates (9) and others for the 
genus. At the time of fertilization the ovule is possessed of an 
outer integument composed of about four layers of cells and an 
inner one of from one to three layers. The integuments at this 
time entirely surround the large nucellus in which the embryo 
sac is deeply imbedded. The arrangement of the parts of the 
