LIFE HISTORY OF PINUS 13 



of Pinus Strobus were again collected regularly, as described 

 above, throughout the spring and early summer of 1899. Collec- 

 tions of the staminate cones of Pinus Strobus and P. rigida 

 were made during May and June 1901, and from May 15 to 

 June 15 of the same year the young pistillate cones of Pinus 

 rigida were gathered daily. 



Material was obtained from different trees and different locali- 

 ties. The practice of collecting all one's material from a single 

 tree, as reported by Murrill (1900), Land (1902) and others, does 

 not seem a safe one to follow, for certain peculiarities of develop- 

 ment which are not characteristic of the species may appear in 

 a*n individual. At the time of each collection, ovules were put 

 up from several cones of each species, these cones being taken 

 not from the tip of one branch but from different branches. 

 The central portion only of the cone was used, the ovules at 

 either extremity being more or less abortive. After collecting, 

 the material was taken at once to the laboratory and preserved. 

 The staminate cones and, in the early stages of development, 

 the pistillate ones were fixed entire or cut into quarters longitu- 

 dinally. Very soon the individual scales of the female cones 

 were removed from the receptacle before fixing, and, when 

 the scales were of sufficient size to admit of such manipu- 

 lation, all superfluous parts were cut away, leaving the two 

 tiny ovules still united by a small portion of the scale. 

 With the renewal of growth in the spring, the ovules were 

 removed from the scales and, as soon as it was feasible, 

 a portion of the integument was cut away from two or 

 more sides of each ovule, thus bringing the fixing fluid into 

 direct contact with the young gametophyte. For later stages, 

 the endosperm was frequently removed from the integument, 

 but such material did not prove to be as satisfactory as that in 

 which the nucellar cap and a small portion of the coat were 

 left in connection with the prothallium. Throughout the entire 

 mechanical process of preparing material for the fixer, the most 

 extreme care was used, as it was found that a very slight pres- 

 sure was sufficient to cause distortions and thus to render the 

 material worthless for cytological studies. 



Fixing. The methods used in fixing and staining do not 



