260 Methods in Plant Histology 



while to keep the ovules from each separate. Stages like that shown 

 in Fig. 90 are rare in miscellaneous collections, but if ovules from 

 each cone are kept separate and this figure is found, the rest of 

 the ovules from that cone will be likely to show some phase of this 

 interesting mitosis. 



Thuja and Juniperus are good types to illustrate the arche- 

 gonium complex and the large, highly organized male cells. In 

 Thuja a series from the appearance of archegonium initials to 

 young embryos may be collected between June 10 and June 20. In 

 Juniperus pollination occurs late in May and fertilization takes 

 place 12J months later. The megaspores are formed late in April 

 and the development of the female gametophyte occupies about 6 

 weeks. 



The Embryo.- -The early stages of the sporophyte, usually 

 designated as the proembryo, have been mentioned already. 



From the time when the suspensors begin to elongate up to the 

 appearance of cotyledons, instructive preparations can be made by 

 mounting the embryo whole. Mr. J. T. Buchholz has developed a 

 method for handling these small objects. Remove the testa and 

 then, under water, hold the endosperm gently with forceps and 

 press the neck and upper part of the archegonium with a needle, 

 pressing, and at the same time drawing the needle away, so as to pull 

 the young embryo out. Some of the embryos will be broken, but 

 by careful manipulation more than half should be entirely uninjured. 

 Fix in formalin (5 per cent in water), stain in Delafield's haematoxy- 

 lin, transfer to 10 per cent glycerin, and continue with the Venetian 

 turpentine method. A preparation made in this way is shown in 

 the photomicrograph, Fig. 91. 



These stages, and all subsequent stages, are easily cut in paraffin 

 without removing the embryo from the endosperm. Cut a thin 

 slab from opposite sides of the endosperm, fix in chromo-acetic acid, 

 with or without a little osmic acid, imbed in paraffin, and stain in 

 safranin and gentian-violet. This will give a good view of the abun- 

 dant starch and other food stuff, and at the same time will bring out 

 sharply the cell walls of the embryo. 



