342 



METHODS IN PLANT HISTOLOGY 



After walls appear there is less danger. From the middle of May to 

 the first of July collections should be made at intervals of two or three 

 days, since during these six weeks the gametophyte completes the free 

 nuclear stage and develops cell walls, the archegonium completes its 

 entire development, the egg is fertilized, and the sporophyte may 

 reach the suspensor stage. 



At the stages shown in Figure 119 B-D, it is a good plan to remove 

 the female gametophyte with its proembryos from the ovule; but at 



z*^^.. 



B 



'/*«^^ ' 



:.i' ^^'t /^ 



z? 



Fig. 119. — Pinus laricio: A, top of prothallium, with an archegonium just before the cutting 

 off of the ventral canal cell; fixed in Flemming's weaker solution and stained in Haidenhain's iron- 

 alum haematoxylin; collected June IS, 1897. B, C, D, early stages in the development of the em- 

 bryo; fixed in chromo-aoetic acid and stained in safranin, gentian violet, orange; collected July 2, 

 1897. X104. 



the stages shown in Figures 119 ^ and 120 the pollen tubes with their 

 contents are rapidly working their way through the nucellus toward 

 the archegonia, and consequently, in some of the material, it is better 

 to retain enough of the tissues of the ovule to keep the nucellus in 

 place. In later stages, after fertilization has taken place, the develop- 

 ing testa should be removed with great care, for a very slight pressure 

 is sufficient to injure the delicate parts within. 



With any fixing agent of the chromic-acid series, the free nuclear 

 stages of the female gametophyte and, later, the archegonia and pro- 



