326 METHODS IN PLANT HISTOLOGY 



4 c.c). Renew the fixing agent. Don't let it dry up. After an hour, 

 run down gradually through the alcohols to water and stain in iron- 

 alum haematoxylin. 



Fix later stages in formalin acetic alcohol. Transverse sections are 

 easier to cut since the peripheral end of a sporophyll can be cut only 

 in early stages. Sporogenous tissue appears first at the base of the 

 cone and last at the top; but pollen ripens first at the top and last at 

 the bottom. 



In all genera of cycads, the microspore germinates while still within 

 the sporangium, the pollen grain, at the time of shedding, consisting of 

 a prothallial cell, a generative cell, and a tube cell. For preparations 

 at the shedding stage, shake the cone over a piece of paper and pour 

 the pollen into water. After 15 or 20 minutes, put it into the fixing 

 agent. In wind-pollinated plants, the pollen would look shriveled if 

 you put it into a fixing agent before it regained its turgidity. 



The pollen, mounted whole, makes beautiful preparations. Fix in 

 formalin-acetic acid (formalin 10 c.c, acetic acid 5 c.c, water 100 

 c.c), and let it remain here for a week, shaking it gently once in a 

 while. Stain in iron-alum haematoxylin and follow the Venetian tur- 

 pentine method. Or, run it up to 85 per cent alcohol and, after 2 days, 

 run it back to water. Then stain and follow the Venetian turpentine 

 method. Since the pollen makes fine preparations when cut at 2 to 5 /x 

 in paraffin, the lot may be run up to 85 per cent alcohol and then part 

 may be taken back to water, while the rest goes on to paraffin. Pow- 

 ers' methods, described on page 217, are satisfactory for pollen to be 

 mounted whole. 



If some material is put into a 5 or 10 per cent sugar solution for 2 or 

 3 days, early stages in the formation of the pollen tube may be added. 

 In a week or two the tubes may reach several times the length of the 

 pollen grain; but, as far as we know, the generative cell has never 

 divided in culture solutions (Fig. 112 A). 



The development of the pollen tube and its structures must be 

 studied in sections of the nucellus. As soon as the integument is re- 

 moved the nucellus is exposed and the position of the pollen tubes is 

 easily determined, since the haustorial portions of the tubes form 

 brownish lines radiating from the nucellar beak. Having learned the 

 location of the pollen tubes, it is better not to remove the integument, 

 but to remove the female gametophyte; then cut from the underside 

 of the nucellus against the hard, stony layer of the integument so as 



