Spermatophytes Gymnosperms 247 



In all the 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 the fixing agent. Use formalin alco- 

 hol, unless you have so much pollen that you can lose most of it 

 and still have enough left. 



The pollen germinates readily. Shake some of it into a 10 per 

 cent solution of cane-sugar in water. In two or three days the pollen 

 tubes will appear and in a week or two may grow to several times the 

 length of the grain; but, as far as we know, the generative cell has 

 not divided in sugar solutions (Fig. 85, A). 



The development of the pollen tube and its structures must be 

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

 removed 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 integu- 

 ment, but to remove the female gametophyte; then cut from the 

 under side of the nucellus against the hard, stony layer of the integu- 

 ment so as to remove a small piece of the nucellus 5 to 7 mm. square, 

 according to the species. Fix in Flemming's weaker solution with 

 the osmic acid somewhat weaker than the formula indicates (Fig. 

 85, Band C, and Fig. 86). 



The pollen tubes, with their sperms, make instructive prepara- 

 tions when mounted whole. Fix the nucellus, with its pollen tubes, 

 as if for paraffin sections. About 6 per cent formalin in water has 

 proved successful. Wash in water for half an hour and stain in 

 aqueous safranin, 1 or 2 hours. Extract the stain until it is satis- 

 factory, and then transfer to 10 per cent glycerin and follow the 

 Venetian turpentine method. When the turpentine becomes thick 

 enough for mounting, tease the pollen tubes from the nucellus and 

 mount with pieces of cover-glass under the cover to prevent crushing. 



A skilful technician, with steady hands, can tease out the pollen 

 tubes before fixing. It is easier to judge the stain if the pollen tubes 

 are removed in this way. 



