768 Botanical Microtechnique 



(1935) for the morphology and seasonal sequence of the reproductive 

 cycle in other genera and orders, and adapt the methods described 

 here to other subjects. 



Staminate cones of Pin us are differentiated during the season prior 

 to the shedding of pollen. Cones can be dissected from buds and the 

 stage of microsporogenesis ascertained by means of acetocarmine 

 smears. Several species of Pin us undergo meiosis early in May, in the 

 Chicago region. Killing fluids do not penetrate readily into large 

 masses of highly resinous tissues. It is therefore necessary to subdivide 

 all but the very smallest cones. Kill in FAA for general morphological 

 studies and in a Nawaschin type, such as Craf II, for more critical 

 details. Nuclei of microspores and mature pollen grains are stained 

 adecpiately in hemalum-erythrosin. For the first gamctophytic somatic 

 mitosis, which takes place in the microspores before they are shed, use 

 iron hematoxylin or safranin-fast green. 



Preparations of the ovule history are much more difficult and 

 time-consuming to make than the pollen history. The time of 

 occurrence of interesting and important states varies with the species, 

 the locality, and probably in a given locality in accordance with the 

 weather conditions. In the Chicago area the megasporocyte of Pimis 

 hirido is evident when the cones emerge from the I)ud. Fertilization 

 has been found toward the end of June, while early embryo stages are 

 obtainable during July (Chamberlain 1935) . 



The deep-seated megasporocyte is not reached readily by killing 

 fluids, necessitating the use of vigorous fluids that produce distortion. 

 The very young cones may be fixed entire in strong chrome-acetic, FAA, 

 or /'Vi/i-bichloride of mercur). Such preparations are of interest 

 principally to the student of developmental morphology. It may be 

 preferable to cut away the young ovules from the sporophyll and 

 strive to }jreserve the sporogenous and gamctophytic feature. Strong 

 chrome-acetic seems to ha\c gi\en the best results lor most students 

 of this group, riie Nawaschin modifications and Alkn-liouin modi- 

 fications deserve further study. 



Staining of ovulate structures is particularly difficult. Resinous 

 materials in the cells tend to make the jMeparations unsightlv, 

 although the essential nuclei may l)c ckarlv differentiated. Safranin- 

 fast green meets the requirements for all but research needs. 



After the first lew di\isions of the zygote, microtome sections are 

 no longer adequate lor the siiul\ of emt)ryology. Ihe development of 

 dissection methods has facilitated great progress in such studies. 



