Chemical structure and biological activity 



substances (Steward and Caplin, 1952). This leads to the general philosophy 

 that in the organized growth of the plant body one factor that suppresses the 

 indefinite growth by cell division is the accumulation of inhibitory substances 

 that poison or regulate the system tending to cause cell multiplication. 

 Conversely, it also leads to the idea that examples in which cells indulge in 

 such proliferative growth, where they would otherwise be expected to 

 remain quiescent, furnish instances where these active growth-promoting 

 systems, uninhibited by antagonists, can be detected. 



Two prominent examples are furnished by (i) the tumors which may be 

 induced by the crown gall organism on plants such as Bryophyllum (Steward, 

 Caplin, and Shantz, 1955) and (ii) the parthenocarpically generated fleshy 

 tissue of the banana fruit (Steward and Simmonds, 1954). In both these 

 cases extracts of the organ in question can be the cause of a growth induction 

 in the carrot tissue explants which is very similar to that normally produced 

 by coco-nut milk. Thus we are led to the idea, not by any means unique to 

 this system, that one of the factors involved in differentiation is that the 

 mechanism which would normally lead to indefinite proliferating growth by 

 cell division is brought under control, either by the entire lack of the growth- 

 promoting substances for this type of growth, or much more probably by 

 the accumulation of inhibitory substances. In short, in the balance between 

 the growth promoters for cell division and their inhibitors lies the clue to 

 the regulatory control of growth in this type of system. 



GROWTH BY CELL DIVISION AND CELL EXPANSION 



The tissue culture system, therefore, presents a powerful tool in the investiga- 

 tion of growth regulation and of the substances that both induce and 

 antagonize growth of an undifferentiated kind. There is, however, a further 

 development necessary to give the technique its full range and usefulness. 

 In much of the work, previously published, the main criterion of growth was 

 the total increment of weight in the growing culture. For a great many 

 general purposes, this suffices. However, for the understanding of the 

 mechanism and for the full exploitation of the technique, it is necessary to 

 differentiate between growth in cell number and growth in cell size. This 

 has long been recognized but the opportunity only presented itself recently 

 for a full investigation of the growth of these tissues at the cellular level. 

 The basis of this will now be described. 



Maceration techniques have been familiar to the plant anatomist for some 

 time. It was, however. Brown and Rickless (1949) who first exploited the 

 maceration technique to trace the course of growth in this way. Following 

 this lead, we have adopted similar procedures to study the growth of these 

 tissue cultures at the cellular level. Since considerable reference will be made 

 to results obtained by this technique, its essential basis is here presented. 



The tissue explants, whether the original explants from the whole organ 

 or those grown under the conditions described, are macerated in a suitable 

 volume of a mixture of 5 per cent chromic and 5 per cent hydrochloric acids. 

 This procedure is normally done in small vials. At the appropriate time, 

 which experience indicates is approximately 5-10 hours at room temperature, 

 the tissue is fragmented as much as possible by agitating the liquid. Following 

 Brown's procedure, a hypodermic needle, through which the fluid is taken 



170 



