Chemical structure and biological activity 



and these were adapted by means that have been described to experiments 

 that can be carried out in a quantitative manner. The essential outlines of 

 these experiments only will be described here because the methods have been 

 published elsewhere (Caplin and Steward, 1949; Steward, Caplin, and 

 Millar, 1952). In making this summary of the technique, however, an 

 opportunity will be taken to mention certain refinements that have not 

 hitherto been published in full. 



TISSUE CULTURE METHODS 



The tissue of which the greatest use is made in these investigations is the 

 secondary phloem tissue of the carrot root. Essentially the same methods 

 have, however, been applied to the culture of artichoke tuber tissue and of 

 potato tuber tissue. All of these tissues are essentially secondary in origin 

 but the extent to which they may embark upon rapid, external, proliferative 

 growth is determined by the conditions to be described: these include the 

 use of certain isolated and now-known substances or the use of extracts that 

 contain growth-promoting substances. 



The tissue of the storage organ is isolated aseptically in the form of small 

 pieces, or cylindrical 'plugs', which weigh approximately 2 mg. Special 

 steps are taken to ensure that the tissue explants are removed from as closely 

 identical positions in the organ as possible. An essential feature of the tech- 

 nique is that, in given experiments, explants are all removed from the same 

 individual organ, be it root or tuber. In this way variability is kept to a 

 minimum. In our experience there is no method by which already cultured 

 tissues can be subdivided in a random fashion to produce populations of 

 explants that are as uniform in their subsequent growth as the tissues isolated 

 from the intact organ by the means which have been described (Caplin, 1 947) . 

 It is not necessary to enlarge here upon the accuracy with which these 

 techniques can be performed although one may refer to the discussion that 

 has taken place regarding the use of relatively large, or relatively small, 

 explants (Heller, 1953). The use of small explants is in these experiments 

 essential to exclude those minute centres of growth that would permit the 

 tissue to respond without the addition of the particular formative substances 

 that are here under investigation. We can merely say that in our hands the 

 use of small explants, and even transfers of isolated floating cells from a 

 suspension, is capable of giving a greater degree of uniformity than any other 

 means known to us. 



The tissue culture procedure which is preferred uses growth of the tissue 

 explants in a liquid medium contained in special tubes arranged around 

 a horizontal shaft revolving about one revolution a minute, so that the tissue 

 spends part of its time in air and part of its time bathed in water. The type 

 of growth curve obtained under these conditions with carrot phloem explants 

 has been described (Caplin and Steward, 1949). The familiar criterion of 

 growth in these experiments has been the change in fresh weight of the 

 individual explant. The medium in which the best growth has occurred has 

 been a standard tissue culture medium (White, 1943), supplemented by 

 whole coco-nut milk, the liquid endosperm from the relatively mature nut 

 (Caplin and Steward, 1948). With suitable modifications of the medium, 

 potato tissue can be successfully cultured in a similar way (Steward and 



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