264 PLANT GROWTH SUBSTANCES 



kinds which provided indirect support for the view that auxin may act 

 as a coenzyme. If this is true, it means that auxin must act in one or 

 perhaps several, but nevertheless definite, metaboUc systems. Further- 

 more, from the fact that quantitative growth responses to appUed 

 indoleacetic acid are obtained only over a restricted range of concentra- 

 tions, it may be deduced that other components of this "auxin reaction" 

 system must also be present in relatively limited quantities in the plant. 

 It was of interest, therefore, to determine what this system and its other 

 components might be. A possible experimental approach to this problem 

 became available with the development by White (14) of callus tissue 

 cultures which could be grown indefinitely on a medium of known 

 composition. White found that callus of the hybrid Nicotiana glauca X 

 N. langsdorffii grown in submerged cultures would differentiate to pro- 

 duce numerous buds (15), and one of us found (9) that the addition of 

 indoleacetic acid (lAA) or naphthaleneacetic acid (NAA) to the culture 

 medium would completely prevent the initiation of buds. Even low 

 concentrations (o.i mg./l. or less), which did not retard the rate of 

 increase in fresh or dry weights of the tissues, effectively prevented 

 bud formation, whereas higher concentrations inhibited also the rate 

 of growth of the callus. In short, the tobacco tissue cultures have a very 

 sensitive response to lAA and are a favorable material for the testing 

 of various organic compounds which might normally interact with auxin. 

 On the above assumption of an auxin reaction system, such compounds 

 should counteract the inhibiting effect of lAA on bud formation and 

 at the same time should increase the growth of the callus in media with 

 added lAA. 



Substances were selected for testing which are, or might be, required in 

 relatively large amounts in respiration. Tests were first carried out with 

 the four-carbon dicarboxylic acids, malic and succinic acids. The results 

 were negative. Such effects on bud formation as were obtained by the 

 addition of these acids could be ascribed entirely to lowering of the 

 pH of the medium. A careful study of the relation of pH to bud forma- 

 tion (16) showed that the optimum was in the range from 4 to 4.5 (9). 



The effects of increased concentrations of phosphate and sucrose were 

 tested with positive results. The data from one experiment are sum- 

 marized in Table i. It may be seen that about half the callus pieces form 

 buds in the control nutrient solution and that none form buds in the 

 cultures with added lAA. With increased KH2PO4 and sucrose contents, 



