696 J. P. Nitsch and C. Nitsch 



Auxin tests. To determine the auxin activity of various fractions, 

 the first internode test and the sensitized coleoptile tests were per- 

 formed as described previously (26). 



Stability of the Tomato Juice Factor 



The tomato juice factor (TJF) was found to be relatively stable 

 when autoclaved with the medium at pH 5.5 for 20 min. at 15 lbs. 

 pressure. On the other hand, an experiment showed that autoclaved 

 TJ (pH 5.0) left at room temperature under sterile conditions but ex- 

 posed to air through a cotton plug had lost its activity one month 

 later. 



Extraction Procedures 



The TJF was extracted from both dried and fresh material. After 

 many preliminary experiments, the two following methods were found 

 to give good results: 



From dried material. Fresh tomatoes were frozen at — 21° C, then 

 broken into pieces while in the frozen state, and lyophilized. The re- 

 sulting dry powder was extracted stepwise with the following sol- 

 vents (Figure?): 



1. Petroleum ether (b.p. 30 to 60° C): This produced a yellow 

 liquid which fluoresced red in UV light and contained an in- 

 hibitory principle which was partially destroyed by autoclaving. 



2. Benzene: The extract did not seem to contain any active sub- 

 stances. 



3. Ethyl acetate: The dark green extract did not seem to contain 

 active substances. 



4. Cold methanol: The light green extract was partitioned between 

 ether and water containing 0.5 per cent concentrated HCl. The 

 ether fraction, which picked up all the chlorophyll, had little 

 biological activity. The golden yellow aqueous fraction, on the 

 contrary, contained both growth-promoting and growth-inhibit- 

 ing substances. 



5. Boiling methanol: This fraction had the greatest growth-pro- 

 moting activity, together with some growth-inhibiting activity. 



6. Boiling water: This fraction still had appreciable growth- 

 promoting activity. 



In short, the stepwise extraction procedure showed that: (a) pe- 

 troleum ether extracts an inhibitor which is partially destroyed by 

 boiling, and (b) the growth-promoting factor is preferentially water- 

 soluble, together with a strong inhibitor which is also water-soluble. 



From fresh material. Fresh tomatoes were stored frozen until needed. 

 They were autoclaved for 10 to 15 min. at 15 lbs. pressure, in order 

 to inactivate all the enzymes. The juice was then pressed out, filtered, 

 and the remaining pulp washed with enough hot water to obtain a 



