WILLIAM S. HILLMAN 681 



with aiul ^\•ithout EDTA were subjected to two further conditions. 

 Some were transterred in toto to fresh medium every third day, while 

 others were left in the original medium. The results of this experi- 

 ment are presented in Table 2, which shows that frequent transfer 

 completely prevents the development of flower primordia in M 

 medium but does not do so in the medium with EDTA. The simplest 

 interpretation is that EDTA replaces rather than hastens the "old" 

 medium effect. Another point to be noted in Table 2 is the signi- 

 ficantly lower frond number in the flow^ering as compared to the 

 non-Howering cultures on M medium. In L. gibba, even more than 

 in L. perpusilla (7) , the onset of flow^ering is followed by a decrease in 

 multiplication rate; see also Fig. 3 and caption. 



Many other questions arise concerning the "old" medium effect 

 and its relationship to that of EDTA. One is whether there is any 

 difference between the action of the two on flowering, once the time 

 required to render the medium "old" is eliminated. A second is 

 whether fronds grown in long days but maintained vegetative by 

 frequent transfers to fresh M medium are at least partially induced, 

 so that they will be more responsive to flow^er-promoting conditions 

 than short-day stocks. A third question concerns the role of growing 

 cultures in rendering the medium "old": wdll simply leaving the 

 medium unplanted under the same conditions have the same effect? 

 A factorial experiment directed at these three questions was performed. 



Four kinds of medium were used: M; M with EDTA; M made 

 "old" by growth under long days for 12 days, followed by removal 

 of all fronds under aseptic conditions; and M from flasks prepared 

 and treated like the flasks of "old" medium but not planted. Into 

 these media, two kinds of stocks were inoculated: the standard short- 

 day stocks, or long-day stocks maintained vegetative for 12 days by 



TABLE 2 



Effects of Frequent Transfer on Flowering of L. gibba G3 on M Medium 



WITH OR without 3 X 10"^ M EDTA under Continuous Light. 



See Experimental, B4. Exp. A, 2-10-60 



" Mean ± standard error, 4 flasks, on day of dissection, 

 ''too fronds taken at random from each of 4 flasks. 



