CHLOROPHYLL FORMATION 427 



by Dangeard. Artari (1902) cultivated Stichococcus bacilaris for 4 years, 

 and Dangeard (1921) cultivated Scenedesmus acutus for 8 years in the 

 dark without the organisms' losing their green color. During their 

 sojourn in the dark the organisms were frequently transferred to fresh 

 culture media. 



Of course, when organisms are grown in the dark, they must be sup- 

 plied Avith an organic source of carbon and suitable nitrogenous constitu- 

 ents. Considerable effort has been spent in determining the nutritional 

 requirements for growing algae in the dark and yet maintaining their 

 green color (see Oltmanns, 1923 ; Kufferath, 1913 ; Ludwig, 1938). Under 

 favorable nutritional conditions many algae grow and maintain their 

 green color in the dark. There are others, however, e.g., Chlorella vari- 

 egata (Beijerinck, 1904), C. luteo-viridis (Kufferath, 1913), C. vulgaris 

 (Finkle et at., 1950), and several species of Euglena (Pringsheim, 1948- 

 1949), which readily lose their chlorophyll in the dark. That such organ- 

 isms lack some specific substance essential for chlorophyll formation 

 which they cannot produce in the absence of light is suggested by the 

 feeding experiments of Pallares et al. (1945). These workers discovered 

 that E. viridis required the addition of limited quantities of vitamins C 

 and H in addition to the usual organic nutrients in order for them to 

 maintain their green color. 



The chlorophylls produced in darkness are the same as those produced 

 in the light. The following organisms have been examined with respect 

 to this property: Scenedesmus acutus (Dangeard, 1921), Chlorella vulgaris 

 (Radais, 1900; Myers, 1940), C. pyrenoidosa (Van Hille, 1938), Nostoc 

 punctiforme (Etard and Bouilhac, 1898), and Protococcus sp. (Myers, 

 1940). Myers demonstrated, in particular, the presence of chlorophyll b 

 both by spectrographic and by chromatographic methods. He stated 

 that the ratio of chlorophyll a to chlorophyll b is of the same order of 

 magnitude in cells grown in the light and in the dark. 



The physiology of dark-grown pine seedlings is very instructive in 

 regard to chlorophyll formation. The researches of Schmidt and of 

 Bogorad have special significance. Schmidt (1924) demonstrated the 

 close relation between embryo and endosperm in the production of green 

 cotyledons. He separated the white embryos from the endosperms of 

 Pinus sylvestris and placed them on moistened filter papers at 27°C in a 

 dark room for 4 days. The embryos grew from 3-mm length to 6 mm 

 but showed no trace of chlorophyll by visual or spectroscopic examination. 

 The embryos when placed in diffuse daylight for 5 days became distinctly 

 green. Embryos of other species, P. strohus, P. pinaster, P. jeffreyi, and 

 Biota orientalis, behaved similarly. The embryos grew, so that it was not 

 the lack of vitality which hindered greening. 



When the embryos were left in contact wuth only a small piece of 

 endosperm, they greened. Any part of the embryo left in contact with 



