POTENTIALLY UNLIMITED GROWTH OF EXCISED PLANT CALLUS IN AN ARTIFICIAL NUTRIENT

ONE OF the ultimate objects of the cultivation of isolated plant or animal organs or tissues has always been, as Haberlandt (1902) long ago pointed out, the investigation of problems concerning the metabolism of the individual cell and its relation to neighboring cells. This object can be fully attained only if the cell can be isolated from its fellows, either anatomically or physiologically. Haberlandt and his immediate pupils sought to isolate cells anatomically. But the same end can be attained approximately if a completely undifferentiated mass of tissue can be grown in which all cells are alike and hence exert like influences on one another. It is for this reason that an "undifferentiated" state has come to be considered one of the irreducible characteristics of a "tissue culture." There is, however, another irreducible characteristic of a true "tissue culture" which must be demonstrated if the metabolism of the cells is to be considered normal. That is unlimited capacity for growth. This question has been repeatedly dis*cussed elsewhere (White, 1931, 1936). As was pointed out in earlier papers (White, 1934, 1936), the root cultures maintained in this laboratory for the past six years satisfy the second requirement, of unlimited capacity for growth, but are by no means undifferentiated. Gautheret's cultures of excised cambium (1934, 1935, 1938a, 1938b), Nobecourt's carrot root cultures (1937, 1938), and Bonner's pea-pod cultures (1936), on the other hand, are relatively undifferentiated and have the appearance of tissue cultures. Gautheret has grown cambium cultures for more than a year, through five or six passages, thus furnishing evidence of a possible capacity for unlimited growth. It seems probable that he has, therefore, fulfilled both these requirements and is to be credited with the first true plant tissue cultures. His observations, however, do leave some doubt on this score. The tissue fragments used in his cultures were relatively large, one to two centimeters in diameter, and contained considerable stored material. The growth rates reported were so low that' transfers could be made only once every 4 to 6. weeks. The cultures may have survived at the expense of the stored nutrients within the explant and may not be capable of unlimited growth. Nobecourt's cultures likewise grew slowly and were of relatively brief duration. Bonner's results were still less satisfactory. A more adequate demonstration is needed than that furnished by the data of these authors. I have for some time been interested in a hybrid tobacco resulting from the cross Nicotiana glauca S X N. langsdorffii cd (Kostoff, 1930; Levine, 1937). This hybrid has an unusual capacity for prolifera1 Received for publication December 23, 1938. tion, producing calluses and galls at any point on stem or leaf which has been mechanically abraded. Callus tissue may also develop in the absence of obvious wounding. It seemed possible that tissues of such a plant might be especially favorable for cultivation in vitro. This paper reports the results of an investigation of this possibility. The nutrient used was the same as that employed for cultures of isolated roots, consisting of a modified Uspenski solution, 2 per cent sucrose and 0.01 per cent of an extract of dried brewer's yeast (White, 1934). It early became evident that most callus tissues would not float on the surface of the nutrient as do roots, so that their oxygen supply