THE INFLUENCE OF THE COMPOSITION OF THE MEDIUM ON GROWTH IN VITRO OF EXCISED TOBACCO AND SUNFLOWER TISSUE CULTURES

PATHOLOGICAL CELL proliferation in plants is of special interest because of the possible relationship to diseased growth in animals and man. Therefore, the crown-gall disease incited by Phytomonas tumefaciens (Smith and Town.) Bergey et al. has been studied extensively. The early literature on crowngall has been reviewed by Riker and Berge (1935) and by Levine (1936). Although much work has been done with crown-gall bacteria, little is known yet about the physiology of the host cells, because of certain technical difficulties involved in such studies. The plant tissue culture technique offers certain opportunities for studies of tissue metabolism not possible with intact plants. For example, isolated cell masses may be cultured in vitro on a medium which, except for traces of impurities, contains only known chemicals. There are no neighboring cells, tissues, and organs to influence development. The tissue cultures either grow or fail to grow because of the materials originally either absent from or present in the medium and because of the tissue metabolites. The influences of temperature, pH# sucrose concentration, certain plant extracts, and crown-gall bacterial products on growth of plant tissue cultures in vitro have been reported earlier (Hildebrandt, Riker, and Duggar, 1945, 1946). Certain of these extracts at low concentrations caused increased growth of tissue cultures, but often they inhibited growth of the cultures at higher concentrations. Since certain extracts stimulated growth of tissue cultures, this stimulation might have been due either to the introduction of more favorable quantities of certain of the constituents of the basic medium or to factors not present in it. For example, certain preliminary experiments suggested that higher concentrations of iron resulted in better growth of the tissue cultures. Therefore, the influence of various concentrations of salts and vitamins in the basal medium on growth of tobacco and sunflower tissue cultures was studied in three salt solutions. A report on part of the work herein reported has already appeared in an abstract (Hildebrandt, Riker, and Duggar, 1944). MATERIALS AND METHODS.-The materials and methods were essentially the same as those described earlier (Hildebrandt, Riker, and Duggar, 1945). 1 Received for publication March 15, 1946. This work was supported in part by the International Cancer Research Foundation and the Wisconsin Alumni Research Foundation. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. 2 The writers are indebted to Eugene Herrling for preparing figures 1 and 2. Tobacco and sunflower tissues were used. The tobacco tissue was from the hybrid Nicotiana gtauca Grah. 9 X N. langsdorfii Weinm. d and was isolated and supplied by Dr. P. R. White. The second tissue was isolated from "secondary" crown galls on sunflower (Helianthus annuus L. var. Giant Russian). Both tissues were free of crown-gall bacteria and were capable of unlimited grctwth in vitro. The composition of the basal medium (White, 1942) is repeated later for comparison with improved media. Reagent grade chemicals, except sucrose, ferric tartrate, boric acid, and potassium iodide, were used, and a record was kept of the impurities in the salts. The agar was leached by -washing in distilled water at least 3 times daily over a 3to 4-day period. All experimental cultures were incubated in 125 ml. Erlenmeyer flasks containing 50 ml. of medium. The flasks were washed previously in potassium dichromate-sulfuric acid cleaning solution and rinsed in tap and distilled water. The experimental cultures were maintained in the dark at 260 + 10C. in a special culture room. Twenty-four tissue pieces (four per flask) were used per treatment, and each experiment was repeated three times unless otherwise noted. To determine the amount of growth the tissue pieces were removed from the flasks after a 6-week incubation period and weighed individually. All data were analyzed statistically to determine significance between treatments.3 Media prepared according to the triangular method were used to study the influence of increasing and decreasing concentrations of each of the constituents of the basal medium. Ferric tartrate, ferric sulfate, and thiamine hydrochloride were sterilized by filtration; all others were autoclaved. The three salts under test were added aseptically to the culture flasks to avoid precipitation. The growth on nineteen different media of representative sunflower tissue and the concentrations of the three salts under test from one such experiment are shown in figure 1, A and B, respectively, as an example of the method. A tissue grown on the unchanged basal control medium is shown at the upper left of the triangle; tissues grown on media without one of the three salts under test are shown at the upper right of the triangle (fig. 1, A). The omission of one nutrient did not mean necessarily that those materials were lacking completely because, for example, of impurities in the washed agar, the salts, the sugar, and in the carry-over of various nutrients with the seed tissue pieces, which were grown on the basal medium.