CHEMICAL CONTROL OF GROWTH AND BUD FORMATION IN TOBACCO STEM SEGMENTS AND CALLUS CULTURED IN VITRO

EARLIER REPORTS (Skoog et al., 1942; Skoog, 1944) have presented evidence for competitive inhibition of auxin effects by compounds structurally related to auxin (indoleacetic acid) and have shown that the formation of buds in tobacco tissue cultures can be prevented by addition of indole-3acetic or naphthalene acetic acids (IAA or NAA) to the nutrient medium. From these results the conclusion could be drawn that auxin exerts its effect on growth through its role as a component of a "reaction system," and that the type of effect is dependent on the concentration of auxin, not per se but in proportion to the concentrations of other components of this quantitatively limited system. It became of interest, therefore, to determine possible components of this system and to study the effects on growth of varying their concentrations. The callus tissue cultures developed by White (1939), in which prevention of bud formation is a very sensitive response to added auxin and which permit long time treatments without complications from micro-organisms, seemed to be favorable material for such studies. Evidence presented by Thimann and Sweeney (1937), Sweeney and Thimann (1938-1942) and by Commoner and Thimann (1941) indicated a definite role of auxin in the respiration of Jvena coleoptiles. Furthermore, data by H. F. Clements (private communication) demonstrated a correlation between phosphate content and ability of lateral buds of sugar cane to grow out. Experiments were therefore set up to test whether increases in the concentrations of substances such as phosphate and sugar, which are known to be required in large amounts for respiration, would affect growth, organ formation and the sensitivity of these processes to added auxin in tobacco callus cultures. The results of such experiments and of their extension to experiments with adenine and related compounds will be presented to show (1) that bud formation in callus tissue and in stem segments of tobacco cultured in vitro can be induced by chemicals added to the medium and (2) that nearly complete control of the type of growth which will occur, i.e., the formation of roots, buds or undifferentiated growth of tissues can be obtained by the application of different proportions of auxin and adenine to the medium. MATERIALS AND METHODs.-In the first series of experiments, carried out from 1941 to 1944, a strain of White's tobacco Callus (Nicotiana glauca X N. langsdorfii) kindly supplied by him in 1939 was used. The work was discontinued in 1944 because of war duties and the strain was lost. On resump1Received for publication August 3, 1948. This work was supported in part by the University Research Committee on funds from the Wisconsin Alumni Research Foundation. tion of the work in 1947 several new cultures from the same original strain were again provided by Dr. White, but these had become contaminated with an anaerobic bacterium and are unsuitable for this work. New Strains of callus have, therefore, been developed from stem internode segments of N. tabacum. Wis. #38 and use has been made of the stem internode segments themselves. The internodes were cut from the upper portions of plants ca. 2 ft. tall and growing rapidly in soil in the greenhouse. They were surface sterilized by swabbing with 70 per cent alcohol followed by immersion for 20 min. in 0.1 per cent HgCl2 solution. The epidermis was then removed and pieces cut into 7 mm. long quadrants which were cultured on nutrient agar in 125 cc. Erlenmeyer flasks. Each segment, therefore, contains all tissues present in the stem except the epidermis and portions of cortex and pith. All materials were grown on White's basic medium of the following composition in mg./1. of water: Ca(NO3)2 100, KNO3 80, MgSO4 35, KCI 65, KH2PO4 12.5, KI 0.75, MnSO4 4.4, Fe2(SO4)3 2.5, ZnSO4 1.5, H3BO3 1.6, and sucrose 20000. In experiments carried out 1941-1944 only glycine (2 mg./l.) and thiamin chloride (0.1 mg./l.) were used as accessory nutrients. -In the experiments 1947-1948 nicotinic acid (0.5 mg./l.) and pyridoxin (0.5 mg./l.) were used in addition to the above. Fifty cc. of medium was used per 125 cc. flask. The pH of all cultures was adjusted with HCI or NaOH to initial values of about 5.0. Pvrex glassware and pyrex redistilled water were used throughout. Solid media contained 0.7 per cent agar. Usually two pieces of tissue were placed in each flask, but in a few experiments either one or three pieces were used. Other details of the culture methods were the same as described (Skoog, 1944). The cultures were kept at room temperature in cabinets without artificial illumination but exposed to very weak diffuse light. Data will be presented for only a few experiments, but general conclusions are drawn from the experience with over 4000 cultures. RESULTS.-Bud formation in callus tissue cultures.-Tobacco callus tissues when cultured submerged in liquid media generally grow as undifferentiated callus for a few weeks and then many pieces produce numerous small buds. The addition of IAA or NAA completely prevents the formation of buds. This is true even with such low concentrations as do not decrease the rate of growth in fresh or dry weight. Effect of increased phosphate and sucrose levels. -Experiments were carried out with increased amounts of KH2PO4 and sucrose. In order to maintain iron in available form the Fe2(SO4)3 content was increased together with the phosphate. The