Influence of some growth-regulating substances on sunflower and tobacco tissue in vitro

THE WELL-KNOWN responses of plant materials to various growth regulators have suggested a study of their effects on plant tissues as they were grown in vitro. This is a part of a larger work on fundamental aspects of pathological growth, in which mu%h attention has been given to the crown-gall disease caused by Phytomonas tumefaciens (Smith and Town.) Bergey et al. The early literature was reviewed by Riker and Berge (1935) and by Riker, Spoerl and Gutsche (1946). These growth regulators may be among the critical factors in the balances resulting, respectively, in normal or pathological growth (Riker, 1942). Tissue cultures have advantages for certain work over studies with whole plants. For example: (1) Simple, callus-like tissue may be grown vegetatively in vitro for unlimited periods free from variations arising from pollination. (2) Since the nutrients consist only of known chemicals, the basal metabolism can be studied with relatively simple and controlled procedures. (3) Changes in both kind and character of cells may be induced under controlled conditions. (4) The tissues may be exposed a long time to a wide range in concentrations of materials, including cell-stimulating and inhibiting agents, while other factors are constant. (5) It is possible to determine how far the tissue develops by itself or is dependent on other parts of the plant. Growth regulators show their presence in whole plants, for example, by epinasty, formation of adventitious roots, inhibition of bud development, stimulation of cambial activity, and delayed abscission of old leaves. A number of natural and synthetic growth regulators may produce various ones of these effects and either stimulate or inhibit abnormal cell proliferation (reviewed by Grieve, 1943; van Overbeek, 1944; Thomson, 1945). The exact role they play in crown gall is not established. Large numbers of growth regulators have been tested on many kinds of whole or decapitated plants (Thompson, Swanson and Norman, 1946,; Zimmerman, 1942, 1943). The influence of certain growth regulators has already been tested on growth in vitro of "normal" plant tissue (reviewed by Nobecourt, 1943; Gautheret, 1945). Indole-3-acetic acid particularly was important for growth of plant tissue cultures from many species and was indispensable in some cases. Gautheret (1945) noted that indole-butyric acid and naphthalene-acetic acid 1Received for publication April 14, 1947. This work was supported in part by the Donner Foundation and by the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. The authors are indebted to Eugene Herrling for preparing fig. 1-10. could be substituted for indole-3-acetic acid when indole-3-acetic acid was needed for tissue cultures. With some species these materials modified the shape and kind of cells in the cultures (Gautheret, 1945). However, the effects of these materials on pathological plant tissue growing in vitro have received only limited attention. Indole-3-acetic acid and alpha-naphthalene-acetic acid -prevented bud, leaf, and stem formation on tobacco callus in a liquid medium (Skoog, 1944). The present paper describes the influence of some representative natural and synthetic growth regulators on excised tissues in vitro. It aims to clarify the stimulating or inhibiting effects on growth of various substances, their critical concentrations, and whether they act alike on two different tissues. An abstract of some of this material has already appeared (Hildebrandt and Riker, 1946b). MATERIALS AND METHODS.-Tissue cultures from two species were used. The sunflower tissue was isolated from a "secondary" crown-gall on Helianthus annuus L. var. Giant Russian and was free of of the crown-gall bacteria. The tobacco tissue (P. R. White strain) was from the hybrid Nicotiana glauca Grah. 9 X N. langsdorffii Weinm. S. Both tissues have already been described and illustrated (Hildebrandt, Riker and Duggar, 1945). The cultural procedure was essentially as given by Hildebrandt, Riker and Duggar (1945). Four tissue pieces were grown in each 125 ml. Erlenmeyer flask on 50 ml. of a basal, mineral-salt, agar medium. The basal medium for the sunflower tissue has been described (Hildebrandt, Riker and Duggar, 1946a). A similar medium (Hildebrandt, 1944) for the tobacco tissue had the following ingredients in milligrams per liter of distilled water: 400 Na2SO4, 800 Ca(NO3)2 4H20, 180 MgSO4 7H20, 160 KNO3, 65 KCI, 33 NaH2PO4 H2O, 9 MnSO4 4H2O, 3 ZnSO4 7H2O, 3 H3BO3, 1 KI, 20 ferric tartarate, 3 glycine, and 0.1 thiamine hydrochloride. All these chemicals were of reagent grade except boric acid and potassium iodide. In addition each liter contained 20 g. of sucrose and 5 g. of Difco agar. The growth-regulating compounds used and their sources were as follows: cysteinehydrochloride, indole-3-acetic acid, indole-butyric acid, parachlorophenoxy-acetic acid, alpha-naphthalene-acetic acid, alpha-naphthalene-acetamide, and beta-naphthoxy-acetic acid (Eastman Kodak Company); 2,4dichlorophenoxy-acetic acid, and 2,4-dichlorophenoxy-butyric acid (Dow Chemical Company); and sodium 2,4-dichlorophenoxy-acetate (E. D. Whitman, Ohio State University). The sodium 2,4-dichlorophenoxy-acetate, indole3-acetic acid, and cysteine hydrochloride, respec-