Histological effects of growth-regulating substances on sunflower tissue of crown-gall origin grown in vitro.

VARIOUS GROWTH-REGULATING substances, each at progressively increasing concentrations, have shown marked effects on the development of sunflower and tobacco callus in tissue cultures (Hildebrandt and Riker, 1947). The sunflower tissue came from crown-gall but was bacteria free; the tobacco tissue came from a natural gall on a hybrid. Except for the amount of growth produced, no conspicuous macroscopic differences appeared in these tissues. However, the possibility remained that histological differences might be present. The inhibition of growth of plant tissue with a strong concentration of growth substance has been reported (K6gl et al., 1934; Marmer, 1937; Levan, 1939). Kraus et al. (1936) have shown the increased activity and proliferation of cells of bean resulting in the formation of adventitious roots when treated in certain ways with indole-3-acetic acid. Since then, many investigators have descrihed responses made by different types of plant cells following applications of different growth substances (reviewed by Skoog, 1947, who includes other reviews). The microscopic structure of tobacco callus cultured in vitro has been described by White (1939) and by Caplin (1947). White considered the two main requirements for a true tissue culture are "an undifferentiated state and the unlimited capacity for growth." The effects of growth-regulating substances on "normal" and pathological plant tissue growing in vitro have already received some attention. Gautheret (1945, 1947a) for example. in a series of papers has shown the concentration to be a critical factor in callus development and differentiation. Similarly the effects of indole-3-acetic acid, indole-butvric acid and alpha-naphthalene-acetic acids, respectively, on "normal" plant tissue and bacteria-free tissue of crown-gall origin of sunflower and periwinkle. grown in vitro, was reported by de Ropp (1947). The "normal" tissues responded at high and low concentrations by proliferation of cells of the cambial zone and by root formation. Tissue of crowngall origin remained unchanged in structure, except at the high concentrations where growth of the tissues was reduced. Gautheret (1947a, 1947b) '-Received for publication December 12, 1948. This work was supported 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. Valuable suggestions were received from Dr. J. B. Torrie on the statistical analysis and from Prof. Emma L. Fisk on the manuscript. studied "normal" plant tissue cultures grown in vitro. At a low concentration of indole-3-acetic acid, considerable cell division appeared; whereas cells without the growth substances failed to grow. As the concentration was increased, the frequency of cell division decreased but a swelling of the cells in all directions followed. The present studies were made to determine what, if any, histological changes were induced in the bacteria-free crown-gall tissue of sunflower by the added growth substances. This extends the work already reported by Hildebrandt and Riker (1947). Representative masses of tissues were fixed, sectioned, stained and examined. MATERIALS AND METHODS.-The sunflower used, the cultural procedures, and the amount of growth secured has been described bv Hildebrandt and Riker (1947). Representative tissues from 13,000 individual pieces were selected for examination. They explained that the sunflower tissue was originally isolated in 1941 from a secondary petiolar crown-gall but no longer contained crown-gall bacteria. The weak concentrations of growth-regulating substances on which sunflower tissue cultures examined here were grown, were 1 X 10-11 g./l. for indole-3-acetic acid and for alpha-naphthaleneacetic acid; I X 10-9 g./l. for indole-butyric acid; and 1 X 10-7 g.jl. for parachlorophenoxy-acetic acid. These concentrations were selected because they represented high points on the growth curves already described. The strong concentration for all of the growth substances was 1 X 10-3 g./l. Control cultures on media lacking a growth substance were also examined. After 6 weeks incubation on the media, fragments of the tissue were weighed and then fixed in formalin-aceto-alcohol. The tissue fragments were dehydrated with n-butyl alcohol and embedded in paraffin. Sections 10 , in thickness were cut on a rotary microtome. The stains used were either ironalum hematoxylin and safranin, or fast green and safranin. Microchemical tests (Johansen, 1940) were made for membrane substances. For statistical analysis, the total number of cells and the number of tracheal elements per unit area (approximately 1/4 sq. mm.) were counted. Cells were counted in ten fields in each of five different tissues at each of the concentrations (control, weak and strong) used for the four growth-regulating substances. OBSERVATIONS.-The kinds of cells present and their development were relativelv simple in this