Expression of Disease Reaction Types in Soybean Callus from Resistant and Susceptible Plants

Abstract

HOLLIDAY, M. J., and W. L. KLARMAN. 1979. Expression of disease reaction types in soybean callus from resistant and susceptible plants. Phytopathology 69: 576-578. Conditions influencing expression of disease reaction types were sections. Differences in colonization rates remained high with inoculum examined in calluses derived from soybean plants resistant (cultivar Cutler doses varying from 50 to 1,000 zoospores per callus section. Sections of 71) or susceptible (cultivar Cutler) to race 1 of Phytophthora megasperma Cutler and Cutler 71 calluses 5 mm thick were colonized equally by race 3 of var. sojae (Pms). Cutler 71 calluses were colonized less than those of Cutler Pms which is pathogenic to plants of both cultivars. No combinations of when both were grown on medium containing 6 or 10 mg of 2,4-D/ L and incubation temperatures, 2,4-D concentrations, sizes of calluses, or numbers incubated at 16 or 20 C prior to and following inoculation with Pms of zoospores used for inoculum resulted in Cutler 71 calluses with the nearly zoospores. Differences between colonization rates of Cutler and Cutler 71 absolute resistance to race 1 of Pms found in whole plants of that cultivar. calluses were greater in callus sections 5 mm thick than in thicker or thinner Plant tissue culture can provide simplified model systems for Soybean calluses, 3-4 wk old, were transferred to fresh B5 studying host-pathogen relationships (6). Helgeson et al (2) medium in Pyrex jars (100 mm X 80 mm) with fitted lids 3 days reported that the single dominant gene which confers resistance to prior to inoculation. Each jar contained five calluses, all from the Phytophthora parasitica var. nicotianae in tobacco plants was same cultivar. Jars were coded and arranged randomly in a expressed in tobacco pith callus cultures. Resistance was expressed, laminar-flow hood and each callus was inoculated with Pms however, only when hormone (2,4-D) concentrations were adjusted zoospores. Zoospores also were placed on V8 agar to check for to yield tight callus, and expression also was influenced by contamination. incubation temperatures and amounts of inoculum (3). Inoculated calluses were rated numerically according to a visual Monogenic resistance to Phytophthora megasperma Drechs. determination of amounts of colonization by Pms. Numerical var. sojae A. A. Hildeb. (Pms) in soybean (Glycine max [L.] Merr.) ratings were: 0 = no mycelial growth; 1 = mycelium colonizing less plants is characterized by a hypersensitive reaction and production than half of the callus; 2 = mycelium colonizing more than half, but of the phytoalexin, glyceollin (7,10). This host-parasite system has not all, of the callus, and 3 = mycelium colonizing the entire callus. been employed as a model in several investigations of disease Ratings were begun 4 days after inoculation and were repeated resistance mechanisms (8). daily for 8 days or until mycelial growth over the agar medium The purpose of this study was to determine if a callus culture made it difficult to rate an individual callus. system might be developed which would allow genotypic expression of soybean cultivars resistant or susceptible to Pms. We RESULTS report conditions which influence expression of these genotypes. Incubation temperature. Calluses of Cutler and Cutler 71 were MATERIALS AND METHODS subcultured three times on B5 medium supplemented with 6 mg of 2,4-D/ L. Thirty calluses of each cultivar were incubated at 16, 20, Callus cultures were obtained from two near-isogenic soybean 24, or 28 C for 24 hr, inoculated with approximately 10 zoospores cultivars, Cutler and Cutler 71. Cutler plants are susceptible and of Pms race 1 per callus, and incubated for 7 days. Calluses of both Cutler 71 plants are resistant to Pms, race 1. Both cultivars are cultivars, incubated at 24 or 28 C, were colonized equally but, at 16 susceptible to Pms, race 3. Callus cultures were grown on the B5 or 20 C, those of Cutler 71 were colonized less than those of Cutler medium of Gamborg et al (1) solidified by addition of 12 g agar per (Fig. 1); maximum differences in colonization occurred on calluses liter. Explants were 5-mm hypocotyl sections obtained from 5-dayincubated at 16 C. This incubation temperature, therefore, was old seedlings grown axenically in the dark at 28 C. Explants were used in succeeding experiments. placed on B5 medium in petri dishes and, after 4 wk, proliferating Concentrations of 2,4-D. Thirty calluses of each cultivar were callus tissue was isolated and transferred to fresh B5 medium. subcultured three times on B5 medium containing 2,6, 10, or 20 mg Subsequent subcultures were made by cutting a callus, which had of 2,4-D/ L and incubated at 16 C for 24 hr. Each callus was grown in 3 wk to about 2.5 cm in diameter, into five pieces which inoculated with about 10 zoospores of Pms, race 1, and incubated were transferred to a single dish of fresh B5 medium. Tissues were at 16 C. Colonization of Cutler 71 calluses was significantly less selected for subculture only on the basis of size and age. All cultures than that of Cutler when they were grown on media with 6 or 10 mg were incubated at 28 C in the dark. of 2,4-D/L (Fig. 2); calluses grown at these concentrations were Cultures of Pms, races 1 and 3, were maintained on V8 agar at24 tighter and less friable than those at other concentrations. C in the dark and zoospores were obtained by the method of Ho Maximum differences in colonization occurred in calluses grown and Hickman (5). Zoospores were counted on a 0.1-ml Palmeron medium with 6 mg of 2,4-D/L and, therefore, that Maloney counting chamber and diluted with sterile distilled water; concentration was used in succeeding experiments. the desired numbers of zoospores were delivered to calluses in Callus sizes and inoculum concentrations. Cylinders were cut single drops from a sterile pipet. with a sterile 16-mm cork borer from centers of 3-wk-old calluses of both cultivars. Cylinders were sliced perpendicular to the long axis 00031-949X/79/000102$03.00/0 into sections 1.0, 2.5, 5.0, and 10-mm thick which were placed in 01979 The American Phytopathologiqal Society Pyrex jars containing fresh B5 medium containing 6 mg of 2,4-D/ L