A Simple Technique for the Establishment of Nitrogenase in Soybean Callus Culture

Abstract

A nitrogen-fixing association can be readily established in vitro between Rhizobium and cultured soybean root cells. Plant cells are grown as a thin callus on the surface of solid defined medium containing low levels of inorganic nitrogen and inocu-lated with bacteria during the active phase of growth. Acety-lene reduction activities up to 275 nmoles ethylene/hr g dry weight of cells have been recorded. The system is compact, uses inexpensive glassware, and facilitates multiple replication of samples. The use of a single medium throughout the experiments avoids unnecessary disturbance to the cells. The development of nitrogenase activity is relatively rapid and its activity can be recorded within a 3-week period. Relatively little is known of the factors controlling infection or the development of nitrogenase in the nitrogen-fixing symbiosis between leguminous plants and bacteria. The sensitive acetylene reduction assay technique for the detection of nitrogenase activity (4) and the demonstration that symbiosis can be established between Rhizobium and plant cell tissue cultures in vitro (5) allow a novel approach to study these problems. The system described by Holsten et al. (5) utilized relatively large volumes of soybean cells grown in a klinostat. The infection process took place in a liquid nutrient medium containing coconut milk and 2,4-D. After 3 to 7 days, the cells were transferred to a similar medium lacking 2,4-D, to allow the establishment of nitrogenase activity. We were unable to detect nitrogenase using the Holsten et al. technique (5), nor could we form a symbiotic association in suspension culture in conventional vessels. Therefore, we tried establishing a symbiosis in soybean callus culture grown on solid nutrient. Nitrogenase activity could not be detected when we used the Lindsmaier and Skoog (6) and Eriksson (1) media or the Murashige and Skoog (7) medium used by Holsten (5). However, positive results were occasionally obtained using Gamborg's (3) B5 and B5c media. These have a lower inorganic nitrogen content than the other media tested, indicating that low levels of fixed nitrogen might promote the symbiotic process. Reduction of the nitrogen level in Gamborg's (3) medium resulted in the establishment of a reproducible nitrogen-fixing system. This paper describes the medium and the experimental conditions used. MATERIALS AND METHODS Plant cell suspension cultures of root explants of eight varieties of soybean (Glycine max) were established by Gamborg's method (2). The cells used as inoculum were grown in 250-ml Erlenmeyer flasks containing 50 ml of B5 medium supplemented with 2 ,ug/ml 2,4-D on a rotatory shaker (150 rpm, 1.25 inch eccentricity) at 26 C in the dark and subcultered into fresh medium (1:5 dilution) after 5 to 7 days incubation. Stock cultures of seven strains of Rhizobium japonicum were maintained under similar growth conditions in a liquid medium (5) and transferred at weekly intervals. For whole plant studies, seeds were germinated singly in pots containing a vermiculite-sand (3:1) mixture under greenhouse conditions. The seedlings were exposed to Rhizobium at the first leaf stage, and nitrogenase activity of intact roots (4) was measured 7 weeks after planting at the time of maximum activity. After the assay, nodules were harvested and weighed, and nitrogenase activity was expressed per gram of nodule tissue.