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A suspension culture of soybean (Glycine max L.) was grown on a defined medium in whichi the nitrogen sources were nitrate (25 mM) and ammonium (2 mM). The source (3, 4, 8, 10, 16). Supplementing the media with mixtures of amino acids or commercial preparations of hydrolyzed proteins frequently enhances the growth (2, 14), although amino acids also may inhibit growth (5,8).The concentration of nitrogen and the relative amounts of ammonium and nitrate may be critical for growth and morphogenesis of plant cells (10,15).In an earlier report from this laboratory it was shown that a soybean cell culture growing on a defined medium required ammonium or glutamine in order to grow on nitrate (8). Growth ceased when nitrate was used without a source of reduced nitrogen. A series of amino acids has now been examined to determine their effectiveness as a replacement for ammonium sulfate. Furthermore, the effect of various nitrogen sources on the growth of soybean cells and cells of five other plants was investigated. Table I (8). The cells were grown in 250-ml DeLong flasks in a total volume of medium and inoculum of 40 ml. The flasks were incubated at 27 C in continuous light on a gyrotory shaker (6). METHODS AND MATERIALSThe amino acids used in the experiments were filter-sterilized and added to the autoclaved medium.Analytical Methods. For determining dry weight, the cells were collected on Miracloth, washed with water, and dried in a vacuum oven at 60 C for 20 hr. Total protein was determined by a micro-Kjeldahl method. RESULTSThe results in Table II serve to illustrate that the lack of growth of soybean cells in nitrate alone is not influenced by the concentration of nitrate. The other important observation is the profound influence of ammonium on the utilization of nitrate reflected in the increased growth rate. Ammonium is only one form of reduced nitrogen, and other sources were subsequently tested. In a series of experiments a number of amino acids were added one at a time at a concentration of 1, 2, and 4 mm. These concentrations were selected since those were the levels at which ammonium sulfate was used. Cell growth was reduced in the presence of both isomers of most amino acids at the three concentrations. The data in Figure 1 show the effect on growth of the soybean cells when various amino acids were substituted for ammonium sulfate in the medium. The medium contained 25 mm nitrate, and the concentration of the amino acids was 4 mM. Addition of L-glutamine resulted in growth equal to that obtained with ammonium sulfate. The L-isomers of alanine and arginine could not replace ammonium, but the cell yield was higher than for cells grown in nitrate alone. Chromatographic analysis showed that the majority of the amino acids disappeared from the medium and were presumably absorbed.The results in Figure 2 show the growth achieved by substituting glutamine, urea, and amino acids related to the urea cycle for ammonium sulfate. The L-glutamine was fully equal to ammonium sulfate in supporting growth. On ...

Suspension cultures of Triticum monococcum L., Triticum vulgare Vill. var. Thatcher, Hordeum vulgare L. var. Gateway, and Hordeum vulgare L. var. Gateway mutant yv2 have been established. The cultures were derived from root sections of seedlings and cultured in a denned medium consisting of mineral salts, sucrose, B vitamins, and 2,4-dichlorophenoxyacetic acid, with nitrate and ammonia as the sources of nitrogen. In the early period of the cultures the cell aggregates readily, differentiated to form roots, but this characteristic diminished after several generations of subculture. The cells and medium contained a number of glucanases. The presence of a laminaranase (endo-β-(1 → 3)-D-glucan glucanohydrolase (EC 3.2.1.99)) that did not attack lichenan was established. The culture media of the wheat contained an oligosaccharide which on acid hydrolysis yielded galactose, arabinose, and xylose. Hydrolysis of a cell-wall fraction yielded the same sugars in addition to glucose and mannose.

Interspecific and intergeneric fusions of plant protoplasts were induced by polyethylene glycol (PEG) 1540 or 4000. The frequency of heterokaryocyte formation (or rate of fusion) was much higher when PEG was eluted with a high pH-high Ca(2+) solution or a salt solution than when it was eluted with a protoplast culture medium. The frequency of heterokaryocyte formation was also affected by the types of enzymes used for wall degradation, duration of enzyme incubation and molality of the PEG solutions.The maximum frequency of heterokaryocyte formation was 23% for V. hajastana Grossh.-soybean (Glycine max L.) and barley (Hordeum vulgare L.)-soybean, 35% for pea (Pisum sativum L.)-soybean, 20% for pea-V. hajastana, 14% for corn (Zea mays L.)-soybean and 10% for V. villosa Roth-V. hajastana.40% of the barley-soybean, corn-soybean and pea-soybean heterokaryocytes divided at least once. Some divided many times and formed clusters of up to 100 cells in 2 weeks. The heterokaryocytes of soybean-V. hajastana, V. villosa-V. hajastana also divided. Of the PEG-treated protoplasts of N. langsdorffii and N. glauca 13.5% developed into tumor-like calli. The morphology of these calli was very much like that of the tumors produced on amphidiploid plants of N. langsdorffii x glauca.Nuclear staining indicated that heterokaryocytes of V. hajastana-soybean, pea-soybean, corn-soybean and barley-soybean could undergo mitosis. Nuclear divisions in a heterokaryocyte were usually synchronized or almost synchronized. Nuclear fusion and true hybrid formation usually occurred during the first mitotic division after protoplast fusion. A hybrid of barley-soybean in third cell division was observed. The frequency of heterokaryocytes which underwent nuclear fusion has not been determined. Multipole formation and chimeral cell colonies were also observed.