Soybean callus cultures

Botran was one of the first xenobiotics reported to be metabolized to an N-malonyl conjugate by this mechanism In plant tissues (120) (Equation 28). The N-malonyl conjugate of botran was the major metabolite In both soybean and soybean callus culture. It was Isolated by chromatographic methods and Identified by synthesis and by chemical and mass spectral methods. 

Quantitative differences in 2t4-D metabolism were also observed as a result of herbicide concentration and tissue age in soybean callus cultures (31). Unmetabolized 2(4-D constituted most of the extracted from three-wedc old callus tissue, and the concentration of free 2.4-D increased 32-fold in the tissue as external 2.4-D concentration was increased from 10 ° to 10 M (Figure 3). Aqueous (glycosides) and ether soluble metabolites (amino acid conjugates) were present in lower amounts, and increased slowly over the 10 ° to 10 M concentration range. In nine-week old callus cultures, ether soluble metabolites (2.4-D amino acid conjugates) were the major components of the tissue, and increased four-fold over a three-fold increase in 2.4-D concentration in the external medium (Figure 3). The concentration of 2.4-D remained low and relatively constant as it was converted to amino acid conjugates. Aqueous soluble metabolites formed a relatively minor component in nine-week old callus. 

Feung, C. S., Hanrilton, R.H., and Mumma, R.O. Metabolism of 2,4-dichlorophenoxyacetic acid. V. Identification of metabolites in soybean callus tissue cultures. J. Agric. Food Chem., 21(4) 637-640, 1973. 

Feung, C.S., R.H. Hamilton, F.H. Witham, and R.O. Mumma. The Relative Amounts and Identification of Some 2,4-Dichlorophenoxyacetic Acid Metabolites Isolated from Soybean Cotyledon Callus Cultures, Plant Physiol, 50 80-86 (1972). 

Biotests for the detection and quantitative measurement of C. measure one of the following 1. stimulation of cell division in tissue cultures, e.g. in tobacco or soybean callus tissue 2. enlargement of cells in the leaf disc test, often with bean or radish leaves 3. inhibition of the loss of chlorophyll from detached leaves 4. promotion of seed germination in the dark. 

Studies of the Mode of Action of Cytokinin in Cytokinin-Dependent Cell Suspension Cultures of Soybean Callus... 

Soybean callus of cotyledonary origin (cv Acme) is a principal bioassay for cytokinins [31] with an unusually stable, absolute requirement of cell division for cytokinin. We choose to use it in suspension culture in order to obtain synchronous activation of all cells, allowing us to investigate early events. 

Fig. 2. Effect of anti-calmodulin drugs and Ca- + -channel blockers on cell proliferation in suspension cultures of soybean callus supplied with cytokinin. Cells in the linear phase of the batch growth cycle were suspended at 0.4 X 10 cells/ml. Cell number and viability were estimated after 6 days of culture using methods described in reference [34]. D-600 and verapamil were a generous gift of Knoll A.G., Ludwigshafen am Rhein, FRG...

Table 2. Effect of the Ca-"-ionophore A23187 on cell number and cell viability in cytokinin-responsive samples of suspension-cultured soybean callus 6 days after addition...

Reductive reactions are not generally of common occurrence in plants and usually are insufficiently rapid to play a significant role in pesticide detoxification and selectivity. A reaction which has been recorded, however, is the reduction of aromatic nitro groups to the corresponding anilines, and the observed reduction of botran (2,6-dichloro-4-nitroaniline) by axenic callus cultures of soybean demonstrated the ability of plant tissue per se to mediate such biotransformations. While such reactions appear not to occur extensively in higher plants, the reductive deamination (Figure 10.3) of the substituted triazinone metribuzin (3) to 4 represents a major detoxification mechanism which may account for the tolerance of soybean cultivars to metribuzin. Thus, deamination in leaves of tolerant Bragg ... 

Fig. 1. Effect of kinetin (-1-) and water (X ) on incorporation of [ -P]Pi into phospholipids of cyto-kinin-responsive samples of soybean cells. Cytokinin-responsive samples of soybean cells are suspension cultured cells of soybean (cv Acme) cotyledonary callus brought to mitotic arrest by cyto-kinin-deprivation. Filter-sterilized kinetin solution (1 ml to 50 ml of cell suspension), or water, was supplied at t = 0. At the same time each sample was supplied with p-P]Pi at 74kBq mV Cells were killed in hot /50-propanol at the times shown on the abscissa. The ordinate represents radioactivity in each phospholipid expressed as a percentage of the total radioactivity recovered from each 2-D TLC plate. The total radioactivity recovered from each plate was 15 min. water = 28 Bq, kinetin = 11 Bq 30 min. water = 12 Bq. kinetin = 12 Bq 60 min, water = 29 Bq. kinetin = 27 Bq...

When the acidic lipids of soybean cells heavily pre-labelled with [ P]Pi were resolved by 2-D TLC, no trace of radioactivity could be detected in the position occupied by a sample of authentic PI(4,5)P2 run on the same plate [34]. However [ P]-label was found at the location of a phosphatidylinositol 4-phosphate (PI(4)P) standard. This is only a preliminary to the process of proper identification. Co-chromatography of radioactivity, whether from p P]-Pi or [ H]-inositol, with a phosphoinositide standard in 1 -D TLC is not acceptable evidence of identity. It has subsequently been shown that suspension cultured cells of tomato and carrot callus also do not appear to contain detectable levels of PI(4,5)P2 [39]. 

The mode of action of cytokinin in the control of cell proliferation in cotyledonary callus of soybean remains unknown. Bevan and Northcote [47] showed that the stimulation by kinetin of the increase in numbers of polyribosomes on transfer of these cells to fresh medium was yet another gratuitous effect of cytokinin, not necessary for cell division. They also showed that treatment with cytokinin, as compared with water, led to no detectable change in the relative levels of any mRNAs, and had no detectable effect on bulk protein synthesis or on the synthesis of any particular polypeptides. These results have always pointed to the cytoplasm, rather than the nucleus, as the site of the cytokinin signal-translating machinery. It seems that on cytokinin-starvation, cells can arrest at a number of positions in the mitotic cycle. This suggests that there are a number of different processes in the cell cycle requiring the presence of cytokinin [48]. This may be the reason for the unusual stability of cytokinin-dependence, a feature characteristic of the differentiated state which these cells would otherwise be expected to lose. If there are a large number of cytokinin-surmountable hurdles in the process of cell proliferation for cultured soybean cells, the mode of action , when we find it, is unlikely to be simple. 

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