A population of 3070 clones derived from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated mesophyll protoplasts of haploid Hyoscyamus muticus was tested for amino-acid auxotrophy without enrichment. One clone (MA-2) was stably and specifically dependent on casein hydrolysate and could be fed also by a number of single amino acids or by other reduced nitrogen sources. MA-2 was found to be chlorate resistant and devoid of in vivo nitrate reductase activity under inductive conditions. Permissive and restrictive growth conditions for MA-2 were investigated more closely and media were found promoting morphogenesis. Selection and testing of clones were complicated by an unspecific growth stimulation of some wild type cultures by amino acids, thiamine and m-inositol.
Growth inhibition of plated cells by antimetabolites present in the culture medium was alleviated by corresponding metabolites supplied on filter paper disks placed on the surface of the medium. The metabolites diffused into the medium producing a halo of growing cells (an exhibition zone). Exhibition zones were recorded for 15 pairs of compounds and with cell cultures of Rosa, Acer pseudoplatanus, Daucus carota, Hyoscyamus muticus and Zea mays. The interaction between the compounds could be quantified by measuring exhibition zone diameters and the method was partially optimised for L-ethionine/L-methionine and p-fluorophenylalanine/L-phenylalanine. The exhibition zone method was compared to two other methods, measurement of growth yield and plating efficiency, for demonstrating .reversion of growth inhibition.
AbbreviationsETH = L-ethionine PFP = p-fluorophenylalanine 5MT= 5-methyl-DL-tryptophan A2C = L-azetidine-2-carboxylic acid PE = Plating efficiency Met = L-methionine Phe = L-phenylalanine Trp = L-tryptophan
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