Selection for resistance to growth inhibition by lysine plus threonine in equimolar concentrations (LT) The heritability and expression in plants of traits selected in tissue culture are of particular concern to those using these methods for genetic modification of plants. Only a few studies have demonstrated expression in regenerated plants and sexual transmission of selected traits to progeny. These include resistance to methionine sulfoximine (1), streptomycin (2, 3), Helminthosporium maydis race T toxin (4), picloram (5), and valine (6).Selection for resistance to growth inhibition by lysine plus threonine in equimolar concentrations (LT) has been proposed as a means ofobtaining feedback-resistant mutants in the lysinethreonine-methionine-isoleucine (aspartate family) biosynthetic pathway (7). Reversal of LT inhibition by the addition of methionine or the methionine precursor, homoserine, indicates that inhibition is the result of methionine starvation (8, 9) and that altered phenotypes which increase methionine synthesis should provide resistance to LT inhibition. Aspartokinase and homoserine dehydrogenase, in the aspartate pathway, are subject to feedback inhibition by lysine and threonine, respectively, in maize (10, 11).In a recent report from this laboratory, we described the selection from tissue cultures ofmaize ofa stable LT-resistant line (D33) that contained aspartokinase with decreased sensitivity to lysine inhibition (12). Analysis offree amino acid pools in D33 cultures showed substantial increases in methionine and threonine and small increases for lysine and isoleucine. Attempts to determine the inheritance of LT resistance failed because of complete sterility in plants regenerated from D33 cultures.Additional LT-resistant lines have now been selected from which we have obtained regenerated plants and progeny. In this report, we describe the inheritance of LT resistance and the expression of this trait in tissue cultures and progeny of regenerated plants. METHODSThe initiation, maintenance, and incubation of tissue cultures from immature embryos were as described (13,14). Immature embryos, 1-2 mm in length, were obtained from a cross between inbred lines A188 and W22. Cultures were grown on Murashige and Skoog (MS) medium (50 ml per 25 X 100 mm Petri dish) containing 0.5 mg of2,4-dichlorophenoxyacetic acid (2,4-D) per liter. The cultures were transferred to fresh medium after 1 month, and after 2 months they were subdivided into pieces of ca. 20 mg of fresh weight and placed on 50 ml of MS medium. One drop of 0.1 M phosphate buffer (pH 3.0) containing 0, 0.1, 1, or 10 mM sodium azide was applied to each explant. This solution was completely absorbed into the tissue or growth medium after 1 hr. Eight days after azide treatment, lysine and threonine were added to the culture plates to give a final concentration of 2 mM for each. At monthly intervals, sectors of cultures which appeared to be alive (nonnecrotic) were transferred to fresh MS medium containing 2 mM LT.Plant regeneration wa...

Twenty‐one amino acids were tested individually for their effects on growth of roots from excised mature embryos of maize (Zea mays L.). Among these, tryptophan, tyrosine, threonine, and homoserine were partially inhibitory to root growth at 1 mM concentrations. Length and dry weight data showed that maize roots both from embryos and from kernels were synergistically inhibited by lysine + threonine but not other paired combinations of aspartate‐derived amino acids. Roots from embryos, however, were more sensitive to inhibition. The synergistic inhibition of embryo root length was near maximal at 1 mM lysine + 1 mM threonine. This synergism was not observed with other paired combinations of aspartatederived amino acids. Methionme and homoserine specifically relieved lysine + threonine inhibition, whereas 17 other amino acids were ineffective. These results are compatible with a model and published enzymology studies which suggest that exogenously added lysine and threonine may act as feedback inhibitors of aspartokinase and homoserine dehydrogenase, respectively, early enzymes in the aspartate biosynthetic pathway.The lysine analog, S‐2‐aminoethyl‐L‐cysteine (AEC) was a potent inhibitor of root growth in maize seedlings. Among 21 amino acids tested, lysine was most effective in relieving AEC inhibition while arginine and methionine were partially effective.