Rescue of lethal defective endosperm mutants by culturing immature embryos

Examination of kernels of EMS-induced lethal defective endosperm mutants has revealed that many of the mutants had partially developed embryos which had been interrupted at some time before kernel maturity, suggesting the possibility that the embryo in those cases had been sustained in its development either because the mutant did not control an earlier developmental stage and therefore was delayed in expression, or because some constituent had been supplied for a limited period by the surrounding cells. If the latter case should be true, there is hope that providing more of the required constituent will bring about more normal development of the embryo and, therefore, restore viability. Removal of the embryo from an immature mutant kernel at an early developmental stage and placing it on culture medium containing nutritional supplements may enable it to overcome the mutant defect, permitting the rescue of such mutants from lethality.

The procedure used to test this hypothesis was to plant stocks known to be segregating for each mutant, self-pollinate the plants and select for culturing at the earliest stage that the mutant kernels were distinguishable. Segregating ears were surface-sterilized and the immature embryos dissected out and placed individually into culture tubes, some containing mineral salts (T. Murashige and F. Skoog, Physiol. Plant., 1962) with 4% sucrose and 0.8% agar (basal medium) and some containing basal medium supplemented with amino acids, vitamins and nucleic acid bases (enriched medium). When possible, 10 mutant embryos from each segregating ear were cultured on each type of medium and 10 normal embryos from the same ear were cultured on the regular basal medium. The cultures were placed in a growth chamber with a mean temperature of 24 C and a light-dark regime of 16 hr: 8 hr for a period of three to five weeks, after which the growth response of the embryo on each medium was observed, the height in the tube measured, and fresh weights obtained of shoots and roots, if they had developed.

During the past two years, 81 lethal defective endosperm mutants have been cultured as described above. Results for the 72 mutants cultured during the summer of 1977 are presented in simplified form in the accompanying table.

Table 1.

About four-fifths of the mutants grew (responses ++ and +++ ) on two kinds of basal media (regular basal and ammonia-free basal) and on enriched medium. At the other extreme in response were nine mutants that did not grow on any of the media (responses 0 and +). Despite our assumption that it was unlikely that any mutants would grow only on basal media, nine mutants did display a preference for the unenriched media. Of particular interest are the few mutants that seem to prefer the ammonium-free basal medium (E627, 874, 1369, and 1394) and the few that grew only, or considerably better, on the enriched medium (E792, 1092?, 1121, 1255, 1162, 1417 and 1430). The mutants in the latter group are of special interest because it is among this type that auxotrophic mutants are expected to be found.

W. F. Sheridan, M. G. Neuffer and E. Bendbow


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