RALEIGH, NORTH CAROLINA
North Carolina State University

The effect of Rf1 on the methomyl sensitivity of cms-T callus
--Mark E. Williams and C. S. Levings, III

The dominant allele of the Rf1 gene substantially reduces the expression of URF13, the protein responsible for the T-toxin and methomyl sensitivity of cms-T maize. Based on a root-tip growth assay, Kuehnle and Earle (Theor. Appl. Genet. 78:672, 1989) showed that at certain concentrations of methomyl, root-tips of the Rf1-containing version of W64Acms-T were significantly less sensitive. We tested for this effect on callus growth using the isogenic background A188cms-T x T204 with and without a single Rf1 allele. As shown in Figure 1, the growth of both versions is completely inhibited at 1.3mM methomyl. However, at 0.65mM methomyl, while growth of A188cms-T x T204 rf1 rf1 is still completely inhibited, the growth of A188cms-T x T204 Rf1 rf1 is substantial (about 75 percent of growth on 0mM methomyl). Normal callus is completely unaffected at either of these methomyl concentrations.

In animal systems, many genes which have a selectable phenotype in cell culture have been cloned by the technique of marker rescue (Watson et al., Recombinant DNA, pp. 180-188, 1983). Since genomic DNA containing a selectable marker has also been shown to transform maize (BMS) protoplasts at a high rate (Antonelli and Stadler, Theor. Appl. Genet. 80:395, 1990), this method could be applied to attempt to clone the Rf1 locus. The donor line of genomic DNA would be homozygous Rf1 Rf1 and the recipient protoplasts would be a cms-T line which is rf1 rf1 followed by selection on 0.65mM methomyl. An ideal recipient line would be BMS; we have backcrossed cms-T into BMS for 4 generations, but have not as yet been able to establish ideal suspension cultures.

Figure 1. Growth of A188cms-T x T204 +/- Rf1 on various concentrations of methomyl.


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