The carbamate insecticide Lannate (active ingredient methomyl) mimics the cytoplasm-specific action of Helminthosporium maydis race T toxin (HmT toxin) on cms-T plants (Humaydan and Scott, Hort. Sci. 12:312, 1977) and isolated mitochondria (Koeppe and Malone, Science 201:1227, 1978). In vitro selection for resistance to H. maydis race T toxin has been successful (Brettell et al., Theor. Appl. Genet 58:55, 1980; Gengenbach et al., Proc. Nat. Acad. Sci. 74:5113, 1977), suggesting that methomyl may also be a useful selective agent. The chemical is more readily available than the purified fungal toxin and is also water soluble, making it convenient to use for in vitro selection. We obtained over 100 maize plants which were resistant to methomyl, and whose progeny also show resistance.
Callus was established from immature embryos derived from cms-T versions of the inbreds W182BN, P39, MDM-1, and of the F1 hybrids of SW-1, IL766A-1 and IL766A-2 with W182BN-N (a non-restoring pollen parent, used here to improve regeneration). All lines except W182BN are sweet maize. Embryos were obtained from fully male sterile plants except for IL766A-2 and MDM-1, which were either unrated or partially fertile. The maternal parents all show methomyl sensitivity regardless of fertility restoration.
Callus was increased over a period of 3 months before selection was initiated. Meanwhile, selection conditions were defined for different genotypes. Addition of 0.6 mM methomyl to modified Murashige-Skoog medium with 5mg/l 2,4-D, 4% sucrose caused about 20% inhibition of fresh weight increase for W182BN and W182BN x IL766A-2. Callus plated on 0.65mM methomyl showed intermediate inhibition of growth and on 0.7mM methomyl showed 80 to 100% inhibition. The other genotypes were cultured on Duncan's Medium D (Duncan et al., Planta 165:322, 1985). Inhibition of fresh weight increase on this medium containing 0.6mM, 0.65mM, or 0.7mM methomyl varied between 50 to 95%, depending on the genotype.
Over 2800 callus pieces were used in selection experiments, with half of these pieces serving as controls not exposed to methomyl. Callus which grew and remained regenerable in appearance was subcultured monthly on selection medium or, for the controls, on maintenance medium. Both gradual and high selective pressures with media containing methomyl were used. Regeneration was started after 3, 4, 5, 6, or 10 subcultures by transfer to MS medium with 10% sucrose and no 2,4-D; in some cases 1mM methomyl was included. Regenerated plantlets were tested for resistance to methomyl at the 3-5 leaf stage by direct swabbing of 0.3M methomyl onto the distal sections of a leaf. In this assay, leaf necrosis was visible after 1-3 days on the treated leaves of cms-T plants while no effects were seen on treated leaves of plants with other cytoplasms (N, C, S). Regenerants showing no necrosis were scored as resistant.
Presently, 288 plants regenerated after 3 to 5 selection cycles have been examined. Of the regenerants from callus exposed to methomyl during subculture, 76% (117/153) showed methomyl resistance; of these, 97 plants were fully male-fertile, 16 had unrated or abnormal tassels, and 4 were male sterile. Only 2% (3/135) of the regenerated controls showed resistance: these were all derived from IL766A-2, which yielded the most control regenerants (100). Several fertile, methomyl sensitive regenerants have also been obtained from sterile starting material subcultured on methomyl. Almost all (40/41) plants regenerated in the presence of methomyl were resistant, while only 69% (77/112) of the plants were resistant after regeneration in the absence of the insecticide. Control callus was generally not capable of plant regeneration on medium containing methomyl. This suggests that high methomyl concentrations in the regeneration medium provide an effective roguing of sensitive material. The influence of time in culture, type of selection pressure, and of genotype on the proportion of resistant and sensitive plants is currently being examined.
The resistant plants varied in their response to injection of HmT toxin into the whorls. Of the 82 resistant plants examined, 39% showed little or no response to toxin and 61% showed intermediate to high sensitivity. The regenerated controls for the corresponding cultures showed 100% sensitivity. It is also interesting that gain in fresh weight of callus was not a clear indication of resistance. A resistant plant was obtained from material on methomyl which showed no weight increase during the last selection cycle before regeneration, and a mix of resistant and sensitive plants resulted from callus which grew even better on methomyl than healthy control tissue.
We are currently doing seed increases in the greenhouse and in the winter nursery in preparation for inheritance studies. Considering the results of the HmT toxin resistance work, it is likely that the shift to methomyl resistance and male-fertility involves alterations in the mitochondria. Progeny of resistant selections show resistance, both in seedling tests with methomyl and in field-grown material subjected to biweekly sprayings of Lannate.
The apparent link of methomyl resistance with male-fertility (seen also in material selected for resistance to the HmT toxin) may limit the usefulness of such methomyl-resistant plants for hybrid seed production. However, selection for methomyl resistance offers a method for rapid cytotype conversion from male-sterile to male-fertile; it eliminates the need for repeated backcrosses after conventional fertility restoration. Also, methomyl resistance could be easily incorporated into lines where fertility changes are less important than the maintenance of specific cytoplasm-nuclear background combinations. One such example is T-Rf sweet corn. In addition, apparent differences in structural requirements and effective dosages of the toxin and insecticide leave open the possibility that different mechanisms of action may be involved. Molecular analysis of methomyl resistant mutants offers distinct potential for providing additional understanding of the mitochondrial genomes and cytoplasmic male sterility
A.R. Kuehnle and E.D. Earle
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