A serious error in recommended method for treatment of pollen with EMS

For some years now (since 1966) we have been conducting highly successful experiments on the induction of mutants in corn pollen using mixtures of EMS and other mutagens in paraffin oil. The results far exceeded our original expectations and led to the cumulation of large numbers of mutations, the analysis of which have become a major undertaking in this laboratory and elsewhere. We reported our method and our progress from time to time in the Maize News Letter and elsewhere, beginning with MNL 42:124-125, 1968, and culminating in a publication entitled "Paraffin oil technique for treating mature corn with chemical mutagens" in Maydica 23:21-28, 1978. Since we were already overwhelmed by a wealth of material with new mutants, no further treatments were carried out after the early 70's. In 1979 we needed a particular kind of treated material, so we made some new treatments using our well-tried method. The treatment was a complete failure. No seed was found on the treated ears. Thinking that we might have miscalculated, we repeated the treatment again a second and a third time. To our dismay, they also were complete failures. We then remembered with mounting embarrassment, reports of other workers trying our method without success. We rechecked all our measurements and calculations and chemical sources. All were correct, but when we retested pollen germination, we found no tube growth. Killing was complete. Many anxious re-checks later, someone discovered only one change in procedure. In the successful experiments we used S/P diSPo P5210-1 glass pipets hand calibrated to 1 ml to make our stock and treatment solutions. In the unsuccessful treatments we used pre-calibrated Corning 7077 disposable 5 ml pipets. The first pipets were calibrated by taking one pipet, finding the 1 ml level, marking, measuring that level and then measuring and marking all the rest of the pipets in a box. A re-check revealed that all pipets so marked delivered 0.75 ml instead of 1 ml. Then it was clear what had happened. In our earlier experiments our stock solution was 0.75% instead of 1% and our treatment solutions were 0.75 ml instead of 1 ml of this solution in 8 ml of oil (the oil was measured in pre-calibrated large pipets). The concentration was actually .0075/9 = 0.83% instead of the published .11%. To obtain this concentration one should take 1 ml of 1% stock solution and mix with 11 ml of oil. Following this discovery we then proceeded to make new determinations of acceptable concentrations of EMS in oil for pollen tube growth and from this we identified and successfully tested new levels of concentration for good seed set and high mutation frequency. We found that one part of a 1% stock solution in 15 parts oil (.063%) gave excellent seed set and a high frequency of mutation events. With sincere apology it is recommended that our methods for pollen treatment outlined in various publications are good and can be followed with the above-described correction in concentration of treatment solution.

M. G. Neuffer

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