--M. James, P. Stinard, D.S. Robertson, and J. Stadler

Regenerant plants from A188/Mutator and H99/Mutator embryogenic callus lines were tested for Mutator activity (James and Stadler, MNL 62:9,1988). All Mutator parents used in the crosses to establish these lines were presumably active second generation inbred Mu parents (Mu2); however, subsequent genetic tests for Mutator activity (Robertson, Mutat. Res. 51:21-28, 1978) revealed one Mu parent (86-657-2) to be transpositionally inactive (a Mu-loss plant). Calli from an embryogenic line (86-657-2B) which was derived from the cross of this Mu-loss plant with an H99 inbred were regenerated to whole plants. Most primary regenerants (R0) exhibited mutant phenotypes (typically, tassel-seed tassels and stunted growth), but two regenerants developed normal tassels and ears and were self-pollinated. The R1 progeny of one of these R0 plants were normal in appearance, while the R1 progeny of the other segregated for a knotted mutant of variable expressivity. This was probably a culture-induced mutation, but whether it was Mu-induced is unknown. The R1 plants were self-pollinated, outcrossed to a standard line, or both. At least 50 F1 progeny from the outcrosses were then planted and self-pollinated. As in Robertson's standard test for Mutator activity (Robertson, Mutat. Res. 51:21-28, 1978), progeny ears were examined for the appearance of new seedling mutants in the sandbench. A new mutant frequency of 8% was observed when one of the R1 plants (87-399-2) was crossed as a female. Four of the 50 ears tested segregated for new seedling mutants of five types (pale green, yellow green, yellow green with zebra banding, pale yellow necrotic, and white). Mutability of these phenotypes (revertant sectors), however, was not observed. No new seedling mutants were observed when this same R1 plant (87-399-2) was crossed as a male. Seeds from the segregating ears were grown in the field the next summer and self-pollinated, and their progeny ears were also observed to segregate for seedling mutants when tested in the sandbench. New mutant frequencies of 2% were seen in two additional R1 plants also crossed as females, but the reciprocal outcrosses were not available. All other R1 plants tested showed no new seedling mutations.

The observation that R1 plant 87-399-2 generated new seedling mutants only when crossed as a female suggests that the events which gave rise to the mutations occurred in the female gametes of this plant, but not in the male. This could be due to reactivation of Mu elements through the culturing and/or regeneration process, with Mu transpositional activity taking place only in the female gametes. The frequency of new mutants generated in this outcross is similar to that seen in other active Mutator lines, and the variety of seedling mutants is also indicative of an active Mutator system. The possibility that the generation of new mutants was due to culture-induced rearrangements (somaclonal variation) seems unlikely for two reasons. First, the data suggest that the mutations occurred as post-culture events in the female gametes of the R1 plant. Second, the variety of mutations observed, as well as the appearance of each mutant phenotype in only one ear apiece, is suggestive of mutations caused by independent events, such as transposable element insertions.

Preliminary molecular analysis of the callus tissue and tissues of plants regenerated from this Mu-loss line (86-657-2) was performed by digestion with HinfI, an enzyme which cuts within Mu element inverted repeats, and hybridization with a Mu1-homologous probe. This showed that DNA from the initial 86-657-2B callus tissue had a mixed population of Mu elements with respect to HinfI modification. Approximately 1/3 to 1/2 of the Mu1-hybridizing elements were unmodified at the HinfI sites, while the remainder were modified. HinfI site modification has been correlated with inactivity of Mu elements following inbreeding (Chandler and Walbot, PNAS 83:1767-1771, 1986). The finding that at least some of the Mu elements in the callus DNA were unmodified leaves open the possibility that, despite the genetic evidence, the original Mu parent may not have been a true Mu-loss plant. Interestingly, HinfI digestion of the DNA of various tissues of two mutant R0 somaclones derived from this callus line indicates that the degree of HinfI modification of the population of Mu elements varied between tissues, with unmodified Mu elements seen only in the immature cob. Molecular analysis of R1 plants and their outcross progeny with HinfI as well as with enzymes which are external to Mu1 may help determine whether active Mu elements were involved in the generation of new mutants.

Genetic tests for Mutator activity in a primary regenerant from an A188/Mutator callus line (86-646-8C) (derived from a cross with a transpositionally active Mu parent, 86-207-8) revealed that Mutator activity in the R0 plant was maintained. Although a limited number of seeds from the outcross of this R0 plant prevented a full-scale test for Mutator activity, two of five ears tested segregated for new seedling mutants in the sandbench, with one of the mutants (striate) showing a mutable phenotype.

These initial results demonstrate that Mutator activity can be maintained through the culture and regeneration process. They also suggest that an in vitro system might possibly be instrumental in the reactivation of a Mu-loss plant, although further molecular and genetic studies are necessary.

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