Brettell and colleagues (Theor. Appl. Genet. 58:55, 1980) reported that malefertile plants were obtained among plants regenerated from tissue cultures of male-sterile T-cytoplasm corn. The male-fertile plants also were resistant to Helminthosporium maydis race T toxin and were regenerated from control cultures not exposed to toxin.
Among 162 plants regenerated from A188T cultures we have now obtained 8 plants (Table 1) which expressed an immediate altered phenotype for male sterility and toxin susceptibility and/or segregated for such alterations in the maternal progeny.
The results indicate that the following situations can arise in regenerated plants from unselected A188T tissue cultures: (1) the entire plant phenotype may be altered, e.g., fertile tassel, resistant leaves and uniform progeny from the ear (T*6 & 7 and possibly T*8 & 9); (2) the tassel may be sectored for fertility and the leaves may be resistant (T*5) or susceptible (T*2); (3) the tassel may be fertile, the leaves susceptible and the ear may segregate (T*1 & 2); and (4) the plant may be uniform but progeny from tassel seeds may segregate (T*4). In this last case, the tassel was entirely pistilloid and had no lateral branches, therefore, male-fertility in the altered sector likely did not have the opportunity to be expressed in the regenerated plant. Brettell observed one plant that was male fertile and toxin susceptible but did not report data on progeny from that plant. The progeny data shown here support the notion that even though male fertility and toxin resistance can be separated spatially in a regenerated plant (probably as the result of a heterogeneous multicellular condition early in the differentiation process), this separation is temporary and does not carry through to progeny of such plants.
Other progeny tests (data not given) in which the male-fertile, regenerated plants were used as males have confirmed that the male fertility and toxin resistance traits are cytoplasmically inherited.
Mitochondrial DNA (mtDNA) was extracted from the variant lines by the procedure of Kemble et al. (Genetics 95:451, 1980) and digested with various restriction enzymes. Fig. 1 shows the patterns obtained when mtDNAs from A188N and A188T controls and the six variants were digested with Xho1. Examination of ethidium bromide-stained gels revealed that a fragment of about 6.6Kb was missing in five of the six variant lines compared to the A188T control pattern. This alteration is the same as found in previous analyses of nine other tissue culture-derived lines selected for toxin resistance (Gengenbach et al., Theor. Appl. Genet. 59:161, 1981; Pring and Gengenbach, unpublished). One male-fertile, toxin-resistant line (T*4) has not exhibited any detectable differences from the A188T control pattern. T*4 mtDNA has a band at the comparable position as the 6.6Kb fragment in A188T mtDNA, but small alterations may not be resolved by this level of comparison. T*5 also exhibits alterations in higher molecular weight fragments indicating the possibility of additional rearrangements in this line.
We have compared mtDNA from 34 male-sterile, toxin-susceptible lines derived from plants regenerated from unselected A188T cultures. None of these lines had detectable alterations in the 6.6Kb fragment (not shown). Thus, 14 of 15 male-fertile, resistant lines but 0 of 34 male-sterile, susceptible lines regenerated from T cytoplasm cultures at Minnesota have an alteration in a specific portion of the mtDNA. We think the circumstantial evidence is strong enough to propose that the 6.6Kb fragment is responsible for the male-sterile, toxin-susceptible phenotype of T cytoplasm corn and that alterations in this sequence (e.g., base substitutions, new restriction sites, rearrangements or deletions) could result in a male-fertile, toxin-resistant phenotype.
Burle Gengenbach and Paul Umbeck
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