The S type of cytoplasmic male sterility (S-cms) is characterized by the presence of two unique plasmid-like mitochondrial DNAs designated as S1 and S2 (Pring and Levings, 1977, PNAS 74:2904). Cytoplasmic mutations leading to restoration of male fertility were associated with integration of these plasmid-like molecules into high molecular weight mt DNA (Levings et al., 1980, Science 209: 1021). Molecular analyses of such high frequency mutations as well as physical characteristics of these plasmid DNAs suggest similarities with transposable elements. Indeed, Levings et al. (1980) referred to these mutations as transpositional events. We report here a similar type of transpositional event involving S1 and S2 under tissue culture propagation of callus tissue. Correlations of this event with a certain callus morphology is of special significance.
Callus cultures were initiated from immature embryos of WF9 inbred line carrying the S-cms trait, according to Green and Phillips (1975, Crop Sci. 15:417). Plants were regenerated from scutellar calli after six months of culture. A portion of the callus was also maintained as a callus line. This callus culture is now approximately two years old and retains its original compact and highly organized morphology (referred to as 'organized' callus); however, plant regeneration from it appears no longer possible. This callus culture has also been used to select and isolate a total of four independent friable types of callus cultures during the past two years, as done previously in the Black Mexican Sweet line (Chourey and Zurawski, 1981, TAG 59:341). The friable and the 'organized' calli have been maintained on the same medium and under identical growth conditions for the analysis for S1 and S2 DNAs.
The 'organized' callus, four independent isolates of friable callus and the leaves of ten plants regenerated from the initial scutellar callus were analyzed for the presence/absence of S1 and S2 DNAs according to Kemble (1980, TAG 57:97). These plasmid-like DNAs were readily detectable in leaf extracts by UV fluorescence of ethidium bromide stained agarose gels. Similar analysis of 'organized' and friable calli extracts, however, showed only trace amounts of S1 and S2 DNAs in the former and no detectable levels in the latter. Southern hybridization using nick-translated radioactive probes of cloned S1 and S2 DNA sequences revealed significant hybridization corresponding to the position of S1 and S2 in the 'organized' callus. However, no hybridization was seen at the position corresponding to S1 and S2 in extracts obtained from the four friable isolates. Instead, these extracts showed intense hybridization at the position of high molecular weight mt DNA, indicative of transposition of S1 and S2 into the mt DNA. It is noteworthy in this analysis of tissue cultured cells that altered callus morphology (i.e., friable in contrast to 'organized' type) was associated with the transpositional event of these DNAs. The significance of such a correlation remains unclear.
Prem S. Chourey and Roger J. Kemble*
*Present address: Dept. Plant Path., Kansas State Univ., Manhattan, Kansas 66506
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