This contribution describes a further study of changes occurring in the mitochondrial DNA (mtDNA) of maize derived from Texas cytoplasm tissue cultures. Changes in the male-sterile, T-toxin sensitive phenotype have been observed in two independent laboratories as the result of a passage through tissue culture (B. G. Gengenbach et al., 1977, PNAS 74:5113 and R. I. S. Brettell et al., 1980, TAG 58:55). Similar phenotypic changes were also described in Texas cytoplasm material subjected to a mutagenic treatment by A. Cornu et al. (1980, MGCNL 54:42). Restriction enzyme analysis led B. G. Gengenbach et al. (1981, TAG 59:161) to remark that increased variation in mtDNA was probably the consequence of passage through tissue culture, and that the change in phenotype was not simply the result of a selection of N-type mitochondrial genomes already present at low frequency in Texas cytoplasm.
Here we have examined four lines obtained from four plants regenerated from a culture carrying Texas cytoplasm. The culture was initiated from an embryo which derived from crossing (WF9T/W22) x A188Nrf with pollen from W22rf. The culture was maintained according to Brettell et al. with and without a selection for resistance to T-toxin imposed by incorporating T-toxin in the culture medium. Four resistant, fertile plants were regenerated to give four lines as follows:
When tested under field conditions these lines were uniformly male-fertile and scored as resistant to T-toxin.
Mitochondrial DNA was extracted from each of the four lines and subjected to a BamHI digest. The fragments were separated on a gel and suggested that a series of rearrangements had occurred, when compared to Texas material that had not passed through tissue culture. Such rearrangements were also observed by Gengenbach et al. in tissue culture-derived lines which continued to express the male-sterile, T-toxin sensitive phenotype, and so are not necessarily related to the appearance of fertile, T-toxin resistant plants. A XhoI digestion similarly revealed a number of apparent rearrangements. However, when taken with the data of Gengenbach et al. it would seem that one of the XhoI fragments present in the Texas cytoplasm controls is altered in all lines so far examined which express the fertile, T-toxin resistant phenotype. This fragment, which may thus relate to the change in phenotype, is about 6.6 kb in length and has now been isolated from a parental T-cytoplasm line, nick-translated and hybridized to the mtDNA of the plants obtained from tissue culture. In three of the lines (V6, V23 & V24) homology appeared at about 6 kb. In V18 on the other hand there was homology at about 20 kb.
HindIII and BamHI digests were also made from the chloroplast DNA of the lines obtained from culture, but in contrast to the digests of mtDNA, they showed a pattern constancy.
A mystery remains as to how and when these rearrangements are occurring. Interestingly, there were similarities between the three lines (V6, V23 & V24) which were derived from the culture maintained in the presence of T-toxin and kept separate from that which gave V18. This might indicate that the rearrangement events are occurring early in the culture sequence (the unselected and selected culture lines were separated four months after the embryo culture was initiated) rather than during regeneration.
Finally, we note that restriction enzyme analysis of mtDNA from plants derived from N cytoplasm cultures also indicates sequence changes (R. J. Kemble and R. B. Flavell, personal communication). This suggests that the apparent 'instability' of mtDNA in tissue culture is not a specific property of Texas cytoplasm.
R. I. S. Brettell, M. F. Conde and D. R. Pring
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