Conclusion

The high degree of diversity of Nicotiana ct-DNA reflects the complex nature of its evolution.Thus，the search for a common mechanism of ct-DNA alteration is both stimulating and challenging.In Nicotiana speciation was primarily achieved by repeated interspecific hybridizations in nature.The study of different cultivars of N.tabacum，several of which originated by a similar mechanism，may provide important insight into the evolutionary events from which the current diversity of ct-DNA was derived.The results presented here are very encouraging.There is a high frequency of ct-DNA alteration in the CMS lines.Such alteration seems to be localized in one region（Kung et al.，1981）.The pattern of alteration ranges from having a single extra fragment to a more complex pattern which can be best described as recombination.For example，in the more complex case noted above，ct-DNA recombination may have occurred（see Figure 3）.Among the SmaI and EcoRI bands in this CMS line（see Figure 3），some are new and not found in either parent，some bands resemble the female and some resemble the male parent，with the rest of the bands common to all three.Several of the new bands are indeed unique and have not been found in any of the 35 species thus far examined.This suggests that ct-DNA recombination may have occurred in this particular hybrid.The restriction map of Jurgenson and Bourque（1980）strongly supports this view.This also，raises some interesting points.ct-DNA must pair in a manner similar to chromosome pairing during meiosis.Although recombination of mitochondrial DNA in somatic hybrids of Nicotiana have been reported（Belliard et al.，1979），caution should still be exercised in interpreting new results.This is the first reported instance that ct-DNA of two different species may have recombined.It is our opinion that these results can be viewed as strong evidence demonstrating that ct-DNA did actually recombine in Nicotiana.However，other alternatives such as deletions，insertions and/or point mutations cannot be ruled out.

The recombination of ct-DNA molecules with each other in a chloroplast has been reported（Kolodner and Tewari，1979）.Recombination events may be essential in the evolution of Nicotiana ct-DNA.Otherwise，by the simple mode of maternal inheritance Nicotiana ct-DNA could not have evolved to such a high degree of diversity（Rhodes et al.，1981）.In some higher plants，including Nicotiana，the male parent contributes mostly nuclear materials and very little cytoplasm during fertilization.The number of organelles coming from the male is extremely small，if any，and they are altered and excluded（Vaughn et al.，1980）.However，it is still possible there are survivals of male origin.Thus，recombination of ct-DNA from different sources is not totally impossible，but rather，infrequent.The possible recombination of ct-DNA reported here can be tested by protoplast fusion experiments.In fact，this has been done and no sign of such recombination was observed（Belliard et al.，1979）.The ct-DNA from the protoplast fusion product of N.glauca and N.langsdorffii was also analyzed in this laboratory and we found only one or the other parental ct-DNA is present and no recombination occurred（data not shown）.It should be pointed out that if ct-DNA recombination occurs only rarely one would not expect to detect it in the few protoplast fusion products examined.

In the second case of CMS line studied here，the ct-DNA was simply mutated to another type.This mutation may involve a simple inversion of a DNA segment containing a restriction site.This suggests mechanisms of mutation involved in chromosomal exchanges can also be applied to organelle DNA.These include translocation，duplication，inversion，and deletion.(https://www.daowen.com)

Alteration of ct-DNA is not found in all CMS cytoplasms.One possible explanation is that alteration（recombination）occurs only in crosses where the ct-DNA of both parents are quite similar，as in the case of N.labacum with N.plumbaginifolia（see Figure 3），but not with N.repanda or N.fragrans which are different.Even with N.plumbaginifolia the recombination is a rare event since no alteration was detected in a similar cross reported by Frankel et al.（1979）.The biological implication of this phenomenon and its association with CMS is currently under investigation in this laboratory.The results will be important in assessing the role of ct-DNA in cytoplasmic male sterility and will be published elsewhere.