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Plant mitochondrial genomes are composed of unusually complex structures, due to active recombination at numerous repeated sequences in the genome. The maintenance of mitochondrial genome stability is under the control of identifiable nuclear genes. In plants, three nuclear genes (MSH1, RECA3 and OSB1) have been shown to participate in recombination surveillance and the suppression of illegitimate recombination in mitochondria. Disruption of these loci in Arabidopsis results in reproducible mitochondrial genome rearrangements. We demonstrated that repeat-mediated de novo recombination was also enhanced in both Arabidopsis and tobacco during passage through in vitro culture. Furthermore, in vitro conditions led to suppression of MSH1 and RECA3 expression. Subsequent regeneration processes restored normal MSH1 transcript levels and mitochondrial DNA configuration in tobacco. Our results show the utility of in vitro culture as an effective means to study the dynamic features of plant mitochondrial genomes and to facilitate more complete mitochondrial sequence assembly in plants. Disruption of the nuclear gene MSH1, which functions in maintaining mitochondrial and chloroplast genome stability, produces an array of unusual plant growth phenotypes and imparts stress tolerance in Arabidopsis. Similarly, transgenic suppression of MSH1 by RNA interference in crop plants (tobacco, tomato and soybean) produces mitochondrial genome alterations and the associated phenotype of cytoplasmic male sterility. We have observed other phenotypes in MSH1-RNAi tobacco, including dwarfism, enhanced branching, altered leaf morphology, and delayed flowering. The dwarfed growth phenotype was partially reversed by application of gibberellic acid (GA). We have characterized these novel phenotypes, and shown them to be heritable in lines lacking the RNAi transgene. We have also investigated their behavior in crossing.