national Institute of Genetics, Mishima, 411 Japan
Isolation of plasmid-like mitochondrial DNA (mtDNA) in maize proved very
useful for plant-genetic research as it provided an opportunity to
characterize the plant organellar DNA and the DNAs have the possibility of
serving as plant vector in genetic engineering. Recently, Yamaguchi and
Kakiuchi (1983) discovered similar mtDNAs, B-1 and B-2, in a cytoplasmic male
sterile (cms) strain of rice and established the universal nature of mtDNA in
cms strains of gramineous plants.
To obtain further information on the behavior of mtDNA, induction of fertile revertant from a cms-strain of rice was investigated using mutagen treatments. Seeds of a cms-strain, (cms-boro)rf\1\rf\1\, were treated with gamma-rays, ethyl methanesulphonate (EMS) and acridine orange(AO) with various intensities or concentrations. Two fertile plants were obtained from amongst 6,500 EMS treated seeds. These plants, designated 80-1 and 81-3, were found to have reversion of cytoplsmic factor from cms to normal state. When these revertants were crossed as pollen parents with the original cms strain, the F\1\'s were completely sterile. However, when crossed as pollen parents with (n- boro)rf\1\rf\1\, the F\1\'s were fertile. Gamma-rays and AO have induced no revertant so far. EMS is a well known chemical mutagen which induces point mutations, and its strong mutagenicity by alkylation of DNA is widely recognized. The present results confirm the mutagenic potential of EMS, but mutagen specificity for alteration of cytoplasmic factors should be noted.
The following 5 rice strains, viz., (cms-boro)rf\1\rf\1\,(cms-boro) Rf\1\Rf\1\, (n-boro)-rf\1\rf\1\, 80-1 and 81-3, were used for examination of mtDNA. Because of the paucity of seeds of the cms strain, tissue culture technique was utilized with B-5 medium, and the callus cells propagated from seeds were analyuzed. Calli were homogenized in mannitol solution and mitochondria were fractionated by repeated centrifigations. DNAs were extracted from the mitochondria in sodium sarkosyl-pronase, followed by purification with phenol, chloroform and RNase treatments. Mitochondrial DNAs were analyzed in agarose gels. The gel electrophoresis revealed a main high- molecular DNA band in preparations from all the 5 strains. Preparations from male sterile cytoplasms, (cms-boro)rf\1\rf\1\ and (cms-boro)Rf\1\Rf\1\, were found to contain two additional fast-migrating bands (B-1 and B-2) whereas no such DNAs were detected in preparations from normal cytoplasms of (n- boro)rf\1\1rf\1\ and 2 revertants, indicating that reversion of cms to fertile cytoplasm was associated with the disappearance of mitochondrial plasmid DNAs. This suggests a specific involvement of these plasmids in the male sterility of rice.
The plasmids from cms cytoplasm were further purified by equilibrium centrifugation in CsCl-ethidium bromide gradients. Both of B-1 and B-2 were recovered from the lower bands while the main mitochondrial DNAs were recovered from the upper band. B-1 DNA treated with a restriction enzyme moved slower than untreated DNA in agarose gel electrophoresis. The same was the case with B-2. From these results, B-1 and B-2 were estimated to be supercoiled circular DNAs (cccDNA) having molecular weight of 2.3 and 1.6 kb, respectively.
Reference
Yamaguchi, H. and H. Kakiuchi, 1983. Electophoretic analysis of mitochondrial DNA from normal and male sterile cytoplasms in rice. Jpn. J. Genet. 58: 607- 611.