Mitochondrial genome variability in Sorghum cell culture protoclones

Sorghum bicolor cv NK300 seedlings, a cell suspension culture, and five protoclone suspension cultures were compared for the occurrence of somaclonal variation by analysis of their mitochondrial DNA (mtDNA). Restriction digests of the mtDNA showed qualitative and quantitative variation of restriction fragments. Southern analyses were performed using a 14.7-kb EcoRI mitochondrial genome fragment and regions carrying mitochondrial protein coding genes, atpA, atp6, cob, and coxI as probes. These analyses revealed part of the 14.7-kb EcoRI region to be present as a repeat in planta, and to be hypervariable when cells were subjected to protoplast culture. All protoclones differed from each other, from the parental cell suspension culture, and from the seedlings in their mitochondrial genome arrangement. Seedlings of five independent sorghum accessions, unrelated to cv NK300, of diverse geographic origin showed conservation of this mitochondrial fragment. Southern analyses of the mtDNA showed no variation for genomic organization of the region carrying coxI, and atpA was identical in all the tissue culture lines. The atp6 gene was present as two copies in the seedlings, and one copy was rearranged upon tissue culture. The region carrying the cob gene was also found to be variant between tissue culture and seedling mtDNA. A substoichiometric 3.3-kb EcoRI cob fragment present in seedlings was amplified in the tissue culture lines. Protoclone S63 differed from the original suspension culture and remaining protoclones in that it had lost the 3.0-kb EcoRI band, the most abundant fragment in seedlings. A new set of fragments was detected in this protoclone. Northern analysis for the cob gene demonstrated altered transcript size in protoclone S63.