Purification of mitochondria and mitochondrial nucleic acids from embryogenic suspension cultures of a gymnosperm,Larix x leptoeuropaea

After a number of attempts to isolate mitochondria from different conifer tissues, embryogenic suspension cultures of hybrid larch (Larix x leptoeuropaea) were developed which enabled the purification of mitochondria using slight modifications to standard techniques. The mitochondrial purity was verified by analysis of the mitochondrial RNA, DNA and proteins. The larch mitochondrial genome size is surprisingly large (> 1000 kbp) and the polypeptide pattern differs greatly from those of wheat or potato mitochondria, suggesting that valuable evolutionary insights will be gained from comparisons between gymnosperm and angiosperm mitochondria. The ease with which embryogenic conifer suspensions can be initiated and used for mitochondrial purification implies that they will be the material of choice for future studies of this type.Abbreviations PCR polymerase chain reaction - mtDNA mitochondrial DNA - 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - Tris tris(hydroxymethyl)aminomethane - EDTA ethylenediaminetetraacetic acid - BSA bovine serum albumin - EGTA ethylene glycol-bis(ß-aminoethyl ether) N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulphate - DEPC diethyl pyrocarbonate - PAGE polyacrylamide gel electrophoresis - IEF iso-electric focusing