Confocal laser Scanning Microscopy of Mitochondria within Microspore Tetrads of Plants Using Rhodamine 123 as a Fluorescent Vital Stain

The present study demonstrates that rhodamine 123 penetrates the callose walls surrounding plant microspores before they are released from tetrads. The stain accumulates in active mitochondria due to the electrical potential across the mitochondrial membrane. Accumulation of dye does not occur in mitochondria of fixed cells and fades quickly when mitochondrial activity is inhibited by exposure to carbonyl cyanide m-chlorophenyl hydrazone. Rhodamine can be used as a viability test for microspores still within tetrads, thus making it possible to determine when during development genes leading to pollen sterility are expressed. Rhodamine 123 is excited by blue (550 nm) light and can thus be used with confocal laser scanning microscopy. Anthers of Nicotiana tabacum, Oenothera villaricae, Silene dioica and Lycopersicum esculentum were studied here.