Confocal optical sectioning for meiotic analysis in Oenothera species and hybrids

We present a photomicrographic technique for analysis of meiosis in Oenothera species and hybrids. In many species, the 14 isomorphic, 1-2 microns chromosomes are organized into a permanent translocation ring. Interspecific hybrid meiotic analysis is complicated by variations in homology between chromosome sets (Renner complexes) of allospecific origin. Though most wild species from complete rings of 14 at meiosis, hybrids often form combinations of smaller rings and bivalents. There is evidence that asynapsis (chain and univalents) also frequently occurs in hybrids. The greater complexity of hybrid meiotic organization can hinder resolution of individual chromosomes in such chains and small rings and encumber photomicrography of all chromosomes in a cell. Confocal laser scanning microscopy (CLSM) offers a new cytogenetic approach in ring-forming Oenothera species and hybrids. Fortunately, crystal violet, the stain classically used with this difficult material, fluoresces at an excitation wavelength of 488 nm and may thus be employed with the CLSM. Serial projection of image maxima from Z-axis confocal optical sections enables the investigator to count chromosomes and establish pairing relationships, regardless of chromosomal distribution.