Meiosis & Karyotyping for Plasmodiophorids
Some of the first images we obtained of Sorosphaera veronicae when I started TEM studies of it back in the early 70s showed synaptonemal complexes (SCs). I was familiar with SCs because my dissertation at Iowa State University was on meiosis in Lilium longiflorum. SCs in Sorosphaera led us to suspect that the noncruciform divisions in transitional sporogenic plasmodia were meiosis. One of my goals was to see how many plasmodiophorids had meiosis in transitional sporogenic plasmodia, and if they did, use the SCs to karyotype them. Since the nuclei of plasmodiophorids are only several micrometers in diameter, conventional light microscopy was insufficient for counting chromosomes. We thought that using serial sections as shown by Moens and Perkins (1969) was the way to get karyotypes.
Suzanne Harris was a MS student in the department who showed the type of perseverance needed to take on the difficult assignment of serial sectioning entire nuclei (no skips were allowed) of Sorosphaera in our first attempt to get an accurate karyotype for a plasmodiophorid. I happened to be on a sabbatical in 1978-79 in Michael Bennett's lab at the then Plant Breeding Institute in Trumpington, Cambridge, UK, where they were serial sectioning entire nuclei of wheat, which are magnitudes larger than nuclei of plasmodiophorids. Suzanne visited me over the Christmas holidays in 1978 and we worked on interpreting her serial sections of Sorosphaera. Her MS thesis was the first accurate karyotype of a plasmodiophorid.
When I returned to Ohio University after my sabbatical, I was able to apply the techniques I learned from Jim Smith in Bennett's lab to the plasmodiophorids. A summary of the method that was based on Wells (1974) and Rowley and Moran (1975) was published in Zoosporic Fungi in Teaching and Research, edited by M.S. Fuller and A. Jaworski. What I did that was a little special was to make serial sections of specific, individual infected cells. I would take a 1/2 µm "thick" section and look at it through light microscopy. When I found an infected cell that had a stage of the plasmodiophorid that I wanted to serial section, I would orient the block from which the thick section was taken in such a way that when viewed with a dissecting microscope, the cells could be seen in the freshly cut surface. I would then trim to the cell I wanted, reposition the block back onto the ultramicrotome, and make serial sections.
Analyzing the serial sections was not as straight forward as it seems when reading the publications. For the SCs in plasmodiophorids the sections needed to be 100-130 nm (gold). If they were thinner (silver), which many microscopists would recommend for improved resolution at higher magnifications, the SCs could be lost when viewed laterally. Also, if one or more ends of SCs did not end at the nuclear envelope in a nucleus that had been serial sectioned, the analysis could not be completed. There were a lot of nuclei that we serial sectioned, but could not be used in the analysis. One interesting aspect of plasmodiophorid meiotic nuclei is how the SCs end near the poles in a double bouquet. The poles are defined in pachynema by centrioles, and the SCs end on the nuclear envelope near the centrioles. This arrangement became clear to us when we started making models of the nuclei. Of course today the computer would make the 3D reconstructions and a 3D printer could be used to construct the models.
Images of Meiosis, Synaptonemal Complexes, & Karyotyping
References for Meiosis, Synaptonemal Complexes, & Karyotyping