Chapter 5 Genetic Analysis of Apomixis - cimmyt

aa"llkatio. 01 ApOIIIidic Medla.lsm, 35Appendix: Methods to ClearAngiosperm OvulesApomictic development can be studied withany of the tools used to study other plantanatomical problems. Particularly useful toolsinclude thick and thin sections, clearings, flowcytometry, and Feulgen microspectrophotometry.Clearings have played aprominent role in recent studies because theyallow characterization of large, reproductivelyheterogeneous samples. Sections and clearingsallow the positions and divisions of nuclei andprotoplasts to be observed directly; sequencesof nuclear divisions and movements can beinferred by observing the set of still images. Inthis respect, both are inferior tocinematography of living embryo sacs inovules suspended in silicone oil (Erdelska etal. 1971, 1979), but the latter technique has notbeen widely applied because of the usuallyinsufficient transparency of the nucellus andinteguments. Flow cytometry providesinformation on the DNA content (ploidy) ofembryonic and endospermatic nuclei and thuson the frequency of apomixis and the role ofthe sperm nuclei in apomictic endospermdevelopment. The older technique of Feulgenmicrospectrophotometry provides this sameinformation for species or developmentalstages in which there are too few nuclei forflow cytometry or for which positiveidentification of all nuclei is desired. Exceptfor cinematography, these methods requirefixation of the specimen to stop all divisionsand preserve the cells of interest in asufficiently "lifelike" condition. The type offixation limits the quality of the resulting data.The researcher's objectives (both scholarly andmicroscopic) determine the image quality thatis needed and therefore the time and labor thatare required; screening a segregating F 2population for the Panicum-type does notrequire ultrastructural preservation orfreedom from plasmolysis.Clearings usually require less effort thanembedding and sectioning, but the overallquality is limited by the amount of visualinformation that can be accrued in a singleoptical section (plane of focus) before the ovuleas a whole is rendered opaque. Objects can beseen because they differ from theirsurroundings in color or refractive index; thelatter causes light to bend at the surface of theobject. Clearing media are designed to closely,but not perfectly, match the refractive index ofcell walls or organelles; an object becomesinvisible when immersed in a medium ofequalrefractive index. Variation in refractive indexamong cellular components assures that someaspect of the specimen will always be visiblewhen the refractive index of the medium isclose to that of the bulk specimen.Furthermore, many structures, such as cellwalls, starch grains, and oxalate crystals, varyinternally in refractive index as the light pathmoves relative to their crystal axes. Thisproperty is termed birefringence, and it can bea severe nuisance in examining cleared ovules.Specimen thickness, refractive index, andinformation content (organelle or nuclearfrequency per unit thickness) determine theoptimal refractive index of a clearing medium.Single cells can be successfully examined inwater, and a movie has been made of nuclearmovements and divisions within viable ovulesofJasione montana (Campanulaceae) immersedin silicone oil (Erdelska et al. 1971). "Normal"ovules and grass ovaries require a considerablycloser matching of refractive index, i.e., greaterloss of visual information from individualplanes of focus, for successful visualization oftheir interiors. Information content reflectsorganellar preservation and thus depends onthe type and duration of fixation. Theorganellar refractive index appears to respondto hydration, and thus the optimum refractiveindex differs between hydrous and anhydrous