Chapter 5 Genetic Analysis of Apomixis - cimmyt

38 (IIarIe,F.e-.The following recipes include examples inwhich the fixative and clearing agent areimmiscible, requiring an intermediarydehydrating agent, and examples in whichthe fixative and clearing agent mix freely. Inboth cases, one must pay close attention tothe solubility of air in each liquid in theprocec;lure. Air appears to be much lesssoluble in aromatic oils and concentrated saltsolutions than in ethanol or acetone. Overrapidinfiltration typically results inexsolution of bubbles or films of air that donot disappear with time. Affected ovulesgenerally float and remain opaque during theinfiltration series. Ifsuch bubbles appear, onemust backtrack, possibly all the way to thefixative, and start again. Adding extra stepsat closer gradations in concentrationeliminates bubbling more effectively thandoes lengthening individual steps.The dehydration and infiltration steps can beadvantageously recombined in many of thefollowing recipes. When the clearing agentand fixative are miscible, a general principleis to progress through a graded series ofmixtures of the two, including enough stepsto avoid exsolution, as discussed above. Agood starting point is 10 gradations fromfixative to the microscope slide. Users shouldfeel free to experiment, especially whenadapting a recipe to novel species or stages.The requirements for dissection must also beconsidered; 70% ethanol is much less noxiousand corrosive than FAA, 2:1 acetone: aceticacid, or salt solutions in dimethyl sulfoxide(DMSO). In many cases, one does not havetime to dissect out ovules before fixation ordoes not want to lose small, nearly invisibleovules when solutions are changed.Microscopy can make or break any clearingprocedure. Confocal laser scanningmicroscopy has been used impressively onthin orchid ovules (Fredrikson 1990), but itsusefulness on thicker specimens remainsuntested, and it is not widely accessiblebecause of its expense. Nomarski interferencecontrasthas been applied successfully toapomictic amaryllids (Crane 1978) andpanicoid grasses (Young et al. 1979). Both ofthese methods create a very shallow depth offocus that helps to eliminate information fromoutside the focal plane. Hoffman modulationcontrastis a less expensive alternative tointerference-eontrast. Phase-contrast provides"optical staining" that can help distinguishnuclei from similarly sized vacuoles, but itrequires a closer match between the sampleand the medium in refractive index. Stainclearedspecimens (and many unstainedspecimens) can be examined satisfactorilyunder brightfield optics once the medium isproperly matched to the specimen in refractiveindex. Brightfield is also insensitive tobirefringent cell walls or crystals in thespecimen, which greatly degrade aninterference-contrast or phase-contrast image.Specimen orientation is frequently critical toeasy and correct interpretation of clearedembryo sacs. Ovaries of pooid grassestypically are laterally compressed and presenta nearly end-on view of the embryo sac whenthey are allowed to lie flat. Standing them onedge in a sawtooth cut in index card oraluminum foil yields the desired sagittaloptical section. Stelly et al. (1984) observed thatsqua~hing cleared ovules was generallycounterproductive; the image got worseinstead of better as more visual informationwas crammed into nearly the same plane.Thus clearings are often examined with "Raj"slides (Herr 1974), with which the cover slipis supported by underlying adjacent coverslips that are conveniently held in place with"Superglue" (G. Hodnett, personal comm.).