Chapter 5 Genetic Analysis of Apomixis - cimmyt

Oo,silk.tlo••f Apamiclic MotH.isll' 37affecting their general properties by partiallysubstituting isoeugenol (n D= 1.574; Windholzet al. 1983) for eugenol (n D = 1.541; Windholzet al. 1983) in the recipes given below.Aromatic oils require complete or nearlycomplete dehydration of the ovule, whichtypically lengthens the clearing procedure andleads to shrinkage and possibly distortion. Theovules normally become hard and brittle,which is good on the microscope slide but noton the dissecting stage. While the oils that havebeen used are only mildly toxic, this is notuniformly true, and some oils (polynucleararomatic hydrocarbons) must be rejected ascomponents ofclearing media because of theircarcinogenicity.Both the lactophenol and aromatic-oil clearingagents offer limited opportunities forcytochemistry. Most of the establishedcytochemical procedures are based onreactions in water and may behave abnormallyin less polar solvents. Opaque reactionproducts can obscure unstained regionsbehind them, and accessibility to reactants isalways more difficult in intact ovules than insections thereof. Reaction products can bemobilized or lost upon dehydration andinfiltration with clearing agents. Nevertheless,there is interest in distinguishing various typesof cell walls (especially callose) and cellularinclusions (e.g., starch grains) in clearedovules, and a medium that can be used to thisend is introduced here for furtherinvestigation.Several inorganic salts, including those listedin Table 3.1, dissolve to a considerable degreein OMSO, glycerol, concentrated sugarsolutions, or polyethylene glycol. None ofthem has proven completely satisfactory as analternative to the aromatic oils or 4-1/2 media,but they might permit additionalcytochemistry to be performed on wholemounts. Some media with KI equal the 4-1/2media in refractive index (Table 3.1), but donot clear the tissue well. This is attributed toiodination of the macromolecules in the cell,which raises its refractive index. The problemwith CaCl 2is the high viscosity of its solutionsin polyols. Its hygroscopicity also causes therefractive index of the preparation to vary withthe relative humidity in the room. Ferric andzinc chlorides are extremely corrosive tometals and human flesh, and they destroynuclei relatively rapidly. Potassiumthiocyanate gives a high refractive index insolution, but is chaotropic (destroys nucleiagain) and hazardous to the mental health ofthe slide user (Handbook of Chemistry andPhysics). Finally, the problem with DMSO forembryological clearings is its denaturantaction on DNA and proteins, resulting in poorquality of nuclei or no nuclei at all. This actionis the basis of a very effective recipe (providedbelow) for destructively clearing leaves forvascular and epidermal study.The following protocols are mostly based onfixation in organic solvents that kill the cellquickly and preserve the chromosomes well.The choices include FAA (37% aqueousformaldehyde, acetic acid, and 50% or 70%ethanol, 1:1:18 by volume); FPA (the samesolution with propionic acid substituted foracetic acid), 3:1 etbanol:acetic acid;, Carnoy'ssolution (6:3:1 ethanol:chloroform:acetic acid,by volume); and 2:1 acetone:acetic acid. Crafand Allen-Bouin-type fixatives can also beused, but require longer dehydrations.Formaldehyde and glutaraldehyde, alone orin combination, also require longdehydrationsand typically leave the ovules yellow. Theyalso contribute to autofluorescence in thedetection of callose with aniline blue. The lesspolar fixatives generally cause severeplasmolysis of embryo sacs with two or morenuclei, but they also extract chlorophyll andcarotenoid pigments more completely fromovary walls. Acetone/acetic acid isparticularly effective at this and often leavestissues snow-white.