Histopathology of Seed-Borne Infections - Applied Research Center ...

262 Histopathology of Seed-Borne Infections10.2 DETERMINATION OF THE IDENTITYOF INTERNAL MYCELIUMThe problem of determining the identity of internally seed-borne mycelium of fungiposes some difficulty. D. Singh, while working at the Danish Government Instituteof Seed Pathology (DGISP), Copenhagen (1973 to 1974), developed the techniquecalled component plating. It has been widely used to detect the internal infection offungi, which readily sporulate or produce structures, permitting their identification(Maden et al., 1975; Singh, Mathur, and Neergaard, 1977, 1980; Singh, 1983).10.2.1 PROCEDURE FOR COMPONENT PLATING• Soak seeds in water at room temperature, just long enough to permitseparation of components, namely, seed coat and pericarp, endosperm andembryo.• Dissect seed aseptically to separate components using sterilized scalpel,needles, and forceps.• Sterilize each component by washing in 1% chlorine solution.• Plate on wet blotters (as in the standard blotter test) or potato dextroseagar medium (PDA).• Incubate for 7 days under near ultra-violet (NUV) or daylight fluorescencetubes.• Examine the different components under stereobinocular microscope onday 8.Note: Period for soaking of seeds in water and for incubation of plates may bedetermined in preliminary tests.Separated components of seeds can also be used to determine bacteria andviruses using infectivity, serological, and other tests (Schaad, 1988; Lange, Wu, andvan Vuurde, 1992).10.3 SEED SOFTENINGDry seeds need to be softened for any histological study. Seeds are usually soakedin water, but prolonged soaking of infected seeds at room temperature or temperaturecongenial for revival and growth of the pathogen may enable the pathogen to spreadto new areas. The following treatments are usually followed.• Hydrofluoric acid treatment: Soak seeds in 5 to 20% hydrofluoric aciduntil rendered soft, then wash in running water for 24 or more hours untiltraces of the acid are removed. Store in 70% ethyl alcohol.• Picric acid treatment: Water-boil seeds for 1 to 2 hours. Cool and transferto aqueous saturated picric acid solution at room temperature or at 40°C(keep in oven) for 2 to 4 weeks, depending on the hardness of seed. Washin running water until traces of the acid are removed. Store in 70% ethylalcohol.