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

10Microtechniques in SeedHistopathologySeeds, after dispersal, are autonomous and exposed to the hazards of the environments.They have a strong protective covering with cuticle, waxy coatings, and seedcoat and pericarp with thick-walled lignified or suberised cells. The usual histologicaltechniques cannot be applied to cut seeds in a dry state. Seeds need to be softenedand cut into small pieces. Immature developing seeds and internal components ofseeds, the endosperm and embryo or the embryo after removing the seed coat andpericarp, can be processed like any other soft material.There are several books on plant microtechnique (Johansen, 1940; Baker, 1958;O’Brien and McCully, 1981; Gerlach, 1984; Neergaard, 1997) and electron microscopy(Glauert, 1974; Aldrich and Todd, 1986; Robards and Wilson, 1993). In thischapter, only some tips and methods, which are useful in the study of seed histopathology,are provided. For detailed information and for SEM and TEM techniquesrefer to the books cited above.10.1 CHOICE OF MATERIAL• Artificially or naturally infected seeds, undamaged or degraded ormechanically injured, have been used (Singh, 1983). Naturally fieldinfectedundamaged seeds should have priority over artificially inoculatedseeds or seeds from field-inoculated plants.• Selection should be made following laboratory screening of seed samplesfor seed-borne fungi.• Select samples with single pathogen infection or predominant infectionof the pathogen under study. In the latter condition, samples with goodinfection percentage of the target pathogen and low infection of otherfungi, which are readily eliminated after chlorine pretreatment, may bepreferred.• Asymptomatic as well as symptomatic seeds, the latter categorized intoweakly, moderately, and heavily infected, should be examined.• Size of the seed sample examined should be large enough to give correctassessment.The above procedure is also useful for selecting seed samples infected bybacteria, but for viruses, half-seeds corresponding to those halves that test positivefor the virus in serological assay and/or other tests are recommended (Carroll, 1969;Alvarez and Campbell, 1978; Hunter and Bowyer, 1993).261