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

Microtechniques in Seed Histopathology 263• Water boiling: Water-boil seeds for 2 to 48 hours on a water bath untilseed coat becomes soft. Treating seeds in an autoclave has also beensuggested but the present authors prefer water boiling on a water bath.Cool and store in 70% ethyl alcohol.• Seeds with mucilage in seed coat (Eruca, Linum) or endosperm (Cyamopsis,Trigonella) should be boiled only for a short period (5 to 10 minutes)or kept overnight at 60°C in an oven to soften them.Seeds boiled in water give satisfactory results because this treatment does notcreate problems with stains and staining procedures. Sections from acid-treated seedsstain well, but on storage the stains often fade.Large seeds with a thick seed coat (Cucurbita, Citrullus, and Hevea) and pericarp(Helianthus) should be boiled and, after cooling, the seed coat and pericarp shouldbe separated from the rest of the seed components. Divide the former into smallpieces and the latter into two longitudinal halves before processing further.10.4 HISTOLOGICAL METHODS10.4.1 WHOLE-MOUNT METHODA general account of preparing whole-mounts of plant parts is given by Johansen(1940), Gardner (1975), and Neergaard (1997). These methods cannot be appliedas such for preparing whole-mounts of seed that consist of a number of componentsand of both hard and soft tissues. Whole-mount preparations are, of course, veryuseful for determining the location of the pathogen in seed components because themethod is quick and gives a total picture of the characteristics of the mycelium andits spread.The procedure for preparing whole-mounts of seed components in some studies(Maden et al., 1975; Singh, Mathur, and Neergaard, 1977, 1980; Agarwal et al.,1985) is as follows:• Water-boil seeds for 30 to 45 minutes.• Cool and separate seed components.• Boil components of one seed in a test tube in 5 or 10% aqueous KOH orNaOH solution or in 5% HCl for 10 or 20 minutes.• Wash thoroughly with tap water to remove traces of alkali or acid.• Stain with cotton blue and mount in lactophenol.In the case of bulky components or those that do not become transparent in theabove treatment, it may be cleared in lactophenol by gently heating the slide on aflame or if prolonged treatment is to be given, in the oven at 80°C (Agarwal et al.,1987).Acid treatment is found superior for seeds with crystals in the seed coat as inthe case of sesame. The calcium oxalate crystals, present in the epidermis, areremoved by acid treatment (Singh, Mathur, and Neergaard, 1977).