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

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Microtechniques in Seed Histopathology 265TABLE 10.1Composition of Tertiary Butyl Alcohol SeriesReagentsAlcohol Percentage50 70 85 95 100Distilled water 50 30 15 — —95% ethyl alcohol 40 50 50 45 —Tertiary butyl alcohol 10 20 35 55 75100% ethyl alcohol — — — — 2510.4.3.3 InfiltrationInfiltration is the process of transfer from TBA to the paraffin wax in tissue. It is animportant but slow process. The authors prefer to carry out the process initiallyunder a bulb (60 W). Add flakes of paraffin wax to a vial. Gradually increase theamount by adding more flakes every 2 hours. Continue until the amount of mixturehas doubled, leave overnight, drain half of the fluid, add melted paraffin wax, andkeep for 6 or more hours. Repeat the process twice and transfer the vials into theoven at 60°C. Drain the fluid and change with melted wax. Make changes every 6or more hours until traces of TBA are removed.10.4.3.4 EmbeddingEmbed the infiltrated material in melted wax. Arrange the material in proper order(for details, see Johansen, 1940).10.4.3.5 Softening of Embedded MaterialThis step is very important for microtomy of seeds. The following protocol isrecommended:• Cut block into small individual blocks with one seed per block.• Trim individual blocks, particularly on the cut side of the seed leavingabout 1 mm of the paraffin wax covering it.• Immerse blocks in 1% aqueous solution of sodium lauryl sulphate for24 hours.• Wash with water.• Transfer to a mixture of glycerin and acetic acid (1:1) for 1 to 4 weeksdepending on the size and hardness of seed.• Wash thoroughly with water and store.10.4.3.6 Sectioning and Mounting of RibbonsMicrotome sections can be cut using any rotary microtome. For more details consultJohansen (1940).
266 Histopathology of Seed-Borne InfectionsThe paraffin ribbons containing sections are cut in pieces of suitable size andfixed on slides using Haupt’s or Meyer’s adhesive (Johansen, 1940). For sections ofseeds, the double adhesive process has given better results. Smear the slides withHaupt’s or Meyer’s adhesive, cover the smeared surface with gloy solution, mountthe ribbons, spread, drain the extra fluid, and dry.10.4.3.7 Staining and Mounting1. Temporary preparations: Deparaffinize sections with xylol, bring to water,stain with cotton blue, and mount in lactophenol. Hyphae are stained blue.2. Permanent preparations: Several standard staining procedures aredescribed by Johansen (1940), Baker (1958), and Gerlach (1984). Safranin–lightgreen and safranin–fast green are found most suitable for histopathology.Only the main steps are given below. For details consultJohansen (1940).Deparaffinize slides in xylol, bring down slides to 70% alcohol, stain in safraninsolution, wash excess stain with water, destain in picric acid–alcohol solution or70% ethyl alcohol until the dye is removed from the thin-walled cells. Dehydrateslides through alcohol grades (30, 50, 70 and 90%). Counter stain with light greenor fast green, differentiate in clove oil, and wash in a mixture of xylol and 100%alcohol (1:1) to remove traces of clove oil. Transfer to pure xylol and mount inD.P.X., Canada balsam, or Caedax solution.Slides must be dried before storing in slide boxes.10.5 PROCEDURES FOR PREPARING SOME REAGENTSAND STAINS10.5.1 FIXATIVEFormalin-Acetic Acid-Alcohol (FAA)70% ethyl alcohol 90 mlGlacial acetic acid 5 mlFormalin 5 mlFor delicate materials, 50% ethyl alcohol may be used in place of 70%.10.5.2 ADHESIVESHaupt’s AdhesivePlain, finely divided pure gelatin 1 gDistilled water 100 mlPhenol crystals 2 gGlycerin 15 ml
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Histopathology ofSeed-Borne Infecti
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PrefaceThe book deals with only one
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The AuthorsDalbir Singh, Ph.D., for
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ContentsChapter 1 Introduction ....
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4.3.2 Routes for Infection from Ova
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8.4 Survival in Seed ..............
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2 Histopathology of Seed-Borne Infe
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4 Histopathology of Seed-Borne Infe
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2Reproductive Structuresand Seed Fo
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Reproductive Structures and Seed Fo
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14 Histopathology of Seed-Borne Inf
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3Structure of SeedsThe seed habit i
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Structure of Seeds 49(1971) of usin
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Structure of Seeds 51is found in as
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Structure of Seeds 53dry seed of Ly
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Structure of Seeds 553.3.2 SEED COA
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Structure of Seeds 57Endosperm: One
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Structure of Seeds 59scendembepsmuA
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Structure of Seeds 61Endosperm: Sca
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Structure of Seeds 63Chalaza simple
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Structure of Seeds 65stystypmerA Bc
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Structure of Seeds 67epsent endCADB
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Structure of Seeds 69perhscendembpe
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Structure of Seeds 71eposgepi′epo
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Structure of Seeds 73(Zea and Sorgh
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Structure of Seeds 75Baker, D.M. an
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Structure of Seeds 77Maheshwari Dev
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Structure of Seeds 79Vries, M.A. de
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100 Histopathology of Seed-Borne In
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TABLE 5.6Location of Mycelium of St
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7Seed Infection by VirusesViruses a
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Seed Infection by Viruses 201TABLE
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Seed Infection by Viruses 205Unlike
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Seed Infection by Viruses 2073. The
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Seed Infection by Viruses 209Wilcox
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Seed Infection by Viruses 213floret
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Page 236 and 237: Seed Infection by Viruses 221AMV fo
Page 238 and 239: Seed Infection by Viruses 223REFERE
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