4/1/25 VATION AND QUANTIFICATION OF PLANT VIRUSES INTRODUCTION:- Plant viruses are obligate intracellulas parasite which cause disease in plante →It contains esthes ENA/RNA as genetic material →Bul, most Plant viruses are classified as Single stranded RNA of Double Stranded RNA Vious poorticles →11 cause various types of plant diseases. These diseases do not typically result in plant death Symptoms:- plant cdiseases produce symptoms such as Ring Spots J. mosaic pattern development Leaf Curling is, Lear yellowing and distantion → Some plant Vlovses care not limited to one particular Ploni host, may Infect different varieties of plants £g: Tobacco mosaic virus (TMV) Infects plants including tomatoes, peppers, cucumbers and tobacco. Examples of plant Diseases:- Tobacco Mosaic virus.. → It is the 1st plant virus discovered → Allacks members of the nightshade / tolanaceae family including tobacco, pepper, potato, eggplant and cucumber. - spreads through entry into breaks of cell wallt cowed by insects of other physical damage. Sugarcane Mosaic VITUS: →Discolors leaves of the Sugarcane plant. →Stunts the growth of young plants Aphids and infected seed spread the virus. Peanut stunt vious:- → causes discolouration and distortion of the leavel of Peanuts and stunting their growth →Aphids and sap spreads the virus Cauliflower Mosale Vious: →The Cmy infed's members of the Brassica family →the members include cauliflower, Cabbage, Broccoli →Il causes a mosaic/mottle on the leaves which Stunts growth. → Aphids & mechanical exposure spread the virus. Cultivation of viruses: par succesfull cultivation of viruses. Albion of viruses There are some steps to follow it, Isolation cultivation Isolation: refers to separation of infected plant materials (tissue, past, celle) harbouring virus particles 11, cultivation:- Growing of wous (plant huwes). → It can be carried out by following methods whole plant 6 protoplast Culture f. Tissue culline a, whole plant → plant viruses can be directly grown on leaves of living Plants - Mechanical damage to the leaves is accomplished with the aid of an abratives such as carborundam The Viove is applied to the abrade surface →After incubations lesions are observed. b protoplast cultures protoplast. It is vefers is entire cell excluding cell wat protoplast culture: Cultwing a plant Cany part) without Cell wall refers to postaplast culture. The plant protoplast cultures are very useful for cultivation of plant viruses as they lack cell walls →Basically method used to produce priitoplasι τηνοίve the enzymatic digestion of cell wall componente. Process Of Plant Virus Cultivation in protoplast protoplast should be freshly washed with 0.7M Mannitol Immediately before Inoculation. → poly L-cornithine (a.o) should be added to Inoculum. → proper pH and osmotic pressure should be maintained during inoculation →Alter infection, protoptast should be washed to remove Excess viruses and inoculum medium → Abore mizivse should be re-suspended in incubation medium → Replication of plant viruses within protoplast may be demonstrated in various ways. Eg: Staining with fluorescent Ab, infectivity assay. using electron microscope serology He C, Tissue culture. It is defined as culluring plant lissue, cells, seeds, organs on a chemically defined Synthetic nutrient media process of plant viruses cultivation in tissue culture → Da. PR white was the first to grow plant viruses in Bissue culture- In this method, aheady suspected (viral) plant is used. This Is because it has been found to be difficult to infect healthy plant tissues cullere freshly with virus So, a systematically vine infected tomato plant is Used/taken Its stem was cut up into segments washed thoroughly and were suspended by threade in conical flask containing little water Segments are allowed to develop roots After a period of time, the root tips were removed and placed in flask containing some of suitable rutrient medium The root Eps were allowed to grow about a week ↓ Afterwards, the cultures were cut into unifam length of cm. These were then subcultured in fresh medium The viruses were found to be multiply in these lips Cauting visible changes in root tips Quantification:- It refers to detecting/or enumerating of plant viruses can done by a, pathogenicity 6. Microscopy t, plaque Assay d, Serological-ELISA e, Molecular-PER pathogenecity:- The Vovses can be detected by localized and systemic infections • Localized-Appearance of local lesions at site of ine ulation Systemic-Infections which produce symptomt on paris liter from the inoculation Site MICROSCOPY-TEM Is used for counting the w i plaque Assay virus concentration can be detected by forming plaques in a hatt Culluse plate. d. Serological-ELISA By wing ELISA Technique the detection of viruses in infected sap of plant can be done. e, Molecular-PCR:- It is use when the whal porrticles in low amount → By this [PCR] viral amount can be amplitted then a known probe is added 11 will detected virus. Use of virus cultivation:- Virus can be cultivated within suitable holl, such aa living cell i, to isolate and identify the viruses in clinical somple , to prepare vaccines and To do detailed research on viral structure multiplication tydes, geutics and effects on host cells.