P-10. In planta distribution of Citrus tristeza virus and standardization simple methods for RNA isolation
Abstract
Determination of in planta movement and distribution of plant viruses is important criteria for isolation of virus nucleic acid proper infected tissues for advanced molecular and genetic studies of viruses and ultimately designing management strategies of viruses in crop plants. Further, for quicker and reliable diagnosis of viral disease, it requires selection infected plant parts having considerable amount of virus titre to be detected and method of isolation. In present studies, selection of infected tissues with good amount virus titre and standardization of cost efficient, fast and simple methods of RNA extraction were studied. Twenty different samples from Citrus tristeza virus (CTV) infected acid lime plant (Citrus aurantifolia) like bark tissues of 6 months to 5 yrs old brances, and 4 parts of leaves different ages, prickles/thorn, buds etc were collected and detected virus titre by direct antibody coated-ELISA (DAC-ELISA) using antisera to CTV. DAC-ELISA determined relative concentration of viral titre in different parts of samples. Petiole of new leaves showed highest virus titre followed by epical buds, petioles of mature leaves, mid veins of new leaves, petioles of old leaves matured leaves and barks of six month old branches by 15, 13, 12, 11, 10, 7.5 folds compared to healthy control, respectively; these results were agreed by quality of amplification of DNA by RT-PCR using templates from same plant parts with specific primers coat protein gene of CTV. Necessity of liquid N2 for tissue grinding, and use of refrigerated centrifuge were studied for isolation RNA template for amplification of DNA through RT-PCR. Two different methods, one by commercial methods, and another by laboratory made modified method using cellulose fibre were also standardized for RNA isolation. Templates obtained by commercial method showed best and similar DNA amplification results that obtained from the template from modified laboratory method where liquid N2 and refrigerated centrifuge was used. The commercial method without N2, and laboratory method at refrigerated centrifuge without N2 gave second highest results.
