Plant Protoplasts Cytoskeleton Observation And Detection Techniques Of Orchids Viruses

Bean broad wilt virus 2(BBWV 2)is reported in many regions caused serious losses in economic crops.For study of virus-host-relationship about BBWV 2,the factors that affect protoplast isolation,includes enzyme,pH value of enzyme,isolation time,osmotic stabilities were studied with young and tender leaves,which came from pea that cultivated in pest proof green house for 20 to 60 days.The isolated protoplasts whose sizes were plumpness and round in size and vigor were nice determined with FDA.The protoplasts both healthy and BBWV 2 infected were marked with primary antibody ofβ-Tubulin(mouse)and rabbit anti\mouse IgG.antiserum as second antibody and detected by laser scanning confocal microscope.It was closer to nature that used protoplasts isolated from fresh leaves instead of callus.In results,special change was not detected in microtubule cytoskeleton between protoplasts healthy and BBWV 2 infected, which shows that BBWV infection may not lead to microtubule cytoskeleton changes of pea or maybe the changes were too little to detect.Cymbidium mosaic virus(CymMV)and Odontoglossum ringspot virus(ORSV) cause large-area occurrences on orchids,which leads to economic and ornamental values losses beyond imagine.For virus disease,prevention and controlling transmission is the core control method.In the paper,detection methods of the two viruses on orchids were studied,and comparative analysis of sensitivity and field practicality were carried out.In the paper,the CymMV and ORSV in the multiple strains orchids collected in several areas were detected by I-ELISA and electrical microscopy and I-ELISA, TAS-ELISA and DAS-ELISA comparative analysis was carried out.The data shows that the incidences were similar and high,while there remained virus-free strains.So it is rigorous to control their transmission for prevent virus-free strains from infection.The sensitivity of TAS-ELISA is the highest in the three ELISA methods due to data results.Dot-ELISA and tissue blot-ELISA with monoclonal antibody to detect CymMV were established.3000-dilution monoclonal antibody could be adopted to detect CymMV infecting leaf extract within a dilution limitation of 1:5120.CymMV could still be detected in the forth print by Tissue blot-ELISA.Similar results were shown between the first print by tissue blot-ELISA and 40-dilution of infected leaf extract by Dot-ELISA and satisfactory result shall be showed before 4 prints.Comparison tests showed that the commonly used tissue blot-ELISA was less sensitive than dot-ELISA,but the tissue blot-ELISA is simpler than dot-ELISA,and is suitable for fast detection in fields.