CLONING AND CONSTRUCTION OF SINGLE-CHAIN VARIABLE FRAGMENTS (SCFV) TO CUCUMBER MOSAIC VIRUS AND PRODUCTION OF TRANSGENIC PLANTS

The variable regions of heavy and light chain genes were cloned from the RNA of mouse hybridoma cells that produce monoclonal antibodies specific to Cucumber mosaic virus (CMV). The cloned genes were constructed into single-chain variable fragments (scFv) in combination with various promoters and transferred into Nicotiana benthamiana leaf tissue using Agrobacterium tumefacien. Transgenic plants were generated and selected using the bar gene that confers resistance to phosphinothricin. From purified genomic plant DNA, the scFv was re-cloned after PCR and sequenced to verify the presence of the transgene. Resistance to CMV infection was evaluated by mechanically inoculating plants with purified CMV. Several R0 transgenic plants exhibited some degree of disease resistance in the first screening, showing no symptoms, reduced symptoms or delayed symptom development. In subsequent challenge inoculations of R1 plants with lower dilutions of inoculum, occasionally plants showed resistance when compared with non-transformed plants, but, overall, results were variable. For each of the four promoters, three or four transgenic plants were generated exhibiting some degree of disease resistance.