Transgenic spinach plants expressing the coat protein of cucumber mosaic virus

Abstract

Transgenic spinach (Spinacia oleracea L.) plants expressing cucumber mosaic virus (CMV) coat protein genes were regenerated. Leaf and hypocotyl explants from two spinach cultivars, ‘Fall Green’ and ‘High Pack,’ were transformed with Agrobacterium tumefaciens strain LBA 4404 carrying the binary vector pBI121 in which the β-glucuronidase (GUS) gene was replaced with the coat protein gene of CMV isolate SP103 or SP104. Calluses were induced from both cultivars at rates of 10-27% on MS selective medium containing 86 µM kanamycin, 237 µM carbenicillin and 209 µM cefotaxime. After 12 wk (three transfers), putatively transformed calluses were transferred to MS medium without kanamycin to promote callus proliferation. Shoots were regenerated at the rates of 23-29% for ‘High Pack’ and less than 0.5% for ‘Fall Green’. Approximately 80% of the regenerated shoots from both cultivars were subsequently induced to establish whole plants. Regenerated plants were screened for transgene incorporation using a polymerase chain reaction (PCR) method using the same primer pair originally used to amplify the CMV coat protein genes for insertion into pBI121. Based on the PCR screening, approximately 12% of the regenerated spinach plants contained a CMV coat protein gene. Southern blot analysis confirmed the presence of viral coat protein genes in the plant genomes. Western blot analysis indicated that the CMV coat protein genes were expressed in transgenic spinach plants.