TRANSGENIC SPINACH PLANTS EXPRESSING THE COAT PROTEIN OF CUCUMBER MOSAIC-VIRUS

Transgenic spinach (Spinacia oleracea L.) plants expressing cucumber m osaic virus (CMV) coat protein genes were regenerated. Leaf and hypoco tyl explants from two spinach cultivars, 'Fall Green' and 'High Pack,' were transformed with Agrobacterium tumefaciens strain LBA 4404 carry ing the binary vector pBI121 in which the beta-glucuronidase (GUS) gen e was replaced with the coat protein gene of CMV isolate SP103 or SP10 4. Calluses were induced from both cultivars at rates of 10-27% on MS selective medium containing 86 mu M kanamycin, 237 mu M carbenicillin and 209 mu M cefotaxime. After 12 wk (three transfers), putatively tra nsformed calluses were transferred to MS medium without kanamycin to p romote callus proliferation. Shoots were regenerated at the rates of 2 3-29% for 'High Pack' and less than 0.5% for 'Fall Green'. Approximate ly 80% of the regenerated shoots from both cultivars were subsequently induced to establish whole plants. Regenerated plants were screened f or transgene incorporation using a polymerase chain reaction (PCR) met hod 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 co at protein gene. Southern blot analysis confirmed the presence of vira l coat protein genes in the plant genomes. Western blot analysis indic ated that the CMV coat protein genes were expressed in transgenic spin ach plants.