Electroporetic transfection of pepper protoplasts with plant potyviruses

Abstract

Potyviruses are a persistent threat to bell pepper (Capsicum annuum L.) production worldwide. Much effort has been expended to study the resistance response of pepper cultivars at whole plant levels but with only limited effort at the cellular level using protoplasts. A pepper protoplast isolation procedure is available but an inoculation procedure is needed that provides consistent and highly efficient infection. An electroporation-based procedure for inoculation of potyviruses was developed using a base procedure developed for Cucumber mosaic virus (CMV). The final parameters identified for efficient potyvirus infection of pepper protoplasts involves two 25ms pulses, 200V each pulse with a 10s interval between pulses. Depending on the method of detection, e.g., ELISA versus RT-PCR, potyvirus RNA inoculum ranged from 10 to 40μg with infection detection occurring with samples of 50,000-100,000 protoplasts.