Department of Entomology and Plant Pathology, 209 Life Sciences Building, Auburn University, AL 36849, USA.
J Virol Methods. 2012 Jan;179(1):154-60. doi: 10.1016/j.jviromet.2011.10.015. Epub 2011 Nov 9.
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.
马铃薯 Y 病毒属是一种世界性的威胁甜椒（Capsicum annuum L.）生产的持续性病毒。人们已经投入了大量的精力来研究辣椒品种在整个植株水平上的抗性反应，但在使用原生质体的细胞水平上只进行了有限的研究。已经有一个辣椒原生质体分离程序，但需要一种提供一致和高效感染的接种程序。利用为黄瓜花叶病毒（CMV）开发的基础程序，开发了一种基于电穿孔的接种马铃薯 Y 病毒属的程序。确定的用于有效感染甜椒原生质体的马铃薯 Y 病毒属的最终参数涉及两个 25ms 脉冲，每个脉冲 200V，脉冲之间间隔 10s。根据检测方法，例如 ELISA 与 RT-PCR，马铃薯 Y 病毒属 RNA 接种物的范围为 10 到 40μg，用 50000 到 100000 个原生质体的样本进行感染检测。
