An Improved System for Clover Yellow Mosaic Virus Infection of Pea Leaf Mesophyll Protoplasts

Abstract

Isolation of pea (Pisum sativum L. cv. Alaska) mesophyll protoplasts by both the ‘one-step’ and the‘modified two-step’ methods resulted in similar yields (1-1.5×10⁷ protoplasts per g fresh weight of leaves). The time of protoplast isolation was considerably reduced, from 2 hr to 35 min. by including Pectolyase Y23 in the digestion medium. Maximum infection, 57%, was obtained at concentrations of 1.0 to 1.4μg/ml for poly-L-ornithine and 3μg/ml for clover yellow mosaic virus (CYMV), using 0.24 M phosphate (K₂HPO₄-KH₂PO₄) buffer pH 6.3 as inoculation buffer. The replication of CYMV-RNA in protoplasts was followed by Northern blotting using specific cDNA probe. Synthesis of genomic RNA was detected from 12 hr after inoculation. Polyethylene glycol-mediated inoculation with CYMV-RNA resulted in about 35% infection of viable protoplasts.