Japanese Journal of Phytopathology
Online ISSN : 1882-0484
Print ISSN : 0031-9473
ISSN-L : 0031-9473
An Improved System for Clover Yellow Mosaic Virus Infection of Pea Leaf Mesophyll Protoplasts
Piara S. BAINSHanumantha R. PAPPUChuji HIRUKI
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1988 Volume 54 Issue 2 Pages 174-182


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.

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