Articles
A DYSFUNCTIONAL CYMMV MOVEMENT PROTEIN GENE CONFERS RESISTANCE TO CYMMV IN DENDROBIUM ORCHID
Article number
836_21
Pages
149 – 155
Language
English
Abstract
A Cymbidium mosaic virus movement protein gene with a site-specific mutation (mut11) under control of a ubiquitin promoter was inserted using biolistics into two Dendrobium varieties with the intention of creating CymMV-resistant orchids.
Presence of the transgene in regenerated plants of D. × Jaquelyn Thomas Uniwai Mist and D. × Jaq Hawaii Uniwai Pearl was confirmed by PCR using genomic DNA, and mut11-positive plants were potted ex vitro.
Twenty-four confirmed transgenic plant groups and 4 non-transgenic controls at the 4 to 6 leaf stage were inoculated with a 1: 1000 dilution of CymMV obtained from infected orchids.
Plants were analyzed for systemic infection using tissue blot immunoassay (TBIA). Seventeen plants from 6 independent transformations remained virus-free, whereas all control plants were infected with CymMV within 1 month.
All 17 plants were re-inoculated twice with 1: 1000 dilution of CymMV. Only nine individual plants still remained free from virus.
Further analysis by RT-PCR showed that the mut11 messenger RNA was detectable in only two of these nine plants.
Nine transgenic plants from both varieties remained free from CymMV 12 months after the third challenge.
Lack of detectable mut11 mRNA in these resistant lines suggests that a post transcriptional gene silencing (PTGS) mechanism may be conferring resistance to CymMV.
Presence of the transgene in regenerated plants of D. × Jaquelyn Thomas Uniwai Mist and D. × Jaq Hawaii Uniwai Pearl was confirmed by PCR using genomic DNA, and mut11-positive plants were potted ex vitro.
Twenty-four confirmed transgenic plant groups and 4 non-transgenic controls at the 4 to 6 leaf stage were inoculated with a 1: 1000 dilution of CymMV obtained from infected orchids.
Plants were analyzed for systemic infection using tissue blot immunoassay (TBIA). Seventeen plants from 6 independent transformations remained virus-free, whereas all control plants were infected with CymMV within 1 month.
All 17 plants were re-inoculated twice with 1: 1000 dilution of CymMV. Only nine individual plants still remained free from virus.
Further analysis by RT-PCR showed that the mut11 messenger RNA was detectable in only two of these nine plants.
Nine transgenic plants from both varieties remained free from CymMV 12 months after the third challenge.
Lack of detectable mut11 mRNA in these resistant lines suggests that a post transcriptional gene silencing (PTGS) mechanism may be conferring resistance to CymMV.
Publication
Authors
K. Obsuwan, D.A. Hieber, R.G. Mudalige-Jayawickrama, A.R. Kuehnle
Keywords
Cymbidium mosaic virus, Dendrobium, movement protein, orchid, resistance
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