Poster: Plant Pathogen/Symbiont Interactions
Abs #
732: Development of transgenic Dendrobium orchid resistant to CyMV
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Presenter: |
obsuwan, kullanart -, obsuwan@hawaii.edu | Authors | obsuwan, kullanart - (A) Borth, Wayne - (B) Hu, John - (B) Barry, Kelly - (A) Kuehnle, Adelheid R (A) | | Affiliations: |
(A): University of Hawaii at Manoa, Dept. of Tropical Plant and Soil Sciences, Honolulu Hawaii 96822
(B): University of Hawaii at Manoa, Dept. of Plant and Environmental Protection Sciences, Honolulu Hawaii 96822
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Cymbidium mosaic virus (CyMV)systemically infects all parts of the orchid plant and causes significant losses. Resistance to CyMV is highly desirable. Genes encoding a dysfunctional Cymbidium mosaic virus (CyMV) movement protein with a site-specific mutation (mut11) or a full-length CyMV coat protein (CP) were first transformed into Nicotiana benthamiana . PCR-positive plants that survived selection on antibiotic medium were grown to the 4-5 leaf stage for challenge by mechanical inoculation with CyMV (1:1000 dilution). ELISA conducted 7 days after inoculation identified 5 of 9 lines with the mut11 transgene that exhibited resistance (OD<0.1) compared to the inoculated non-transformed N. benthamiana (OD~0.3). Four lines were highly resistant (OD<0.05). Only 1 of 27 lines with the CP (CCP5) or CP antisense (CCP11) transgenes showed high resistance (OD<0.05) compared to the inoculated non-transformed controls (OD~0.3). Therefore, the mut11 construct was used to transform protocorm-like bodies (PLBs) of 2 varieties of Dendrobium x Jaquelyn Thomas, UH800 and UH306, via particle bombardment. Bombardment conditions used 1.6 mm gold microcarriers, 1100 psi, 26 mm Hg vacuum, and a 1:1 (Hyg: mut11) plasmid co-transformation concentration ratio. A total of 420 PLBs of UH800 and 180 PLBs of UH306 were bombarded. Ninety PLBs of UH800 (21%) and 18 PLBs of UH306 (10%) survived after selection for 3 wk in liquid VWC plus 7 mg/L hygromycin for UH800 and 20 mg/L hygromycin for UH306. PCR analyses of plants recovered 6 months later indicated that 50 individual plants of UH800 (12% recovery rate) and 25 individual plants of UH306 (14% recovery rate) contained the mut11 transgene. PCR-positive plants are being grown in the greenhouse for evaluation of resistance to CyMV infection.
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