scholarly journals Transferring Cucumber Mosaic Virus-White Leaf Strain Coat Protein Gene into Cucumis melo L. and Evaluating Transgenic Plants for Protection against Infections

1994 ◽  
Vol 119 (2) ◽  
pp. 345-355 ◽  
Author(s):  
Carol Gonsalves ◽  
Baodi Xue ◽  
Marcela Yepes ◽  
Marc Fuchs ◽  
Kaishu Ling ◽  
...  
Keyword(s):  
New York ◽  
Coat Protein ◽  
Transgenic Plants ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Microprojectile Bombardment ◽  
Leaf Tissue ◽  
White Leaf ◽  
Npt Ii ◽  
Transfer Procedures

A single regeneration procedure using cotyledon explants effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens (using `Burpee Hybrid' and `Hales Best Jumbo') or microprojectile bombardment (using `Topmark') methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), β-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 μm 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg·liter-1 and carbenicillin (Cb) at 500 mg·liter-1. Our comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R0 plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes. This analysis showed that plants transformed by A. tumefaciens contained all three genes, although co-transferring the genes into bombarded plants was not always successful. R1 plants were challenge inoculated with CMV-FNY, a destructive strain of CMV found in New York. Resistance levels varied according to the different transformed genotypes. Somaclonal variation was observed in a significant number of R0 transgenic plants. Flow cytometry analysis of leaf tissue revealed that a significant number of transgenic plants were tetraploid or mixoploid, whereas the commercial nontransformed cultivars were diploid. In a study of young, germinated cotyledons, however, a mixture of diploid, tetraploid, and octoploid cells were found at the shoot regeneration sites.

Effectiveness of coat protein and defective replicase gene-mediated resistance against Australian isolates of cucumber mosaic virus

1998 ◽  
Vol 38 (4) ◽  
pp. 375 ◽  
Author(s):  
Z. Singh ◽  
M. G. K. Jones ◽  
R. A. C. Jones
Keyword(s):  
Coat Protein ◽  
Transgenic Plants ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Transgenic Tobacco ◽  
Extreme Resistance ◽  
Tobacco Plants ◽  
Systemic Movement ◽  
Cp Gene ◽  
Subgroup Ii

Summary. Transgenic tobacco (Nicotiana tabacum) plants of (i) cv. Samsun NN containing the cauliflower mosaic virus 35S constitutive promoter linked to a defective replicase (DR) gene derived from cucumber mosaic virus (CMV) subgroup I isolate Fny, and (ii) cv. Xanthi containing the CaMV 35S promoter linked to the coat protein (CP) gene of CMV subgroup I isolate C were tested for resistance to various Australian isolates of CMV. The tobacco plants were challenged with 3 CMV subgroup 1 isolates (BNRR, BMR and B6) using sap inoculation. When used to challenge non-transgenic tobacco plants with 5 subgroup II CMV isolates from lupins (LY, LCH, LAcc, LGu and LD), this inoculation method did not result in systemic infection so graft inoculation was used instead to challenge transgenic plants with these 5 isolates. When plants of the line with the DR gene were challenged with the 3 subgroup I isolates, extreme resistance was revealed as none showed symptoms and CMV was not detectable by ELISA. When the same 3 isolates were inoculated to the 3 lines with the CP gene, resistance was characterised by fewer plants becoming virus infected, delayed systemic movement and, in the plants that were infected, partial remission of symptoms plus somewhat decreased virus concentration. Challenge of transgenic plants with DR or CP with the 5 subgroup II isolates resulted in fewer plants becoming infected. Actual numbers of plants infected varied with line and subgroup II isolate and the DR gene was as effective as the CP gene at decreasing infection. With subgroup II isolate LY, infection was associated with remission of symptoms and with the other 4 isolates with delayed systemic movement. Thus the DR gene approach was more effective than the CP approach in obtaining extreme resistance against Australian subgroup I isolates of CMV. These results suggest that introducing a similar DR gene construct made from a subgroup II isolate from lupins into commercial lupin cultivars may be a suitable strategy for obtaining extreme resistance to subgroup II isolates from lupins.

scholarly journals Transfer and Expression of Cucumber Mosaic Virus Coat Protein Gene in the Genome of Cucumis sativus

1991 ◽  
Vol 116 (6) ◽  
pp. 1098-1102 ◽  
Author(s):  
Paula P. Chee ◽  
Jerry L. Slightom
Keyword(s):  
Coat Protein ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Cucumis Sativus ◽  
Blot Analysis ◽  
Binary Vector ◽  
Enzyme Linked Immunosorbent Assay ◽  
Embryogenic Calli ◽  
Mature Embryos ◽  
Npt Ii

Cotyledon explants of cucumber (Cucumis sativus L. cv. Poinsett 76) seedlings were cocultivated with disarmed Agrobacterium strain C58Z707 that contained the binary vector plasmid pGA482GG/cpCMV19. The T-DNA region of this binary vector contains plant-expressible genes for neomycin phosphotransferase II (NPT II), β -glucuronidase (GUS), and the coat protein of cucumber mosaic virus strain C (CMV-C). After infection, the cotyledons were placed on Murashige and Skoog medium containing 100 mg kanamycidliter. Putative transformed embryogenic calli were obtained, followed by the development of mature embryos and their germination to plants. All transformed RO cucumber plants appeared morphologically normal and tested positive for NPT IL Southern blot analysis of selected cucumber DNAs indicated that NPT II, GUS, and CMV-C coat protein genes were integrated into the genomes. Enzyme-linked immunosorbent assay and Western blot analysis indicated that the CMV-C coat protein is present in the protein extracts of progeny plants. These results show that the Agrobacterium-mediated gene transfer system and regeneration via somatic embryogenesis is an effective method for producing transgenic plants in Cucurbitaceae.

scholarly journals First Report of Cucumber mosaic virus Subgroup II Infecting Lycopersicon esculentum in India

Plant Disease ◽  
2006 ◽  
Vol 90 (11) ◽  
pp. 1457-1457 ◽  
Author(s):  
N. Sudhakar ◽  
D. Nagendra-Prasad ◽  
N. Mohan ◽  
K. Murugesan
Keyword(s):  
Coat Protein ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Tamil Nadu ◽  
Indian Subcontinent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infected Leaf ◽  
Polymorphism Analysis ◽  
First Report ◽  
Subgroup Ii

During a survey in January 2006 near Salem in Tamil Nadu (south India), Cucumber mosaic virus was observed infecting tomatoes with an incidence of more than 70%. Plants exhibiting severe mosaic, leaf puckering, and stunted growth were collected, and the virus was identified using diagnostic hosts, evaluation of physical properties of the virus, compound enzyme-linked immunosorbent assay (ELISA) (ELISA Lab, Washington State University, Prosser), reverse-transcription polymerase chain reaction (RT-PCR), and restriction fragment length polymorphism analysis (DSMZ, S. Winter, Germany). To determine the specific CMV subgroup, total RNA was extracted from 50 infected leaf samples using the RNeasy plant RNA isolation kit (Qiagen, Hilden, Germany) and tested for the presence of the complete CMV coat protein gene using specific primers as described by Rizos et al. (1). A fragment of the coat protein was amplified and subsequently digested with MspI to reveal a pattern of two fragments (336 and 538 bp), indicating CMV subgroup II. No evidence of mixed infection with CMV subgroup I was obtained when CMV isolates representing subgroups I (PV-0419) and II (PV-0420), available at the DSMZ Plant Virus Collection, were used as controls. Only CMV subgroup I has been found to predominantly infect tomato in the Indian subcontinent, although Verma et al. (2) identified CMV subgroup II infecting Pelargonium spp., an ornamental plant. To our knowledge, this is the first report of CMV subgroup II infecting tomato crops in India. References: (1) H. Rizos et al. J. Gen. Virol. 73:2099, 1992. (2) N. Verma et al. J. Biol. Sci. 31:47, 2006.

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