Abstract
Transgenic Citrus sinensis (L.) Osb. plants, cvs. Valencia and Hamlin, expressing Citrus tristeza virus (CTV) derived sequences were obtained by genetic transformation. The gene constructs were pCTV-CP containing the 25 kDa major capsid protein gene (CTV-CP), pCTV-dsCP containing the same CTV-CP gene in an intron-spliced hairpin construct, and pCTV-CS containing a 559 nt conserved region of the CTV genome. The transgenic lines were identified by PCR and the transgene integration was confirmed by Southern blot. Transgene mRNA could be detected in most transgenic lines containing pCTV-CP or pCTV-CS transgene. The mRNA of pCTV-dsCP transgene was almost undetectable, with very light bands in most analyzed plants. The transgene transcription appears to be closely linked to the type of gene construct. The virus challenge assays reveals that all transgenic lines were infected. However, it was possible to identify propagated clones of transgenic plants of both cultivars studied with a low virus titer, with values similar to the noninoculated plants (negative control). These results suggested that the transgenic plants present some level of resistance to virus replication. The higher number of clones with low virus titer and where mRNA could not be detected or was presented in a very light band was found for pCTV-dsCP-derived transgenic lines.
This is a preview of subscription content, log in via an institution to check access.
Abbreviations
- BAP:
-
6-benzylaminopurine
- CTV:
-
Citrus tristeza virus
References
Ananthakrishnan, G., Orbović, V., Pasquali, G., Ćalović, M., Grosser, J.W.: Transfer of Citrus tristeza virus (CTV)-derived resistance candidate sequences to four grapefruit cultivars through Agrobacterium-mediated genetic transformation. — In Vitro cell. dev. Biol. Plant 43: 593–601, 2007.
Baptista, C.R., Vega, J., Stach-Machado, D.R., Targon, M.L.P.N., Müller, G.W., Machado, M.A.: Método simplificado de purificação do vírus da tristeza dos citros e obtenção de anti-soro de alta especificidade. — Summa Phytopatol. 22: 234–238, 1996. [In Port.]
Bar-Joseph, M., Marcus, R., Lee, R.F.: The continuous challenge of Citrus tristeza virus control. — Annu. Rev. Phytopathol. 27: 291–316, 1989.
Batuman, O., Mawassi, M., Bar-Joseph, M.: Transgenes consisting of a dsRNA of an RNAi suppressor plus the 3′UTR provide resistance to Citrus tristeza virus sequences in Nicotiana benthamiana but not in citrus. — Virus Genes 33: 319–327, 2006.
Clark, M.F., Lister, R.M., Bar-Joseph, M.: ELISA techniques. — Method. Enzymol. 118: 742–766, 1986.
Domínguez, A., Guerri, J., Cambra, M., Navarro, L., Moreno, P., Peña, L.: Efficient production of transgenic citrus plants expressing the coat protein gene of Citrus tristeza virus. — Plant Cell Rep. 19: 427–433, 2000.
Domínguez, A., Mendoza, A.H., Guerri, J., Cambra, M., Navarro, L., Moreno, P., Peña, L.: Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic mexican lime (Citrus aurantifolia (Christ.) Swing.) plants expressing its p25 coat protein gene. — Mol. Breed. 10: 1–10, 2002.
Doyle, J.J., Doyle, J.L.: Isolation of plant DNA from fresh tissue. — Focus 1: 13–15, 1991.
Fagoaga, C., López, C., Mendoza, A.H., Moreno, P., Navarro, L., Flores, R., Peña, L.: Post-transcriptional gene silencing of the p23 silencing suppressor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime. — Plant mol. Biol. 60: 153–165, 2006.
Febres, V.J., Lee, R.F., Moore, G.A.: Transgenic resistance to Citrus tristeza virus in grapefruit. — Plant Cell Rep. 27: 93–104, 2008.
Gamsey, S.M., Cambra, M.: Enzyme linked immunosorbent assay for citrus pathogens. — In: Roistacher, C.N. (ed.): Graft-Transmissible Diseases of Citrus: Handbook for Detection and Diagnosis. Pp. 193–216. IOCV FAO, Rome 1991.
Grosser, J.W., Gmitter, F.G.: Protoplast fusion and citrus improvement. — Plant Breed. Rev. 8: 339–374, 1990.
Moreno, P., Ambrós, S., Albiach-Martí, M.R., Guerri, J., Peña, L.: Citrus tristeza virus: a pathogen that changed the course of the citrus industry. — Mol. Plant Pathol. 9: 251–268, 2008.
Sanford, J.C., Johnston, S.A.: The concept of parasite-derived resistance-deriving resistance genes from the parasite’s own genoma. — J. theor. Biol. 113: 395–405, 1985.
Tavano, E.C.R., Stipp, L.C.L., Muniz, F.R., Mourão Filho, F.A.A., Mendes, B.M.J.: In vitro organogenesis of Citrus volkameriana and Citrus aurantium. — Biol. Plant. 53: 395–399, 2009.
Waterhouse, P.M., Wang, M.B., Lough, T.: Gene silencing as an adaptative defence against viruses. — Nature 411: 834–842, 2001.
Acknowledgements
The authors acknowledge Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundo de Defesa da Citricultura (Fundecitrus) for research financial support and research fellowships; Sanicitrus Mudas Cítricas (Araras, SP, Brazil) and Horticitrus Mudas Cítricas (Cordeirópolis, SP, Brazil) for kindly providing rootstock material and non-transgenic budwood; Centro APTA Citros Sylvio Moreira (Cordeirópolis, SP, Brazil) for kindly providing seeds of the sweet orange cultivars and certified CTV infected budwood. The first two authors contributed equally to this work.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Muniz, F.R., De Souza, A.J., Stipp, L.C.L. et al. Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to CTV infection. Biol Plant 56, 162–166 (2012). https://doi.org/10.1007/s10535-012-0035-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10535-012-0035-0