Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to CTV infection
Muniz, F.R., Escola Superior de Agricultura Luiz de Queiroz, Piracicaba (Brazil) | De Souza, A.J., Escola Superior de Agricultura Luiz de Queiroz, Piracicaba (Brazil) | Stipp, L.C.L., Universidade de Sao Paulo, Piracicaba (Brazil). Centro de Energia Nuclear na Agricultura | Schinor, E., Escola Superior de Agricultura Luiz de Queiroz, Piracicaba (Brazil) | Freitas, W., Escola Superior de Agricultura Luiz de Queiroz, Piracicaba (Brazil) | Harakava, R., Instituto Biologico de Sao Paulo (Brazil) | Stach-Machado, D.R., Universidade Estadual de Campinas (Brazil). Instituto de Biologia | Rezende, J.A.M., Escola Superior de Agricultura Luiz de Queiroz, Piracicaba (Brazil) | Mourao Filho, F.A.A., Escola Superior de Agricultura Luiz de Queiroz, Piracicaba (Brazil) | Mendes, B.M.J., Universidade de Sao Paulo, Piracicaba (Brazil). Centro de Energia Nuclear na Agricultura
Transgenic Citrus sinensis 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.
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