Avaliação de plantas transgênicas de laranja doce (Citrus sinensis) e transformação genética de laranja azeda (Citrus aurantium) para resistência ao Citrus tristeza virus (CTV)

Abstract

Evaluation of sweet orange (Citrus sinensis) transgenic plants and genetic transformation of sour orange (Citrus aurantium) for the resistance to Citrus tristeza virus (CTV) Citrus tristeza virus (CTV) occurs in almost all citrus-growing areas of the world. Control of citrus tristeza relies mainly on the use of tolerant rootstocks and scion cross protection. Obtaining transgenic sweet oranges cultivars or sour orange resistant to CTV would allow a better use of this excellent rootstock. This way, the aim of this work was to evaluate transgenic sweet orange (Citrus sinensis) lines and to obtain transgenic sour orange (Citrus aurantium) for the resistance to CTV, in order to offer another alternative for the control of the disease in citrus. Transgenic sweet orange cv. 'Valencia' and cv. 'Hamlin' containing three different genetic constructs were evaluated. One gene construct contains a sense sequence (684 pb) of the coat protein gene of CTV (pCTV-CP), another contains a conserved sequence (559 pb) of CTV (pCTV-SC), and the last one a hairpin type, containing the sense and antisense sequences of the coat protein gene separated by an intron (pCTV-dsCP). Ten transgenic lines of each gene construct and each cultivar were previously confirmed by Southern blot and RT-PCR analysis, totalizing 60 transgenic lines. These lines were cloned and grafted into C. limonia and into C. aurantium, totaling 360 plants. The plants, along with non-transgenic plants used as control, were challenged four times with the CTV by means of viruliferous Toxoptera citricida. Indirect ELISA using monoclonal antibody against the CTV coat protein or the Realtime PCR using primers to amplify the CTV genes p20 and p23 were used to detect the virus in the tested plants, 4 weeks after inoculation. Variation in the virus resistance was observed among different transgenic constructs and different clones of the same plant. Some clones were not infected with the virus even after the fourth inoculation, indicating a possible resistance to the pathogen. A total of 30 genetic transformation experiments of sour orange were performed, using as explants internodal segments, epicotyl segments and cotyledon fragment with hypocotyl attached. GUS reaction detected two shoots positive (transformation efficiency of 0,13%). These shoots were in vitro grafted in 'Carrizo' citrange, but only one shoot developed. The plant obtained was acclimatized in greenhouse.