Transformation of sweet orange [Citrus sinensis (L.) Osbeck] with pthA-nls for acquiring resistance to citrus canker disease

Abstract

The COOH terminal of pthA encoding three nuclear localizing signals (NLS) was amplified by polymerase chain reaction (PCR) from the plasmid of Xanthomonas axonopodis pv. citri, the pathogen of citrus canker disease. Then the sense and antisense strands of the nls were cloned into pBI121 vector. pthA-nls driven by the CaMV35 s promoter was transferred into sweet orange via Agrobacterium -mediated transformation. Successful integration was confirmed by PCR and Southern blotting, and 12 sense-nls (nls (+)) and 9 antisense-nls (nls (-)) transgenic clones were obtained. The expression of nls fragment was analyzed by RT-PCR, Real time q-PCR and Western blotting, in which the specific NLS protein was detected only in nls (+) transgenic clones. In an in vitro assay, when pin-puncture inoculation was performed with 2.5 × 10(7) cfu/ml of bacterial solution, the nls (+) transgenic clones showed no typical lesion development, while typical symptoms were observed in the wild types and the nls (-) transgenic clones. In vivo assay results indicated that the nls (+) transgenic clones showed less disease incidence, in comparison with the wild types and the nls (-) transgenic clones, when pin-puncture inoculation was performed with 10(4)-10(5) cfu/ml. The minimum disease incidence was 23.3% for 'Sucarri' sweet orange and 33.3% for 'Bingtang' sweet orange. When 10(4)-10(7) cfu/ml of pathogen was spray inoculated, the nls (+) transgenic clones did not show any symptom, and even the concentration raised to 10(9) cfu/ml, the disease incidence was 20-80%, while the wild types and the nls (-) transgenic clones had 100% disease development with whatever concentration of inoculum. Two transgenic clones were confirmed to be resistant to citrus canker disease in the repeated inoculation. The results suggested that the transformation of nls sense strands may offer an effective way to acquire resistance to citrus canker disease.

Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars

“…Internodal stem segments (1 cm in length) from 30 day old citrus seedlings were incubated in the Agrobacterium cell suspension basically as described by Yang et al (2011). After being blotted dry on sterilized filter paper, explants were placed horizontally in Petri dishes containing solid CCM and incubated in the dark.…”

Antibacterial Activity of Alkyl Gallates against Xanthomonas citri subsp. citri

“…Current research is focused on developing new strategies to minimize losses and to make citriculture profitable. The use of a combination of disease control measures is recommended, which includes the induction of innate plant defenses (9,10), the generation of more resistant citrus varieties (11,12), and, perhaps, the development/amelioration of chemical inhibitors to fight the bacterium and yet display reduced environmental impacts.…”

Transformação genética de laranja doce (Citrus sinensis L. Osbeck) com o gene D4E1 dirigido pelos promotores CaMV35S ou AtPP2

“…Em plantas de citros transgênicas normalmente se faz a enxertia, pois as plântulas obtidas não produzem raízes, ou as produzem muito lentamente (PEÑA et al, 1995;YANG et al, 2011). Esta dificuldade de enraizamento também é relatada na transformação genética de outras espécies lenhosas como macieira e ameixeira (JAMES et al, 1989).…”

“…São classificadas como moderadamente resistentes as cultivares "Valência" e "Pêra" premunizada, moderadamente suscetível a cultivar "Natal" e como suscetíveis as cultivares "Hamlin", "Bahia", "Baianinha" e "Seleta" (FEICHTENBERGER et al, 2005). Para a prevenção e controle do cancro cítrico, além do uso de mudas sadias e eliminação de plantas doentes, é recomendado o uso de quebra-ventos para reduzir riscos de introdução e disseminação do patógeno, fazer aplicações de cobre em fluxos vegetativos novos em pomares próximos a áreas contaminadas e realizar a Devido à inexistência de genótipos resistentes, a transformação genética de variedades comerciais de citros é uma das mais promissoras estratégias para combater esta doença (YANG et al, 2011).…”