Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp. citri

Abstract

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and sprayinoculated with an Xcc suspension (10 6 cfu mL −1 ). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (nontransgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.

Effect of interaction between different plant growth regulators on in vitro shoot multiplication of Citrus latifolia Tan. (persian lime)

“…Also micropropagation systems with high multiplication rates are not only an important asexual method that can be used for the production of clonal plants, but also form the basis for the introduction of genetic variation by genetic transformation or mutagenesis. The genetic transformation of Citrus has been widely studied as a tool to generate transgenic plants with enhanced tolerance of biotic (Cardoso et al, 2010;He et al, 2011 andAli et al, 2012) and/or abiotic stresses (Bunnag and Tangpong 2012). In both cases, is necessary to be able to regenerate viable shoots, which can be propagated, by either organogenesis or somatic embryogenesis (Perez -Tornero et al, 2010).…”

Bacterial Citrus Diseases: Major Threats and Recent Progress

“…Examples of transgenes or genes whose overexpression confer enhanced resistance are shown in Table 4 [124][125][126][127][128][129][130][131][132][133][134][135][136][137][138]. Nonetheless, a main limitation of the transgenic approach is that adoption of genetically modified plants takes many years because of the rigorous field testing required and the long regulation process.…”

Transformação genética de laranjas 'Pera' e 'Natal' (Citrus sinensis L. Osbeck) com o gene atacina A (attA), dirigido por promotores preferencialmente expressos no floema, para resistência a Candidatus Liberibacter spp.

“…Entre as estratégias da engenharia genética de plantas para a resistência a doenças, a utilização de peptídeos antimicrobianos, como as atacinas, vem sendo reportada com resultados promissores (REYNOIRD et al, 1999;KO et al, 2000;BESPALHOK FILHO et al, 2001;BOSCARIOL et al, 2006;CARDOSO, et al, 2010;TAVANO, 2013).…”

“…Atuam aumentando a permeabilidade ou interferindo na síntese da membrana externa de bactérias Gram-negativas. Tem se mostrado eficiente em promover resistência a infecções causadas por bactérias e fungos em plantas de citros (BOSCARIOL et al, 2006;CARDOSO et al, 2010;MONDAL et al, 2012).…”

“…Estudos envolvendo a introdução de genes relacionados ao controle de doenças, tem resultado na redução de sintomas causados por bactérias (BOSCARIOL et al, 2006;CARDOSO et al, 2010) Seis atacinas isoladas foram divididas em dois grupos de acordo com a composição de aminoácidos e sequências amino-terminais. As atacinas A, B, C e D constituem um grupo básico e as atacinas E e F um grupo neutro-ácido.…”