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Citrus canker, caused by Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al., is one of the main problems affecting citrus production. In order to obtain resistance to phytopathogenic bacteria, insect genes, coding for antimicrobial proteins, have been used in plant genetic transformation. In this study, transgenic Citrus sinensis (L.) Osb. `Hamlin' plants expressing the antimicrobial insect-derived attacin A gene (attA) were obtained by Agrobacterium tumefaciens (Smith and Towns.) Conn-mediated transformation. Initially, the cDNA clone was used to construct a binary plasmid vector (pCattA 2300). The construction included the native signal peptide (SP) responsible for directing the insect protein to the extracellular space where bacteria is supposed to accumulate in vivo. In order to investigate the native SP effectiveness in a plant model system, onion (Allium cepa L.) epidermal cells were transformed, via biobalistics, using plasmids containing the attA gene with or without SP, fused with the green fluorescent protein gene (pattA 1303 and pSPattA 1303). Fluorescence accumulation surrounding the cells was observed only in tissues transformed with the plasmid containing the gene with SP, indicating the protein secretion to the apoplast. Citrus transformation was confirmed by PCR and Southern blot hybridization analysis in 12 regenerated plants. Transcription of attA gene was detected by Northern blot analysis in all transgenic plants. Eight selected transgenic lines were propagated and inoculated with a 106 cfu/mL suspension of the pathogen X. axonopodis pv. citri. Compared to control (non-transformed plant), seven transgenic lines showed a significant reduction in susceptibility to citrus canker. The results obtained here indicate the potential use of antibacterial proteins to protect citrus from bacterial diseases.

Somatic embryogenesis from nucellus-derived callus cultures of five cultivars, including three (Caipira, Seleta Vermelha, and Valencia) of sweet oranges (C. sinensis L. Osbeck), Rangpur lime (C. limonia L. Osbeck), and Cleopatra mandarin (C. reticulata Blanco) (lines I and II), were studied. Callus lines maintained on MT medium supplemented with 50 g l 21 sucrose were transferred to MT medium supplemented with different carbohydrate sources: galactose, glucose, lactose, maltose, or sucrose at 18, 37, 75, 110, or 150 mM, or glycerol at 6, 12, 24, 36, or 50 mM. Globular embryos were observed after approximately 4 wk, in several treatments. Cultures of Valencia and Caipira sweet oranges and Cleopatra mandarin (line I) showed high numbers of embryos on medium containing galactose, lactose, and maltose. Histological studies showed somatic embryos in all developmental stages with a normal histodifferentiation pattern. The other two cultivars (Rangpur lime and Cleopatra mandarin, line II) formed very few embryos, which did not develop further following the globular stage. Some of the abnormalities observed were lack or dedifferentiation of protoderm and absence of apical meristems and procambial strands. Embryos that followed the normal sequence of development were easily converted into plants. Non-embryogenic cultures continued as proliferating callus cultures, eventually forming a few embryos which did not convert into plants. Statistical analyses of the callus response to carbohydrate treatments was done using an overdispersion Poisson model.

The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic Hamlin sweet orange (Citrus sinensis (L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 µM of acetosyringone. Genetic transformation was confirmed by performing ß-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUS genes.Index terms: Citrus sinensis, epicotyls, seedlings, transgenics, breeding methods. Transformação genética de laranja Hamlin via AgrobacteriumResumo O desenvolvimento e otimização de protocolos eficientes de transformação genética é essencial nos programas atuais de melhoramento de citros, tanto para porta-enxertos, como para copas de valor comercial. Plantas transgênicas de laranja Hamlin (Citrus sinensis (L.) Osbeck) foram obtidas pela transformação genética de segmentos de epicótilo, coletados de plântulas germinadas in vitro, com Agrobacterium tumefaciens. Foram avaliados fatores que influenciam a eficiência da transformação genética, como: condições de incubação das plântulas utilizadas para coleta de explantes, tempo de inoculação com Agrobacterium e condições de co-cultivo. A regeneração de plantas a partir de segmentos de epicótilo ocorreu em alta freqüência, por organogênese direta. A maior porcentagem de plantas transgênicas foi obtida utilizando-se explantes coletados de plântulas germinadas no escuro e posteriormente transferidas, por 2-3 semanas, para condições de 16 horas de fotoperíodo, e infectados com Agrobacterium por um período de 15-45 minutos. As melhores condições de co-cultivo foram a incubação dos explantes no escuro, por três dias, em meio de cultura suplementado com 100 µM de acetoseringona. A transformação genética foi confirmada pelo teste histoquímico para ß-glucoronidase (GUS) e, posteriormente, pela amplificação de DNA, por PCR, para detecção dos genes nptII e GUS.Termos para indexação: Citrus sinensis, epicótilo, plântulas, transgênicos, métodos de melhoramento.

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Exogenous genes can be introduced in plants by genetic transformation techniques. However, an efficient tissue culture system with high rates of plant recovery is necessary for gene introduction. This work aimed to define organogenesis and plant regeneration protocols for sweet orange varieties Natal, Valencia and Hamlin (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck) which can be used in plant transformation experiments. Seeds of which teguments were removed, were germinated in vitro and maintained in the dark for three weeks, followed by one week at 16-h photoperiod (40 µmol m -2 s -1 ) and 27 ± 2°C. Organogenesis induction was done by introducing epicotyl segments in MT medium with 25 g L -1 sucrose and different BAP concentrations. After adventitious bud growth, the shoots were transferred to MT medium with either NAA or IBA (1 mg L -1 ), or absence of auxin, for rooting. The best results were obtained with 1 mg L -1 BAP for bud induction and 1 mg L -1 IBA for rooting for all three sweet orange cultivars. The use of 0.5-2.5 mg L -1 BAP, followed by 1 mg L -1 IBA were the best growth regulator combinations for bud induction and rooting, respectively, for 'Rangpur' lime. The protocols presented in this work are suitable for associations with genetic transformation experiments for these cultivars.

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.

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the β-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.