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A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used. Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source. Genetic transformation was confirmed by PCR and Southern blot. The transgene expression was evaluated using a chlorophenol red assay and isoenzymes. The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar. This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides.

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.

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 allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol for genetic transformation of Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck). Epicotyl segments of germinated in vitro plantlets (three weeks in darkness and two weeks in a 16-h photoperiod) were used as explants. These were cocultivated with Agrobacterium tumefaciens strain EHA-105 and different experiments were done to evaluate the transformation efficiency: explants were co-cultivated with Agrobacterium for one, three or five days; explants were incubated with Agrobacterium suspension for 5, 10, 20 or 40 minutes; co-cultivation medium was supplemented with acetosyringone at 0, 100 or 200 µmol L -1; Explants ends had a longitudinal terminal incision (2-3 mm); co-cultivation temperatures of 19, 23 or 27°C were imposed. The experimental design was completely randomized in all experiments with five replications, each consisted of a Petri dish (100 x 15 mm) with 30 explants and resulted in a total of 150 explants per treatment. Longitudinal terminal incision in the explant ends did not improve shoot regeneration. However, transgenic plants of all three cultivars were confirmed from explants that had been subjected to inoculation time of 20 minutes, co-culture of three days at 23-27°C, in the absence of acetosyringone. Key words: genetic transformation, organogenesis, micrografting, improvement TRANSFORMAÇÃO GENÉTICA EM Citrus sinensis E Citrus limonia MEDIADA POR Agrobacterium tumefaciens A PARTIR DE SEGMENTOS DE EPICÓTILORESUMO: A transformação genética permite produzir cultivares com características específicas e pode, dessa forma ser associada a programas de melhoramento de citros. O objetivo deste trabalho foi estabelecer protocolos de transformação genética para as laranjas doce 'Valência' e 'Natal' (Citrus sinensis L. Osbeck), bem como para o limão 'Cravo'(Citrus limonia L. Osbeck). Segmentos de epicótilo de plântulas germinadas in vitro (três semanas no escuro e duas semanas sob fotoperíodo de 16h) foram utilizados como explantes. Estes foram co-cultivados com Agrobacterium tumefaciens (EHA-105), realizando-se vários experimentos para avaliar a eficiência do processo de transformação genética: explantes co-cultivados por um, três e cinco dias; tempo de inoculação com a bactéria de 5, 10, 20 e 40 minutos; co-cultivo em meio de cultura contendo 0, 100 e 200 µmol L -1 de acetoseringona; Incisão longitudinal (2-3 mm) nas extremidades do explante; temperatura de co-cultivo 19, 23 e 27°C. Todos os experimentos consistiram de cinco repetições por tratamento, sendo cada repetição representada por uma placa de Petri contendo 30 explantes, perfazendo um total de 150 explantes por tratamento. Plântulas transgênicas dos três cultivares foram obtidas utilizando-se tempo de inoculação de 20 minutos, co-cultivo com Agrob...