Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters.

KEY MESSAGE: Regulatory sequences from the citrus constitutive genes cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1) were isolated, fused to the uidA gene, and qualitatively and quantitatively evaluated in transgenic sweet orange plants. The 5' upstream region of a gene (the promoter) is the most important component for the initiation and regulation of gene transcription of both native genes and transgenes in plants. The isolation and characterization of gene regulatory sequences are essential to the development of intragenic or cisgenic genetic manipulation strategies, which imply the use of genetic material from the same species or from closely related species. We describe herein the isolation and evaluation of the promoter sequence from three constitutively expressed citrus genes: cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1). The functionality of the promoters was confirmed by a histochemical GUS assay in leaves, stems, and roots of stably transformed citrus plants expressing the promoter-uidA construct. Lower uidA mRNA levels were detected when the transgene was under the control of citrus promoters as compared to the expression under the control of the CaMV35S promoter. The association of the uidA gene with the citrus-derived promoters resulted in mRNA levels of up to 60-41.8% of the value obtained with the construct containing CaMV35S driving the uidA gene. Moreover, a lower inter-individual variability in transgene expression was observed amongst the different transgenic lines, where gene constructs containing citrus-derived promoters were used. In silico analysis of the citrus-derived promoter sequences revealed that their activity may be controlled by several putative cis-regulatory elements. These citrus promoters will expand the availability of regulatory sequences for driving gene expression in citrus gene-modification programs.

The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck).

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.

EFEITO DA COMPOSIÇÃO DO SUBSTRATO NA FORMAÇÃO DE MUDAS DE LARANJEIRA `PERA'

To verify the effect of five substrate mixtures on the development of young sweet orange trees (Citrus sinensis cv. `Pera'), this study used the rootstocks Ranpur lime (Citrus limonia L. Osbeck), Cleopatra mandarin (Citrus reticulata, Blanco), and Swingle citrumelo (Citrus paradisi x Poncirus trifoliata). The experimental design was in randomized blocks, 5 x 3 factorial (substrates x rootstocks). The different materials and their proportion studied were, respectively: 1) Soil (100%); 2) Soil (33%) + fine sand (33%) + manure (33%); 3) Soil (33%) + vermiculite (33%) + manure (33%); 4) Soil (33%) + wood chips (33%) + manure (33%); 5) Soil (25%) + wood chips (25%) + fine sand (25%) + manure (25%). Plants of Rangpur lime and Swingle citrumelo presented better growth rate as expresed in plant height and trunk diameter than those of Cleopatra mandarin on most substrates. `Pêra' sweet orange budded on those rootstocks also presented better fresh weight when compared to Cleopatra mandarin. All substrate mixtures were better than the control (100%) in inducing plant development. Different substrate mixtures affected each rootstock differently, but in general, the compositions with 1/3 soil and 1/3 manure in volume were the best to produce young sweet orange trees in containers.

O efeito de cinco composições de substratos na produção de mudas de laranjeira `Pêra' (Citrus sinensis, L. Osbeck) foi estudado sobre três porta-enxertos (limoeiro `Cravo', Citrus limonia, L. Osbeck; tangerineira `Cleópatra', Citrus reticulata, Blanco; e citrumeleiro `Swingle', Citrus paradisi x Poncirus trifoliata). O experimento obedeceu ao delineamento estatístico de blocos aleatorizados em esquema fatorial 5 x 3 (substratos x porta-enxertos). As composições de misturas utilizadas como substrato e respectivas combinações volumétricas foram: 1) Terra (100%); 2) Terra (33%) + areia fina (33%) + esterco bovino curtido (33%); 3) Terra (33%) + vermiculita (33%) + esterco bovino curtido (33%); 4) Terra (33%) + raspas de madeira (33%) + esterco bovino curtido (33%); 5) Terra (25%) + raspas de madeira (25%) + areia fina (25%) + esterco bovino curtido (25%). A análise dos resultados revelou que plantas de limoeiro `Cravo' e citrumeleiro `Swingle' apresentaram maior desenvolvimento do que as de tangerineira `Cleópatra'. A mistura de materiais com solo mostrou-se vantajosa e induziu à formação de plantas mais desenvolvidas do que aquelas em substrato constituído de terra exclusivamente. A composição do substrato influenciou diferentemente no desenvolvimento de cada porta-enxerto, mas de uma forma geral, composições contento 1/3 de volume de solo e 1/3 de volume de esterco bovino proporcionaram bons resultados para os três porta-enxertos estudados.

Efeito da composição do substrato na formação de mudas de laranjeira `pera'

To verify the effect of five substrate mixtures on the development of young sweet orange trees (Citrus sinensis cv. `Pera'), this study used the rootstocks Ranpur lime (Citrus limonia L. Osbeck), Cleopatra mandarin (Citrus reticulata, Blanco), and Swingle citrumelo (Citrus paradisi x Poncirus trifoliata). The experimental design was in randomized blocks, 5 x 3 factorial (substrates x rootstocks). The different materials and their proportion studied were, respectively: 1) Soil (100%); 2) Soil (33%) + fine sand (33%) + manure (33%); 3) Soil (33%) + vermiculite (33%) + manure (33%); 4) Soil (33%) + wood chips (33%) + manure (33%); 5) Soil (25%) + wood chips (25%) + fine sand (25%) + manure (25%). Plants of Rangpur lime and Swingle citrumelo presented better growth rate as expresed in plant height and trunk diameter than those of Cleopatra mandarin on most substrates. `Pêra' sweet orange budded on those rootstocks also presented better fresh weight when compared to Cleopatra mandarin. All substrate mixtures were better than the control (100%) in inducing plant development. Different substrate mixtures affected each rootstock differently, but in general, the compositions with 1/3 soil and 1/3 manure in volume were the best to produce young sweet orange trees in containers.

O efeito de cinco composições de substratos na produção de mudas de laranjeira `Pêra' (Citrus sinensis, L. Osbeck) foi estudado sobre três porta-enxertos (limoeiro `Cravo', Citrus limonia, L. Osbeck; tangerineira `Cleópatra', Citrus reticulata, Blanco; e citrumeleiro `Swingle', Citrus paradisi x Poncirus trifoliata). O experimento obedeceu ao delineamento estatístico de blocos aleatorizados em esquema fatorial 5 x 3 (substratos x porta-enxertos). As composições de misturas utilizadas como substrato e respectivas combinações volumétricas foram: 1) Terra (100%); 2) Terra (33%) + areia fina (33%) + esterco bovino curtido (33%); 3) Terra (33%) + vermiculita (33%) + esterco bovino curtido (33%); 4) Terra (33%) + raspas de madeira (33%) + esterco bovino curtido (33%); 5) Terra (25%) + raspas de madeira (25%) + areia fina (25%) + esterco bovino curtido (25%). A análise dos resultados revelou que plantas de limoeiro `Cravo' e citrumeleiro `Swingle' apresentaram maior desenvolvimento do que as de tangerineira `Cleópatra'. A mistura de materiais com solo mostrou-se vantajosa e induziu à formação de plantas mais desenvolvidas do que aquelas em substrato constituído de terra exclusivamente. A composição do substrato influenciou diferentemente no desenvolvimento de cada porta-enxerto, mas de uma forma geral, composições contento 1/3 de volume de solo e 1/3 de volume de esterco bovino proporcionaram bons resultados para os três porta-enxertos estudados.

Variação de matéria seca e de nutrientes nas folhas e nos frutos, produção de ácido ascórbico e suco, em seis cultivares de citros, durante um ciclo

The experiment was carried out in a commercial citrus orchard located in Mogi-Guaçu (22° 22'S., 46° 56'WGr.), State of São Paulo, Brazil. Five orange types were studied: Cravo (Citrus reticulata Blanco); Hamlin (Citrus sinensis (L.) Osbeck); Natal (Citrus sinensis (L.) Osbeck); Valencia (Citrus sinensis (L.) Osbeck) and Pera (Citrus sinensis (L.) Osbeck) and a tangerine Murcott (Citrus reticulata Blanco x Citrus sinensis (L.) Osbeck). Fruits were picked from 30 days after flowering to the peak of commercial harvest. Leaves nearest the fruits were collected at the same time as the fruits. Sampled material was used to determine: fruit dry weight, macronutrient and micronutrient concentration in the leaves, nutrient extraction by the fruits, juice production per fruit and ascorbic acid concentration in the juice. Fruit dry weight increased following the second month after flowering. Except for Cravo orange fruit dry weight decreased at the end of the cycle. Macronutrient and micronutrient concentrations in the leaves showed variations during fruit development. Rate of nutrient extraction in decreasing order was: K, N, Ca, Mg, P = S, Fe, B, Zn, Mn and Cu. The cultivar that exported the largest amount of nutrients was Pera followed by Hamlin = Cravo, Murcott, Valencia and Natal. Juice per fruit ranged between 43 and 95 mililiters whereas ascorbic acid concentration ranged between 30 and 95 miligrams per 100 mililiters of juice.

De uma plantação de citros, com os cultivares T. Cravo (Citrus reticulata Blanco), L.Hamlin (Citrus sinensis (L.) Osbeck), T. Murcott (Citrus reticulata Blanco x Citrus sinensis (L.) Osbeck), L. Natal (Citrus sinensis (L.) Osbeck, L. Valencia (Citrus sinensis (L.) Osbeck) e L. Pera (Citrus sinensis (L.) Osbeck), situada na "Fazenda Sete Lagoas", no município de Mogi-Guaçu (22° 22% 46° 56'W.Gr.), em Latossolo Vermelho amarelo, fase arenosa, foram coletados frutos 30 dias após florescimento, até a idade da coleta comercial. No material coletado, foram determinadas a variação da matéria seca, a concentração dos macro e micronutrientes nas folhas adjacentes ao fruto, a extração de macro e micronutríentes pelos frutos, a produção de suco (ml) por fruto e a concentração de ácido ascórbico (mg/100 ml de suco). Concluiu-se que: 1. O aumento da matéria seca, intensifica-se a partir do segundo mês apos o florescimento; 2. Com exceção da T. Cravo, ocorre uma diminuição na produção de matéria seca no final do ciclo; 3. A concentração dos macro e micronutrientes nas folhas apresenta oscilações durante o desenvolvimento do fruto; 4. A ordem decrescente de extração de nutrientes é: K, N, Ca, Mg, P = S, Fe, B, Zn, Mn, Cu; 5. A capacidade de exportação de nutrientes pelos cultivares é, em ordem decrescente: L. Pera, L. Hamlin = T. Cravo, T. Murcott, L. Valencia, L. Natal; 6. A quantidade de suco produzido por fruto, oscila entre 43 a 95 ml; 7. A concentração de ácido ascórbico (mg/100 ml de suco), varia entre 30 a 95.

Variação de matéria seca e de nutrientes nas folhas e nos frutos, produção de ácido ascórbico e suco, em seis cultivares de citros, durante um ciclo

The experiment was carried out in a commercial citrus orchard located in Mogi-Guaçu (22° 22'S., 46° 56'WGr.), State of São Paulo, Brazil. Five orange types were studied: Cravo (Citrus reticulata Blanco); Hamlin (Citrus sinensis (L.) Osbeck); Natal (Citrus sinensis (L.) Osbeck); Valencia (Citrus sinensis (L.) Osbeck) and Pera (Citrus sinensis (L.) Osbeck) and a tangerine Murcott (Citrus reticulata Blanco x Citrus sinensis (L.) Osbeck). Fruits were picked from 30 days after flowering to the peak of commercial harvest. Leaves nearest the fruits were collected at the same time as the fruits. Sampled material was used to determine: fruit dry weight, macronutrient and micronutrient concentration in the leaves, nutrient extraction by the fruits, juice production per fruit and ascorbic acid concentration in the juice. Fruit dry weight increased following the second month after flowering. Except for Cravo orange fruit dry weight decreased at the end of the cycle. Macronutrient and micronutrient concentrations in the leaves showed variations during fruit development. Rate of nutrient extraction in decreasing order was: K, N, Ca, Mg, P = S, Fe, B, Zn, Mn and Cu. The cultivar that exported the largest amount of nutrients was Pera followed by Hamlin = Cravo, Murcott, Valencia and Natal. Juice per fruit ranged between 43 and 95 mililiters whereas ascorbic acid concentration ranged between 30 and 95 miligrams per 100 mililiters of juice.

De uma plantação de citros, com os cultivares T. Cravo (Citrus reticulata Blanco), L.Hamlin (Citrus sinensis (L.) Osbeck), T. Murcott (Citrus reticulata Blanco x Citrus sinensis (L.) Osbeck), L. Natal (Citrus sinensis (L.) Osbeck, L. Valencia (Citrus sinensis (L.) Osbeck) e L. Pera (Citrus sinensis (L.) Osbeck), situada na "Fazenda Sete Lagoas", no município de Mogi-Guaçu (22° 22% 46° 56'W.Gr.), em Latossolo Vermelho amarelo, fase arenosa, foram coletados frutos 30 dias após florescimento, até a idade da coleta comercial. No material coletado, foram determinadas a variação da matéria seca, a concentração dos macro e micronutrientes nas folhas adjacentes ao fruto, a extração de macro e micronutríentes pelos frutos, a produção de suco (ml) por fruto e a concentração de ácido ascórbico (mg/100 ml de suco). Concluiu-se que: 1. O aumento da matéria seca, intensifica-se a partir do segundo mês apos o florescimento; 2. Com exceção da T. Cravo, ocorre uma diminuição na produção de matéria seca no final do ciclo; 3. A concentração dos macro e micronutrientes nas folhas apresenta oscilações durante o desenvolvimento do fruto; 4. A ordem decrescente de extração de nutrientes é: K, N, Ca, Mg, P = S, Fe, B, Zn, Mn, Cu; 5. A capacidade de exportação de nutrientes pelos cultivares é, em ordem decrescente: L. Pera, L. Hamlin = T. Cravo, T. Murcott, L. Valencia, L. Natal; 6. A quantidade de suco produzido por fruto, oscila entre 43 a 95 ml; 7. A concentração de ácido ascórbico (mg/100 ml de suco), varia entre 30 a 95.