Francisco de Assis Alves Mourão Filho

Genetic transformation and plant recovery from mature tissues of Citrus sinensis L. Osbeck

Abstract Genetic transformation using mature tissue as explant is important for the precocious evaluation of the genetically modified characteristic. This research presents a protocol for adventitious organogenesis and genetic transformation of sweet orange varieties ( Citrus sinensis L. Osbeck) using as explants internodal segments from adult plants maintained in the greenhouse. Organogenesis was induced in DBA3 medium supplemented with benzilaminopurine (1.0 mgxa0l −1 )+naphthaleneacetic acid (0.5 mgxa0l −1 ), in the dark at 27±2xa0°C until adventitious buds had formed and then transferred to a 16-h photoperiod. Histological analysis showed an indirect formation of the adventitious buds from callus that had proliferated at the cut ends of the explants. Transformation was done using the Agrobacterium tumefaciens strain EHA-105 containing the plasmid p35SGUSINT as a vector. The explants were co-cultivated with Agrobacterium for 24 h, at 24xa0°C. Adventitious shoots were tested histochemically for GUS expression and micrografted into plantlets of the rootstock Carrizo citrange ( C. sinensis L. Osbeck× Poncirus trifoliata L. Raf.). Genetic transformation was confirmed by PCR and Southern blot analysis of the recovered plants.

Transformação genética de laranja 'Hamlin' via Agrobacterium

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 sinensisxa0(L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated inxa0vitro. 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 16xa0hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45xa0min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100xa0mM of acetosyringone. Genetic transformation was confirmed by performing b-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUSxa0genes.

Somatic embryogenesis in Citrus SPP.: Carbohydrate stimulation and histodifferentiation

SummarySomatic embryogenesis from nucellus-derived callus cultures of five cultivars, including three (Caipira, Seleta Vermelha, and Valencia) of sweet oranges (C. sinesis 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−1 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 histodiffeentiation 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.

Citrus somatic hybridization with potential for improved blight and CTV resistance

SummaryThe production of five new somatic hybrids with potential for improved disease resistance is reported herein. Protoplast isolation, fusion, and plant regeneration was achieved from Caipira sweet orange (Citrus sinensis L. Osbeck) as an embryogenic parental source and Volkamer lemon (C. volkameriana Pasquale), Cleopatra mandarin (C. reticulata Blanco), and Rough lemon (C. jambhiri Lushington) as non-embryogenic parental sources. Fusion involving Cleopatra mandarin and Rangpur lime (C. limonia L. Osbeck) as embryogenic parental sources with Sour orange (C. aurantium L.) also resulted in somatic hybrid plants. Somatic hybridization was confirmed by leaf morphology evaluation, chromosome counting, and randomly amplified polymorphic DNA (RAPD) analyses. Somatic hybrids may combine complementary characteristics from both parental sources and have potential for tolerance to blight and citrus tristeza virus (CTV).

Funções e ordem da razão dos nutrientes no estabelecimento de normas DRIS em laranjeira 'Valência'

(3) Resumo†n†O sistema integrado de diagnose e recomendaAao DRIS (Diagnosis and Recommendation Integrated System) tem sido uma boa alternativa para a avaliaAao do estado nutricional de diversas culturas. O†objetivo deste trabalho foi calcular Indices DRIS mediante o uso de trŒs mOtodos (Beaufils, Elwali & Gascho e Jones) de cAElculo das funAies das razies dos nutrientes e de dois critOrios (Letzch e Nick) para a escolha da ordem da razao dos nutrientes, em pomares comerciais irrigados de laranjeira eValŒnciai. InformaAies sobre a produtividade, porta-enxerto e teor de N, P, K, Ca, Mg, S, Fe, Mn, Cu, Zn e B em folhas de ramos nao-frutIferos de cada talhao foram processadas nos anos de 1994 a 1998. O†Indice de balanAo nutricional (IBN) foi calculado pela mOdia da soma dos valores em mUdulo dos Indices nutricionais e do Indice de matOria seca. Os†Indices DRIS e IBN foram calculados e os valores de r 2 (produtividade em funAao de IBN) foram obtidos para cada mOtodo. O†mOtodo Jones para cAElculo do DRIS apresentou melhor correlaAao com a produtividade, e o critOrio Nick mostrou-se mais eficiente na escolha da ordem da razao dos nutrientes. PopulaAies especIficas, com pequeno nœmero de observa- Aies, padronizadas quanto ao porta-enxerto e referentes a um ou dois anos de amostragem foliar e produAao foram bancos de dados eficientes para a obtenAao das normas DRIS. Termos para indexaAao: Citrus sinensis, diagnose foliar, equilIbrio de nutrientes, estado nutricional, mOtodo de anAElise. Functions and ratio order of the nutrients at the establishment of DRIS norms in eValenciai sweet orange Abstract†n†Diagnosis and Recommendation Integrated System (DRIS) has been a useful tool for nutrient diagnosis of several crops. This research aimed to establish adequate norms for DRIS through three methods (Beaufils, Elwali & Gascho and Jones) to calculate ratio functions and two criteria (Letzsch and Nick) for choosing the ratio order of the nutrients for eValenciai sweet orange budded on three rootstocks, in Mogi GuaAœ, SP, Brazil. Data referred to yield, rootstock and concentrations of N, P, K, Ca, Mg, S, Fe, Mn, Cu, Zn and B in leaves of non fruiting terminals, for each plot, were processed for 1994 through 1998. Nutritional Balance Index (NBI) was calculated by the average of the sum (disregarding the sign) of all nutritional index values and dry matter index. DRIS and NBI indexes were calculated and the r 2 †values (yield related to NBI) were obtained for each calculation method. DRIS calculation method of the ratio function, recommended by Jones, presented better correlation with yield. The criterion for choosing the ratio order of the nutrients recommended by Nick was more efficient. Specific populations, with reduced number of observations, standardized by rootstock and referred to one or two sampling years were more efficient data for calculation of DRIS norms.

DRIS norms for 'Valencia' sweet orange on three rootstocks

(2) Abstract - Diagnosis and Recommendation Integrated System (DRIS) applies nutrient ratios instead of the isolated concentration values of each nutrient in interpretation of tissue analysis. The objectives of this research were to establish adequate DRIS norms for Valencia sweet orange irrigated commer- cial groves budded on three rootstocks and correlate indexes of nutrition balance with yield. Experi- ments were conducted in Sao Paulo State, Brazil. Rootstocks Rangpur lime, Caipira sweet orange, and Poncirus trifoliata, with more than six years old and yield above 40 ton ha -1 were utilized. Data re- ferred to yield, tree spacing, rootstock and foliar concentrations of N, P, K, Ca, Mg, S, Fe, Mn, Cu, Zn, and B in non fruiting terminals for each grove were processed for the years 1994 through 1998. DRIS indexes were calculated by Nick criterion for choosing the ratio order of the nutrients and Jones calcu- lation method of the ratio functions. Indexes of nutritional balance calculated from DRIS norms pre- sented high correlation with yield for the three scion/rootstock combinations. DRIS norms defined in this research are valid, since leaf sampling is done on non fruiting terminals and the grove is irrigated.

Efeito do porta-enxerto na qualidade do cacho da videira 'Niágara Rosada'

n†The aim of this paper was to evaluate the cluster quality of eNiAEgara Rosadai table†grape (Vitis labrusca†L.) grafted on different rootstocks and evaluated during seven crops, at TaubatO, SP, Brazil. The†clusters harvested in plants grafted with eIAC†313i, eIAC†766i and eTraviœi presented size (length and width), mass and number of berries significantly higher than those verified in plants grafted with eKober†5BBi and eSchwarzmanni. The†berry presented mass significantly higher in the eIAC†766i and eTraviœi if compared to the eSchwarzmanni. Fruit soluble solid content was higher in eKober†5BBi (15.46 o Brix) and eSchwarzmanni (15.28 o Brix) if compared to the eIAC†313i (14.18 o Brix).

Estado nutricional e produção de laranjeira 'Pêra' em função da vegetação intercalar e cobertura morta

The objective of this work was to evaluate the effects of cover crops composed of grass and perennial leguminous in the orchard inter-rows and mulch on the rows, on soil chemical characteristics and nutritional status of Citrus sinensis (L.) Osbeck cv. Pera sweet orange budded on Rangpur lime (Citrus limonia Osbeck). The treatments were: T1, Cover crop of Brachiaria ruziziensis R. Germ. & Evrard, without mulch on the row; T2, B. ruziziensis and mulch on the row; T3, B. ruziziensis and Arachis pintoi and mulch on the row; T4, B. ruziziensis and stylosanthes and mulch on the row; T5, Brachiaria plantaginea and mulch on the row; T6, Arachis pintoi and mulch on the row; T7, stylosanthes and mulch on the row. Stylosanthes increased N leaf content compared to B. ruziziensis. Grass mulch had no significant effect on N leaf content. There was significant interaction between root density into superficial soil layer, P leaf content and yield.

GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange

The objective of this work was the transformation of tobacco and Valencia sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL) gene promoter (CsPP). Transformation was accomplished by co-cultivation of tobacco and Valencia sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.

Transformação genética de laranja 'Valência' com o gene cecropin MB39

The objective of this work was to produce Valencia sweet orange transgenic plants with the cecropin MB39 gene controlled by a phenylalanine ammonia-lyase gene promoter from citrus in order to direct gene expression in xylem vessels. The genetic transformation was mediated by Agrobacterium tumefaciens with the co-culture of epicotyl segments. Eleven transgenic plants were selected by PCR with the amplification of a 189 bp fragment, which were acclimatized to greenhouse. The integration of the transgene was confirmed in three plants by Southern blot analysis.