Somatic hybridization between Citrus sinensis (L.) Osbeck and C. grandis (L.) Osbeck

This work had as objective to produce citrus somatic hybrids between sweet oranges and pummelos. After chemical fusion of sweet orange embryogenic protoplasts with pummelo mesophyll-derived protoplasts, plants were regenerated by somatic embryogenesis and acclimatized in a greenhouse. The hybrids of 'Hamlin' sweet orange + 'Indian Red' pummelo and 'Hamlin' sweet orange + 'Singapura' pummelo were confirmed by leaf morphology, chromosome counting and molecular analysis. These hybrids have potential to be used directly as rootstocks aiming blight, citrus tristeza virus, and Phytophthora-induced disease tolerance, as well as for rootstocks improvement programs.

Olfactory mediated interactions between Citrus aurantium Toxoptera citricida and Lysiphlebus testaceipes

The objective of this study was to establish whether there are olfactory interactions in the Lysiphlebus testaceipes Toxoptera citricida and Citrus aurantium tritrophic system. The response of male and female L. testaceipes to different odour sources of the host plant C. aurantium, the aphid host T. citricida and aphid-plant complex were investigated using a Y-tube olfactometer. Laboratory experiments were conducted by exposing individually aged male and female L. testaceipes to eight different odour treatments. Response of the parasitoids was taken after 15 min exposure to the volatiles from the different odour sources and based on their orientation to the particular chamber. Seventy percent of both male and female L. testaceipes showed high attractivity to aphid infested leaves. There was no significant difference based on age and sex of the parasitoid on their choice of odour. The organic compounds released by these combinations acted as semiochemicals in the tritrophic interactions and it is suggested that insect feeding induced attraction of the parasitoid L. testaceipes.

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 'Valência' 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.

Validation of microsatellite markers for assisted selection of soybean resistance to cyst nematode races 3 and 14

The objective of this work was to validate microsatellite markers associated with resistance to soybean cyst nematode (Heterodera glycines Ichinohe) races 3 and 14, in soybean (Glycine max L.) genotypes, for use in marker-assisted selection (MAS) programs. Microsatellites of soybean linkage groups A2, D2 and G were tested in two populations, and their selection efficiencies were determined. The populations were 65 F2:3 families from Msoy8001 (resistant) x Conquista (susceptible) cross, and 66 F2:3 families of S5995 (resistant) x Renascença (susceptible) cross, evaluated for resistance to races 3 and 14, respectively. Families with female index up to 30% were considered moderately resistant. Markers of A2 and G linkage groups were associated with resistance to race 3. Markers Satt309 and GMENOD2B explained the greatest proportion of phenotypic variance in the different groups. The combinations Satt309+GMENOD2B and Satt309+Satt187 presented 100% selection efficiency. Resistance to race 14 was associated with markers of G linkage group, and selection efficiency in the Satt309+Satt356 combination was 100%. The selection differential obtained by phenotypic and marker assisted selection showed that both can result in similar gains.

Inheritance pattern and selection criteria for resistance to soybean cyst nematode races 3 and 9

The objective of this work was to determine soybean resistance inheritance to Heterodera glycines Ichinohe (soybean cyst nematode - SCN) races 3 and 9, as well as to evaluate the efficiency of direct and indirect selection in a soybean population of 112 recombinant inbred lines (RIL) derived from the resistant cultivar Hartwig. The experiment was conducted in a completely randomized design, in Londrina, PR, Brazil. The estimated narrow-sense heritabilities for resistance to races 3 and 9 were 80.67 and 77.97%. The genetic correlation coefficient (r g = 0.17; p<0.01) shows that some genetic components of resistance to these two races are inherited together. The greatest genetic gain by indirect selection was obtained to race 9, selecting to race 3 due to simpler inheritance of resistance to race 9 and not because these two races share common resistance genes. The resistance of cultivar Hartwig to races 3 and 9 is determined by 4 and 2 genes, respectively. One of these genes confers resistance to both races, explaining a fraction of the significant genetic correlation found between resistance to these SCN races. The inheritance pattern described indicates that selection for resistance to SCN must be performed for each race individually.

Inheritance of resistance to soybean cyst nematode races 3 and 14 in soybean RIL and F2 populations

The objective of this work was to evaluate the soybean inheritance of resistance to cyst nematode races 3 and 14. The following populations where evaluated: one population of recombinant inbred lines (RILs) [Hartwig (resistant) x Y23 (susceptible line)] for races 3, 14 and 9; one population of families F2:3 [M-SOY 8001 (resistant) x MB/BR 46 - Conquista (susceptible)] for race 3; and one population of families F2:3 [(S5995 (resistant) x BRSMG Renascença (susceptible)] for race 14. In RIL populations, four epistatic genes were identified which conditioned resistance to race 14, and three epistatic ones for resistance to races 3 and 9. The lack of one gene provided moderate resistance under all situations. The highest number of genes for resistance to race 14 points out that genes responsible for lower effects might be involved. In population F2:3 from M-SOY 8001 x MB/BR 46 - Conquista, one recessive gene for moderate resistance and two recessive genes complete resistance to race 3 were identified. Two recessive genes conditioning moderate resistance to race 14 were identified in population F2:3 from the crossing S5995 x BRSMG Renascença. These results will be useful in designing crossings, involving these parentals, with higher possibility to accumulating genes that provide resistance to several SCN races.

Citrus asymmetric somatic hybrids produced via fusion of gamma-irradiated and iodoacetamide-treated protoplasts

The objective of this study was to produce citrus somatic asymmetric hybrids by fusing gamma-irradiated protoplasts with iodoacetamide-treated protoplasts. Protoplasts were isolated from embryogenic suspension cells of grapefruit (Citrus paradisi Macfad.) cultivars Ruby Red and Flame, sweet oranges (C. sinensis Osbeck) 'Itaboraí', 'Natal', Valencia', and 'Succari', from 'Satsuma' (C. unshiu Marcow.) and 'Changsha' mandarin (C. reticulata Blanco) and 'Murcott' tangor (C. reticulata x C. sinensis). Donor protoplasts were exposed to gamma rays and receptor protoplasts were treated with 3 mmol L-1 iodoacetamide (IOA), and then they were fused for asymmetric hybridization. Asymmetric embryos were germinated, and the resulting shoots were either grafted onto sour orange, rough lemon or 'Swingle' (C. paradisi x Poncirus trifoliata) x 'Sunki' mandarin rootstock seedlings, or rooted after dipping their bases in indol-butyric acid (IBA) solution. The products were later acclimatized to greenhouse conditions. Ploidy was analyzed by flow cytometry, and hybridity was confirmed by amplified fragment length polymorphism (AFLP) analysis of plantlet DNAsamples. The best treatment was the donor-recipient fusion combination of 80 Gy-irradiated 'Ruby Red' protoplasts with 20 min IOA-treated 'Succari' protoplasts. Tetraploid and aneuploid plants were produced. Rooting recalcitrance was solved by dipping shoots' stems in 3,000 mg L-1 IBA solution for 10 min.

Synthetic sex pheromone of citrus leafminer in Brazilian citrus groves

The objective of this work was to determine the best conditions of use of the synthetic sex pheromone of Phyllocnistis citrella Stainton for monitoring this species in citrus groves in northeastern Brazil. Pheromone doses (0.0, 0.1, 1, 10 and 100 μg) and longevity (1, 15, 29, 43 and 57-day-old lures) and trap height (0.5, 1.5 and 2.5 m), color (green, red, and white) and model influence on P. citrella males capture were evaluated. The doses of 10 and 100 μg of the synthetic sex pheromone - a 3:1 blend of (Z,Z,E)-7,11,13-hexadecatrienal and (Z,Z)-7,11-hexadecadienal - attracted the greatest number of P. citrella males. Traps baited with these two both dosages continued to capture P. citrella males at a comparable rate for over eight weeks in citrus groves. Although there was no significant decrease in activity of both dosages until 57 days of exposure to the environment, the higher dose, as time passed, attracted significantly more P. citrella males than the lower dose. There were no significant differences in male capture in traps with synthetic sex pheromone placed at 1.5 and 2.5 m height, wich had the better results. Trap color and model did not affect male capture.

Fusion of protoplasts with irradiated microprotoplasts as a tool for radiation hybrid panel in citrus

The objective of this work was to combine asymmetric somatic hybridization (donor-recipient fusion or gamma fusion) to microprotoplast-mediated chromosome transfer, as a tool to be used for chromosome mapping in Citrus. Swinglea glutinosa microprotoplasts were irradiated either with 50, 70, 100 or 200 gamma rays and fused to cv. Ruby Red grapefruit or Murcott tangor protoplasts. Cell colonies were successfully formed and AFLP analyses confirmed presence of S. glutinosa in both 'Murcott' tangor and 'Ruby Red' grapefruit genomes.

Growth regulators, culture media and antibiotics in the in vitro shoot regeneration from mature tissue of citrus cultivars

The objective of this study was to evaluate the effects of 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) combinations, basal media and beta-lactam antibiotics on in vitro organogenesis from mature stem segments of 'Pêra', 'Valência' and 'Bahia' sweet oranges and 'Cravo' rangpur lime. For induction of shoot regeneration, the segments of the four cultivars were placed on Murashige and Skoog (MS) medium containing the following BAP/NAA concentrations: 0.0/0.0; 0.25/0.0; 0.25/0.25; 0.5/0.0; 0.5/0.5; 1.0/0.0; 2.0/0.0; 2.0/0.25; 2.0/0.5; and 2.0/1.0 mg L-1. In order to test the influence of the culture media on shoot-bud induction, (MS), Murashige and Tucker (MT), and woody plant medium (WPM) formulations were evaluated, associated with the best combination of plant growth regulators obtained in the previous experiment. The influence of four beta-lactam antibiotics (timentin, cefotaxime sodium salt, meropenem trihydrate and augmentin) on shoot regeneration was determined. Better regeneration responses were achieved when internodal segments were cultured onto MS-based medium with 500 mg L-1 cefotaxime with the following BAP/NAA concentrations: 0.5 + 0.25 mg L-1 for 'Cravo', 1.0 + 0.25 mg L-1 for 'Valência' and 'Bahia', and 1.0 + 0.5 mg L-1 for 'Pêra'. Genotype, growth regulators, basal media and beta-lactam antibiotics affect the morphogenetic response in mature tissues of citrus.

Stability of Citrus tristeza virus protective isolates in field conditions

The objective of this work was to monitor the maintenance of Citrus tristeza virus (CTV) protective isolates stability in selected clones of 'Pêra' sweet orange (Citrus sinensis), preimmunized or naturally infected by the virus, after successive clonal propagations. The work was carried out in field conditions in the north of Paraná State, Brazil. Coat protein gene (CPG) analysis of 33 isolates collected from 16 clones of 'Pêra' sweet orange was performed using single strand conformational polymorphism (SSCP). Initially, the isolates were characterized by symptoms of stem pitting observed in clones. Then viral genome was extracted and used as template for the amplification of CPG by reverse transcription polimerase chain reaction (RTPCR). RTPCR products electrophoretic profiles were analyzed using the Jaccard coefficient and the UPGMA method. The majority of the clones had weak to moderate stem pitting symptoms and its CTV isolates showed alterations in the SSCP profiles. However, the stability of the protective complex has been maintained, except for isolates from two analised clones. Low genetic variability was observed within the isolates during the studied years.

QTLs for resistance to soybean cyst nematode, races 3, 9, and 14 in cultivar Hartwig

The objective of this work was to identify major and minor-effect quantitative trait loci (QTL) for resistance to races 3, 9, and 14 of soybean cyst nematode (SCN) in Hartwig cultivar; to map new resistance QTLs for these races; and to check for the existence of epistatic interactions between QTLs. Cultivar Hartwig is an important resistance source to SCN. Recombinant inbred lines (RIL) obtained from a cross between 'Hartwig' (resistant) and Y23 (susceptible) were evaluated regarding resistance to the three races. New genomic regions for resistance to SCN were identified by microsatellites. Four QTLs, which explained between 12 and 34% of phenotypic variance, were detected for resistance to race 3 in linkage groups (LG) A2, G, J, and M. The QTL in LG G is also important for resistance to race 9. Epistatic interactions were detected between loci, which indicate resistance to races 9 and 14. There are high and low-effect resistance QTLs to SCN.

CALOGÊNESE EM ÓVULOS DE ESPÉCIES E VARIEDADES DE CITRUS

A Embrapa Mandioca e Fruticultura vem realizando ações de pesquisa visando à obtenção de híbridos somáticos de citros, particularmente porta-enxertos, melhor adaptados às condições tropicais de cultivo brasileiras que as variedades atualmente em uso. Como objetivo principal, busca-se a seleção de genótipos tolerantes à seca e ao alumínio, tolerantes/resistentes a gomose de Phytophthora e tristeza dos citros, além de adaptados a altas densidades populacionais. Como fontes de protoplastos, vêm sendo utilizadas as tangerinas-'Cleópatra', 'Sunki' e 'Swatow', limões-'Cravo Santa Cruz' e 'Santa Bárbara', 'Volkameriano' e 'Rugoso Mazoe', laranja 'Hamlin CNPMF 04' e 'CNPMF 20', laranjas-azedas 'Comum' e 'Narrow Leaf', citrange-'Troyer', Citrus amblycarpa e Microcitrus papuana. O estudo concentrou-se na etapa de obtenção e cultivo de calos embriogênicos. Foram utilizados óvulos extraídos de frutos imaturos, empregando-se como meio de cultura o MT, adicionando-se 50 g.L-1 de sacarose e 500 mg.L-1de extrato de malte e solidificando com 7 g.L-1 1de ágar. Em geral, a indução de calos nas variedades estudadas ocorreu entre a 6ª e a 8ª semana de cultivo, com maior precocidade na laranja-‘Hamlin’, sendo que, em limão-‘Cravo’, laranja-‘Hamlin’ , tangerinas-‘Cleópatra’ e ‘Swatow’ e citrange-‘Troyer’, a porcentagem de formação de calos foi igual ou superior a 50%, destacando-se a tangerina-‘Cleópatra’ com um porcentual próximo a 70% de calogênese.

ORGANOGÊNESE IN VITRO DE Citrus EM FUNÇÃO DE CONCENTRAÇÕES DE BAP E SECCIONAMENTO DO EXPLANTE

O sucesso de técnicas biotecnológicas no melhoramento in vitro de Citrus depende diretamente do desenvolvimento de protocolos eficientes para regeneração de plantas. Objetivou-se avaliar o efeito de concentrações de 6-benzilaminopuria (BAP) na organogênese in vitro de limão-'Cravo' e laranja-'Pêra', bem como o efeito do seccionamento do explante em laranja-'Valência'. Para o limão-'Cravo', foram utilizados como explante, segmentos internodais de plântulas germinadas in vitro, cultivados em meio MT e variando-se as concentrações de BAP em 0; 2,5; 5; 7,5 e 10 mg.L-1. Nas laranjas-'Pêra' e 'Valência' os explantes foram segmentos do epicótilo de plântulas germinadas in vitro. Os explantes de laranja-'Pêra' foram cultivados em meio MT variando-se as concentrações de BAP em 0; 1; 2; 3 e 4 mg.L-1. Para a laranja-'Valência', metade dos explantes foram seccionados e cultivados em meio MT acrescido de 1,0 mg.L-1 de BAP. Todas as brotações obtidas foram alongadas no meio de cultura MT + 25 g.L-1 de sacarose + 1 mg.L-1 de ácido giberélico (GA3) e enraizadas no meio MT + 25 g.L-1 de sacarose + 0,5 g.L-1 de carvão ativado + 1 mg.L-1 de ácido naftaleno acético (ANA). O melhor resultado para o número de brotações adventícias foi obtido na concentração 2,5 mg.L-1 de BAP para limão-'Cravo', e nas concentrações 1,0 e 2,0 mg.L-1 de BAP para laranja-'Pêra'. O seccionamento dos explantes favoreceu a organogênese in vitro da laranja-'Valência', porém as brotações apresentaram menor índice de enraizamento.

CARACTERÍSTICAS DO FRUTO DA VARIEDADE SPAN AMERICANA (Citrus reticulata Blanco): UMA TANGERINA DO TIPO 'PONCÃ' DE MATURAÇÃO PRECOCE

A tangerina-'Poncã' é bastante apreciada pelo consumidor brasileiro. No Estado de São Paulo, a maturação de seus frutos ocorre nos meses de abril a agosto. Com o mercado ávido por frutas de mesa e agravado pelo problema da ocorrência da clorose variegada dos citros (CVC), que afeta principalmente as variedades de laranjas-doces, tem havido um aumento considerável no plantio dessa variedade. Dessa forma, um excesso de produção, num mesmo período, faz com que os preços caiam, desestimulando assim os produtores. O Centro de Citricultura Sylvio Moreira (CCSM) vem buscando outras alternativas no sentido de obter, durante o ano todo, esse tipo de tangerina. Assim sendo, foram realizados estudos de caracterização de frutos, em diversas épocas do ano, com o acompanhamento dos níveis de maturação, envolvendo acessos do Banco de Germoplasma do CCSM. Dentre as variedades estudadas, quanto à precocidade e qualidade dos frutos, destacou-se a Span Americana. A tangerina-'Poncã' tem seu período de maturação para as condições edafoclimáticas do CCSM, nos meses de maio e junho. Já a variedade Span Americana, que apresenta frutos similares à 'Poncã' tradicional, tem maturação bastante precoce, podendo ser iniciada a sua colheita no mês de março.