Characterization of mandarin citrus germplasm from Southern Brazil by morphological and molecular analyses

The objective of this work was to characterize mandarin (Citrus spp.) germplasm from Southern Brazil by morphological and molecular analyses. Thirty seven cultivars from 34 distinct mandarin varieties were evaluated by morphological and agronomic traits of leaves, flowers and fruits, and by microsatellite markers. The morphological and agronomic characteristics suggested that almost all varieties can be produced for commercial use, and some, as the Satsuma variety, are recommended for breeding programs. Pooled DNA samples from 1-5 plants belonging to each cultivar were tested. Eight of the nine primers detected polymorphisms. Specific markers were found for some accessions. The dendrogram constructed with the morphological results divided the 37 cultivars into four groups, while that obtained with the microsatellites clustered 35 of the 37 cultivars into three groups only. Generally, intervarietal differences are not high, and this lack of agreement in the two multifactorial analyses indicates that diverse evolutionary factors are acting at these two levels of investigation.

Selection processes in a citrus hybrid population using RAPD markers

The objective of this work was to evaluate the processes of selection in a citrus hybrid population using segregation analysis of RAPD markers. The segregation of 123 RAPD markers between 'Cravo' mandarin (Citrus reticulata Blanco) and 'Pêra' sweet orange (C. sinensis (L.) Osbeck) was analysed in a F1 progeny of 94 hybrids. Genetic composition, diversity, heterozygosity, differences in chromosomal structure and the presence of deleterious recessive genes are discussed based on the segregation ratios obtained. A high percentage of markers had a skeweness of the 1:1 expected segregation ratio in the F1 population. Many markers showed a 3:1 segregation ratio in both varieties and 1:3 in 'Pêra' sweet orange, probably due to directional selection processes. The distribution analysis of the frequencies of the segregant markers in a hybrid population is a simple method which allows a better understanding of the genetics of citrus group.

Stomatal analysis of citrus somatic hybrids obtained by protoplast fusion

The objective of this work was to evaluate leaf epidermis morphological characteristics of three citrus somatic hybrids, compared to their parents. Parental and somatic hybrid young leaves were collected and processed for scanning electron microscope observations. Citrus polyploid hybrids have fewer stomata per area and these are larger compared to their diploid parental parents. No differences in internal arrangement of the stomatal cells were detected between parental plants and somatic hybrids. Additional studies may determine if these differences will influence physiological behavior of the plants in the field.

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.

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.

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.

SELEÇÃO E CARACTERIZAÇÃO DE LARANJEIRAS-DOCES (Citrus sinensis (L.) OSBECK) NO ESTADO DO ACRE

Objetivando selecionar laranjeiras exploradas no Estado do Acre, que apresentem características desejáveis, tais como produção e período de oferta de frutos, realizou-se, em 1999, a seleção e caracterização de plantas superiores. A identificação e a caracterização das plantas foram concentradas na época principal de colheita, considerando-se a idade e os aspectos fenológicos e fisiológicos das laranjeiras. Foram selecionadas 54 plantas, com idade variando de 12 acerca de 100 anos (segundo informações dos produtores), e média de 22 anos. As médias de altura, circunferência do caule e diâmetro da copa foram de 7,42 m, 0,99 m e 6,00 m, respectivamente. A época de colheita da maioria das laranjeiras selecionadas no Acre distribui-se de fevereiro a outubro, com concentração nos meses de abril, maio a julho, e apenas 5,45% delas apresentaram produção contínua. Dentre os genótipos de laranjeiras selecionados, existem materiais promissores para utilização como porta-enxerto e como variedade-copa, para o consumo local.

EFEITO DO ÁCIDO GIBERÉLICO (AG3) NA FLORAÇÃO E PRODUÇÃO DA LIMA ÁCIDA 'TAHITI' (Citrus latifolia Tan.)

A floração nos citros, assim como em outras fruteiras, é um dos fatores determinantes para a produção. A possibilidade de inibição ou redução da florada normal e alteração da época de produção para a lima 'Tahiti' é fator primordial para determinar sua rentabilidade econômica. O presente trabalho teve como objetivo avaliar o efeito do ácido giberélico sobre a florada, número de flores formadas, da lima ácida 'Tahiti', nas condições do Estado de São Paulo durante dois anos, buscando inibir a florada normal e observar o efeito desta inibição sobre produções temporãs. O delineamento experimental foi em blocos casualizados (5 blocos) com 3 plantas por tratamento. Foi utilizada uma planta como bordadura entre os tratamentos e uma linha de plantas entre os blocos. Os tratamentos foram: testemunha, 20, 40 e 80 mg/L de ácido giberélico (AG3) + 10 ml/L de espalhante adesivo, e o pH da água utilizada foi 6,2. As plantas foram tratadas com ácido giberélico durante o inverno (estação seca) após 50 dias sem irrigação no primeiro ano e 60 dias no segundo. O ácido giberélico reduziu o número de flores formadas (-81%) e aumentou a produção de frutos temporões (+59,77% ou +16,04 kg/pl).