In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm(- 3) 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm(-3) BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm(-3) BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.

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.

Effects of limonene and essential oil from Citrus aurantium on gastric mucosa: Role of prostaglandins and gastric mucus secretion

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Citrus aurantium L. essential oil exhibits anxiolytic-like activity mediated by 5-HT1A-receptors and reduces cholesterol after repeated oral treatment

Background: The current treatments for anxiety disorders and depression have multiple adverse effects in addition to a delayed onset of action, which has prompted efforts to find new substances with potential activity in these disorders. Citrus aurantium was chosen based on ethnopharmacological data because traditional medicine refers to the Citrus genus as useful in diminishing the symptoms of anxiety or insomnia, and C. aurantium has more recently been proposed as an adjuvant for antidepressants. In the present work, we investigated the biological activity underlying the anxiolytic and antidepressant effects of C. aurantium essential oil (EO), the putative mechanism of the anxiolytic-like effect, and the neurochemical changes in specific brain structures of mice after acute treatment. We also monitored the mice for possible signs of toxicity after a 14-day treatment.Methods: The anxiolytic-like activity of the EO was investigated in a light/dark box, and the antidepressant activity was investigated in a forced swim test. Flumazenil, a competitive antagonist of benzodiazepine binding, and the selective 5-HT1A receptor antagonist WAY100635 were used in the experimental procedures to determine the mechanism of action of the EO. To exclude false positive results due to motor impairment, the mice were submitted to the rotarod test.Results: The data suggest that the anxiolytic-like activity observed in the light/dark box procedure after acute (5 mg/kg) or 14-day repeated (1 mg/kg/day) dosing was mediated by the serotonergic system (5-HT1A receptors). Acute treatment with the EO showed no activity in the forced swim test, which is sensitive to antidepressants. A neurochemical evaluation showed no alterations in neurotransmitter levels in the cortex, the striatum, the pons, and the hypothalamus. Furthermore, no locomotor impairment or signs of toxicity or biochemical changes, except a reduction in cholesterol levels, were observed after treatment with the EO.Conclusion: This work contributes to a better understanding of the biological activity of C. aurantium EO by characterizing the mechanism of action underlying its anxiolytic-like activity. © 2013 Costa et al; licensee BioMed Central Ltd.

Estudo farmacológico e neuroquímico dos óleos essenciais de Cymbopogon citratus (D.C) Stapf e Citrus ayrantium L

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The antimicrobial effects of Citrus limonum and Citrus aurantium essential oils on multi-species biofilms

The aim of this study was to evaluate the effects of Citrus limonum and Citrus aurantium essential oils (EOs) compared to 0.2% chlorhexidine (CHX) and 1% sodium hypochlorite (NaOCl) on multi-species biofilms formed by Candida albicans, Enterococcus faecalis and Escherichia coli. The biofilms were grown in acrylic disks immersed in broth, inoculated with microbial suspension (106 cells/mL) and incubated at 37°C / 48 h. After the biofilms were formed, they were exposed for 5 minutes to the solutions (n = 10): C. aurantium EO, C. limonum EO, 0.2% CHX, 1% NaOCl or sterile saline solution [0.9% sodium chloride (NaCl)]. Next, the discs were placed in sterile 0.9% NaCl and sonicated to disperse the biofilms. Tenfold serial dilutions were performed and the aliquots were seeded onto selective agar and incubated at 37°C / 48 h. Next, the number of colony-forming units per milliliter was counted and analyzed statistically (Tukey test, p ≤ 0.05). C. aurantium EO and NaOCl inhibited the growth of all microorganisms in multi-species biofilms. C. limonum EO promoted a 100% reduction of C. albicans and E. coli, and 49.3% of E. faecalis. CHX was less effective against C. albicans and E. coli, yielding a reduction of 68.8% and 86.7%, respectively. However, the reduction of E. faecalis using CHX (81.7%) was greater than that obtained using C. limonum EO. Both Citrus limonum and Citrus aurantium EOs are effective in controlling multi-species biofilms; the microbial reductions achieved by EOs were not only similar to those of NaOCl, but even higher than those achieved by CHX, in some cases.

Effect of essential oil from Citrus aurantium in maternal reproductive outcome and fetal anomaly frequency in rats

Citrus aurantium L., commonly known as bitter orange, is widely used in folk medicine, but there is little data in the literature about the s on pregnancy. The aim of the present study was to evaluate the influence of essential oil obtained from fruits of Citrus aurantium on the maternal reproductive outcome and fetal anomaly incidence in rats. Pregnant Wistar rats were randomized into four groups (n minimum = 12 animals/group): G1 = control, G2 to G4 = treated with essential oil from C. aurantium at dose 125, 250 and 500 mg/kg, respectively. Rats were orally treated, by gavage, with plant essential oil or vehicle during pre-implantation and organogenic period (gestational day 0-14). On gestational day 20 the rats were anaesthetized and the gravid uterus was weighed with its contents and the fetuses were analyzed. Results showed that the treated group with 500 mg/kg presented decreased placental weights and placental index, although the treatment with bitter orange essential oil did not show any alteration in maternal reproductive performance, toxicological , changes in ossification sites, and malformation index. In conclusion, the treatment of Citrus aurantium essential oil was not teratogenic and did not alter the maternal reproductive outcome.

Citrus aurantium L. essential oil exhibits anxiolytic-like activity mediated by 5-HT1A-receptors and reduces cholesterol after repeated oral treatment

Abstract Background The current treatments for anxiety disorders and depression have multiple adverse effects in addition to a delayed onset of action, which has prompted efforts to find new substances with potential activity in these disorders. Citrus aurantium was chosen based on ethnopharmacological data because traditional medicine refers to the Citrus genus as useful in diminishing the symptoms of anxiety or insomnia, and C. aurantium has more recently been proposed as an adjuvant for antidepressants. In the present work, we investigated the biological activity underlying the anxiolytic and antidepressant effects of C. aurantium essential oil (EO), the putative mechanism of the anxiolytic-like effect, and the neurochemical changes in specific brain structures of mice after acute treatment. We also monitored the mice for possible signs of toxicity after a 14-day treatment. Methods The anxiolytic-like activity of the EO was investigated in a light/dark box, and the antidepressant activity was investigated in a forced swim test. Flumazenil, a competitive antagonist of benzodiazepine binding, and the selective 5-HT1A receptor antagonist WAY100635 were used in the experimental procedures to determine the mechanism of action of the EO. To exclude false positive results due to motor impairment, the mice were submitted to the rotarod test. Results The data suggest that the anxiolytic-like activity observed in the light/dark box procedure after acute (5 mg/kg) or 14-day repeated (1 mg/kg/day) dosing was mediated by the serotonergic system (5-HT1A receptors). Acute treatment with the EO showed no activity in the forced swim test, which is sensitive to antidepressants. A neurochemical evaluation showed no alterations in neurotransmitter levels in the cortex, the striatum, the pons, and the hypothalamus. Furthermore, no locomotor impairment or signs of toxicity or biochemical changes, except a reduction in cholesterol levels, were observed after treatment with the EO. Conclusion This work contributes to a better understanding of the biological activity of C. aurantium EO by characterizing the mechanism of action underlying its anxiolytic-like activity.

Evaluation of citrus somatic hybrids for tolerance to Phytophthora nicotianae and citrus tristeza virus

Somatic hybridization is a biotechnology tool that can be used in citrus breeding programs to produce somatic hybrids with the complete genetic combination of both parents. The goal of this work was to test the reaction of citrus somatic hybrids that may be useful as rootstocks to trunk and root infections caused by Phytophthora nicotianae van Breda de Haan (P parasitica Dastur) and to citrus tristeza virus (CTV). The somatic hybrids evaluated were `Caipira` sweet orange (Citrus sinensis L. Osbeck) + `Rangpur` lime (C. limonia Osbeck), `Caipira` sweet orange + `Cleopatra` mandarin (C. reshni hort. ex Tanaka), `Caipira` sweet orange + `Volkamer` lemon (C. volkameriana V Ten. & Pasq.), `Caipira` sweet orange + rough lemon (C. jambhiri Lush.), `Cleopatra` mandarin + `Volkamer` lemon, `Cleopatra` mandarin + sour orange (C. aurantium L.), `Rangpur` lime + `Sunki` mandarin (C. sunki (Hayata) hort. ex Tanaka), `Ruby Blood` sweet orange (C. sinensis L. Osbeck) + `Volkamer` lemon, `Rohde Red` sweet orange (C. sinensis L. Osbeck) + `Volkamer` lemon, and `Valencia` sweet orange + Fortunella obovata hort. ex Tanaka. For P. nicotianae trunk and root infection assays, plants of the somatic hybrids, obtained from 9-month semi-hardwood cuttings, were evaluated and compared with diploid citrus rootstock cultivars after mycelia inoculation in the trunk or spore infestation in the substrate, respectively. `Cleopatra` mandarin + sour orange, `Rangpur` lime + `Sunki` mandarin, `Cleopatra` mandarin + `Volkamer` lemon, `Ruby Blood` sweet orange + `Volkamer` lemon, `Rohde Red` sweet orange + `Volkamer` lemon, and `Caipira` sweet orange + `Volkamer` lemon had less trunk rot occurrence, whereas the somatic hybrids `Cleopatra` mandarin + `Volkamer` lemon, `Cleopatra` mandarin + sour orange, `Caipira` sweet orange + `Volkamer` lemon, and `Caipira` sweet orange + `Rangpur` lime were tolerant to root rot. For CTV assays, plants of the somatic hybrids along with tolerant and intolerant rootstocks were budded with a mild strain CTV-infected or healthy `Valencia` sweet orange budwood. Differences in average scion shoot length indicated that the hybrids `Cleopatra` mandarin + sour orange and `Valencia` sweet orange + Fortunella obovata were intolerant to CTV (c) 2007 Elsevier B.V. All rights reserved.

Comportamento de catorze porta-enxertos para o limão eureka km 47 na região de Araraquara-SP

Foi avaliado o comportamento de 14 porta-enxertos para o limão 'Eureka km 47' [Citrus limon (L.) Burm. f.], clone nucelar, em experimento instalado em 1991, na Fazenda Fittipaldi, Araraquara-SP. Os porta-enxertos foram: tangelos 'Orlando' e 'Seminole' (C. reticulata Blanco x C. paradisi Macfad.), tangerinas 'Cleópatra' (C. reshni hort. ex Tanaka) e 'Sunki' [C. sunki (Hayata) hort. ex Tanaka], limões 'Cravo' (C. limonia Osbeck), 'Rugoso da África' (C. jambhiri Lush.) e 'Volkameriano Catania 2' (C. volkameriana V. Ten. & Pasq.), laranja 'Caipira DAC' [C. sinensis (L.) Osbeck], laranja 'Azeda Doble C��lice' (C. aurantium L.), trifoliatas 'EEL'e 'Kryder 8-5' [Poncirus trifoliata (L.) Raf.], citrange 'Morton' [P. trifoliata (L.) Raf. x C. sinensis (L.) Osbeck], C. karna Raf. e C. pennivesiculata (Lush.) Tanaka. As produções foram avaliadas no período de 1998 a 2002. As maiores produções médias em peso de frutos estiveram relacionadas aos porta-enxertos C. pennivesiculata e tangerina 'Cleópatra' (>220 kg/planta). Entre os porta-enxertos que induziram as mais baixas produções, estão C. karna, os dois tangelos, os dois trifoliatas e a 'Caipira DAC' (<180 kg/planta). As características de qualidade apresentadas pelos frutos e referentes ao ano de 2002 indicaram não haver diferenças expressivas devido à influência dos tratamentos. São também apresentados dados relativos à suscetibilidade das plantas à gomose de Phytophthora, no período estudado.

Potencial de obtenção de novos porta-enxertos em cruzamentos envolvendo limoeiro 'Cravo', laranjeira 'Azeda', tangerineira 'Sunki' e híbridos de Poncirus Trifoliata

O limoeiro 'Cravo' e a laranjeira 'Azeda' são importantes porta-enxertos de citros. Com o objetivo de explorar seu potencial genético, distintas seleções de limoeiro 'Cravo', empregadas como parentais femininos, foram cruzadas com seleções de laranjeira 'Azeda', tangerineira 'Sunki Maravilha' e híbridos de Poncirus trifoliata. Semelhantemente, seleções de 'Azeda' foram hibridadas com seleções de 'Cravo' e híbridos de P. trifoliata. Avaliaram-se a taxa de vingamento de frutos, a porcentagem de híbridos no conjunto total de indivíduos obtidos (zigóticos e nucelares), a altura e o diâmetro do caule destes, em torno dos 12 meses de idade. O cruzamento entre limoeiro 'Cravo', seleções comum e 'Santa Cruz', com tangerineira 'Sunki 'Maravilha' resultou nas maiores taxas de vingamento de frutos (média de 87,5%) e maior percentual de híbridos (média de 64,0%), sendo estes relativamente vigorosos, segundo comparações com seedlings nucelares desse limoeiro. Quanto às hibridações envolvendo a laranjeira 'Azeda', mostraram-se promissoras 'Azeda Jacarandá' x (tangerineira 'Cleópatra' x P. trifoliata seleção 'Swingle 245'), pela relativamente elevada taxa de vingamento de frutos (50%), boa porcentagem de híbridos (32,3%) e possibilidade de obtenção de híbridos vigorosos, e laranjeira 'Azeda' comum x limoeiro 'Cravo' seleções comum e 'Santa Cruz', pela possibilidade de obtenção de híbridos vigorosos e em quantidades relativamente altas, em torno de 40% de plantas híbridas.

Reação de híbridos somáticos de citros à infec��ão por Phytophthora nicotianae

Este trabalho avaliou a resistência à infec��ão de tronco e de raízes por Phytophthora nicotianae em híbridos somáticos de citros com potencial para serem utilizados como porta-enxertos. Os híbridos somáticos avaliados foram laranja 'Hamlin' (Citrus sinensis) + toranja 'Indian Red' (Citrus grandis) (plantas 1 e 2) e laranja 'Hamlin' (C. sinensis) + toranja 'Singapura' (C. grandis). Plantas de limão 'Cravo' (Citrus limonia), laranja 'Caipira' (C. sinensis), laranja-azeda (C. aurantium) e Poncirus trifoliata 'Davis A' (Poncirus trifoliata) foram utilizadas como plantas-controle devido à reação conhecida à infec��ão pelo patógeno. Avaliações realizadas entre 30 e 60 dias após as inoculações com o patógeno incluíram o comprimento das lesões no tronco e a massa seca do sistema radicular nas plantas avaliadas. O híbrido somático laranja 'Hamlin' + toranja 'Indian Red' (planta 1) mostrou-se tolerante a P. nicotianae, indicando potencial para continuidade nas suas avaliações como porta-enxerto para citros.

In vitro organogenesis in some citrus species

In vitro organogenesis of Citrus was studied for the genotypes Citrus sinensis cv. 'Natal', C. limonia, C. volkameriana, and C. aurantium, with the use of epicotyl segments-derived explants, cultured in MT salts and vitamins medium supplemented with different concentrations of 6-benzylaminopurine (BAP - 0.0; 0.5; 1.0; 1.5 or 2.0 mg L-1). For the recalcitrant genotypes C. limonia and C. aurantium the in vitro organogenesis was also studied with internodal segments-derived explants, cultured in MT salts and vitamins medium supplemented with 0; 0.5; 1.0; 2.0, or 4.0 mg L-1 of BAP. The efficiency of culture medium supplementation with the combination of BAP (0.0; 1.0, or 2.0 mg L-1) and NAA (1-naphthaleneacetic acid - 0.0; 0.3, or 0.5 mg L-1) in the development of adventitious shoots was evaluated for C. aurantium. Culture medium supplementation with BAP is not essential for the adventitious shoots development in the four genotypes studied when epicotyl segments-derived explants are used. In general, culture media supplementation with BAP decreased the percentage of responsive explants excepted for C. sinensis cv. 'Natal' and C. limonia when the concentrations of 1.5 and 2.0 mg/L were used. The presence of cytokinin, in concentrations up to 2 mg/L, stimulated the in vitro organogenesis when internodal segments-derived explants were used for C. limonia and C. aurantium. For C. aurantium no adventitious shoots developed in explants (internodal segments) cultured in basal culture medium, without BAP supplementation. Although no statistic differences could be detected, culture media supplementation with the combination of BAP and NAA favored the development of adventitious shoots in C. aurantium. The best concentration of NAA varied according to BAP concentration. The results presented herein, show that Citrus in vitro organogenesis depends on the interaction of culture medium composition, explant differentiation level, and genotype.

Desempenho da tangerineira 'Span Americana' em diferentes porta-enxertos

Este trabalho objetivou avaliar o crescimento vegetativo, a produção e a qualidade de frutos da tangerina 'Span Americana' em diferentes porta-enxertos, nas condições edafoclimáticas de Bebedouro-SP. O plantio foi realizado em junho de 2003, em espaçamento de 6,0 m x 3,0 m, sendo utilizada irrigação por gotejamento a partir de 2006. Os porta-enxertos avaliados foram: citranges [Citrus sinensis (L.) Osbeck × Poncirus trifoliata L. Raf] 'Carrizo' e 'Troyer', tetraploides; trifoliatas (P. trifoliata) 'Davis A' e 'Flying Dragon'; limão Volkameriano Catania 2 (C. volkameriana Tenn. et Pasq.), HRS 849 [(C. aurantium L. cv. 'Smooth Flat Seville' x P. trifoliata cv. 'Argentina')], tangelo 'Orlando' (C. reticulata Blanco × C. paradisi Macf.) e limão 'Cravo' (C. limonia Osbeck). Foram avaliadas a produção acumulada, a eficiência produtiva e a precocidade de entrada em produção, no período de 2007 a 2009. Avaliaram-se, também, as dimensões das plantas e a taxa média de crescimento das plantas no período de 2005 a 2008, além da qualidade dos frutos em 2006 e 2007. Em pomares irrigados de tangerineira 'Span Americana', os porta-enxertos trifoliata 'Davis A' e HRS 849 apresentam desempenho horticultural satisfatório. Para plantio em alta densidade, a melhor performance da tangerineira 'Span Americana' é obtida com a utilização dos porta-enxertos trifoliata 'Flying Dragon' e citranges 'Troyer' e 'Carrizo'.

Resistência de clones e híbridos de porta-enxertos de citros à gomose de tronco causada por Phytophthora parasitica

Sete grupos de híbridos entre porta-enxertos elite de citros (Citrus sp.) e seus genitores foram estudados quanto a reação à infec��ão de tronco por Phytophthora parasitica, em plantas adultas no campo. Inocularam-se artificialmente, em duas posições do tronco, 132 plantas nucelares dos genitores e 486 híbridos entre limão (Citrus limonia) Cravo 'Limeira' (C), Poncirus trifoliata 'Davis A' (T), tangerina (C.sunki) 'Sunki' (S) e laranja Azeda (C. aurantium) 'São Paulo' (A). A classificação do grau de resistência à gomose de tronco em plantas adultas no campo foi possível somente quando baseada na média do tamanho de lesões de mais de 30 plantas nucelares, devido às grandes variações observadas em plantas individuais do mesmo clone, impossibilitando a seleção precoce de híbridos baseada em valores de plantas individuais. O trifoliata e a laranja Azeda mostraram-se bastante resistentes, desenvolvendo, em geral, lesões de tamanho reduzido. A tangerina Sunki e o limão Cravo desenvolveram lesões maiores, embora a tangerina Sunki tenha mostrado uma tendência em desenvolver lesões maiores que às do limão Cravo. Híbridos de trifoliata apresentaram, no geral, lesões intermediárias. Os híbridos de Azeda apresentaram lesões de tamanhos bastante variáveis, porém a maioria, com lesões grandes. Da mesma forma comportaram-se os híbridos rec��procos entre Sunki e Cravo.