Ana Maria Viana

Analgesic Effects of Callus Culture Extracts from Selected Species of Phyllanthus in Mice

Abstract— The aim of this study was to evaluate the analgesic effect of the methanolic extract from callus culture of Phyllanthus tenellus, P. corcovadensis and P. niruri in several models of pain in mice. The extracts (medium containing 2,4‐dichlorophenoxyacetic acid) of P. corcovadensis, P. niruri and P. tenellus (3–90 mg kg−1, i.p.) caused graded inhibition of abdominal constrictions induced by acetic acid (0·6%), with ID50 (i.e. dose that reduced response of control by 50%) values of about 30, 19 and >30 mg kg−1, respectively. The extract of callus of Phyllanthus obtained in indole‐3‐butyric acid and indole‐3‐acetic acid media (3–90 mg kg−1, i.p.) caused a similar analgesic effect. In the formalin test, the extract of P. tenellus obtained in indole butyric acid medium (3–100 mg kg−1, i.p.) inhibited only the second phase of formalin‐induced pain with an ID50 value of about 100 mg kg−1. Both the indole acetic acid and indole butyric acid methanolic extracts of P. tenellus and P. corcovadensis (10–100 mg kg−1, i.p.) dose‐dependently inhibited both phases of formalin‐induced pain (ID50 values for the second phase were approx. 100 and 52 mg kg−1, respectively). However, the extract of callus from Phyllanthus failed to affect formalin‐induced paw oedema, as well as the response to radiant heat in the tail‐flick test. In addition, the analgesic effect of morphine, but not the analgesic effects caused by Phyllanthus callus extract, was fully antagonized by naloxone. Preliminary phytochemical analysis revealed the presence of several compounds having no apparent relationship with alkaloids or flavonoids but showing the presence of phenols. These results indicate that, similar to previous reported data from the extract of P. corcovadensis, the methanolic extracts of callus culture of P. niruri, P. corcovadensis and P. tenellus exhibit potent analgesic properties against neurogenic and inflammatory pain that seem to be unrelated to the activation of opioid mechanisms.

SERK gene homolog expression, polyamines and amino acids associated with somatic embryogenic competence of Ocotea catharinensis Mez. (Lauraceae)

In the present work, we investigate the association of SERK gene homolog expression, polyamines (PAs) and amino acids related to putrescine synthesis (arginine and ornithine) and polyamines degradation (γ-aminobutiric acid) or S-adenosylmethionine synthesis (methionine), with the embryogenic competence in cell aggregates of Ocotea catharinensis Mez. (Lauraceae). Cell aggregates were cultivated during 7 days in woody plant medium (WPM) supplemented with 20 g l−1 sucrose, 22 g l−1 sorbitol, 400 mg l−1 glutamine and 2 g l−1 phytagel, and in Murashige and Skoog medium (MS) supplemented 20 g l−1 sucrose, 3 g l−1 activated charcoal, 2 g l−1Phytagel with and without 40 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). The cell aggregates cultivated in MS plus 2,4-D and in the WPM medium showed hybridization with a SERK gene homolog both in northern and in situ hybridization experiments. Cell aggregates cultivated in an MS basal medium, without 2,4-D, did not exhibit any hybridization signal to the SERK probe used, thus they were considered potentially non-embryogenic cells. In all three media only free polyamines were detected. The higher putrescine levels occurring in WPM callus were associated with a higher arginine and ornithine content, lower γ-aminobutiric acid level, and SERK homolog expression. Putrescine was also the major polyamine in the MS medium. In the MS plus 2,4-D medium, the levels of putrescine, spermidine and spermine were similar. Spermine exhibited similar and the lowest levels in all media. Spermidine intermediary levels occurred in the WPM and MS media. In cell aggregates methionine level was lowest in the MS plus 2,4-D medium, but similar in the MS and WPM media.

IAA, ABA, polyamines and free amino acids associated with zygotic embryo development of Ocotea catharinensis

The aim of this work was to study morphological and biochemical aspects during zygotic embryogenesis in O. catharinensis, by measuring changes in the endogenous concentrations of proteins, amino acids, polyamines (PAs), indole-3-acetic acid (IAA) and abscisic acid (ABA). Buffer-soluble and insoluble protein contents were determined by spectrometry, and amino acids, PAs, IAA and ABA concentrations were determined by high performance liquid chromatography. Total amino acid accumulation, predominantly asparagine, occurred when the embryo showed completely developed cotyledons, with posterior reduction in the mature embryo. This decrease in total amino acid concentration in the mature embryo may result from their use in storage␣as well as for LEA protein synthesis. Free putrescine (Put) concentration decreased, while free spermine (Spm) increased during embryo development. This suggest a role for Put in the initial phases of embryogenesis when high rates of cell division occur, while elevated concentration of Spm are essential from the middle to the end of embryo development, when growth is mainly due to cell elongation. An IAA peak in zygotic embryos occurred during initial development, suggesting a link between growth and cellular division as well as with the establishment of bilateral symmetry. ABA concentration declined during initial stages of development then increased at the mature embryo stage, suggesting a possible relationship with dormancy and recalcitrance characteristics. Our results show that changes in the phytohormones (IAA, ABA and PAs) concentrations in combination with amino acids are likely important factors determining the developmental stages of O.␣catharinensis zygotic embryos.

In vitro culture of Cedrela fissilis Vellozo (Meliaceae)

An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments from juvenile origin for axillary shoot proliferation. Shoot proliferation was significantly affected by salt formulation, explant origin and 6-benzyladenine concentration. Maximum multiplication rates (6–7 new plants were produced in the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoog media supplemented with 1.25–5.0 μM 6-benzyladenine. Addition of α-naphthaleneacetic acid to these media caused significant inhibition on shoot proliferation and growth and stimulated callus formation. High frequency callus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium supplemented with 6-benzyladenine at either 1.25, 2.5 or 5.0 μM combined, respectively, with 2.5, 1.25–5.0 or 5.0 μM α-naphthaleneacetic acid. Rooting was achieved, after 10–12 days, with 87–100% of the node cuttings on half strength Murashige and Skoog medium either without growth regulators or supplemented with 2.5 μM indole-3-butyric acid. Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but for further plant development the sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets under ex vitro conditions was 100% after 3 months. The optimized micropropagation and callus culture protocols offer the possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondary metabolism studies.An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments from juvenile origin for axillary shoot proliferation. Shoot proliferation was significantly affected by salt formulation, explant origin and 6-benzyladenine concentration. Maximum multiplication rates (6–7 new plants were produced in the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoog media supplemented with 1.25–5.0 μM 6-benzyladenine. Addition of α-naphthaleneacetic acid to these media caused significant inhibition on shoot proliferation and growth and stimulated callus formation. High frequency callus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium supplemented with 6-benzyladenine at either 1.25, 2.5 or 5.0 μM combined, respectively, with 2.5, 1.25–5.0 or 5.0 μM α-naphthaleneacetic acid. Rooting was achieved, after 10–12 days, with 87–100% of the node cuttings on half strength Murashige and Skoog medium either without growth regulators or supplemented with 2.5 μM indole-3-butyric acid. Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but for further plant development the sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets under ex vitro conditions was 100% after 3 months. The optimized micropropagation and callus culture protocols offer the possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondary metabolism studies.

In vitro plantlet regeneration of Ocotea catharinensis, an endangered Brazilian hardwood forest tree

A successful system of somatic embryogenesis is described for the forest tree Ocotea catharinensis Mez., which used mature zygotic embryo explants cultured on a modified Murashige and Skoog (MS) medium with activated charcoal, at 25°C in the dark. A medium composed of MS supplemented with 2% (w/v) sucrose, 0.3% (w/v) activated charcoal (AC), 362 μM 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.8% (w/v) Technical Agar Grade III was used for multiplication of embryogenic cultures. Development up to the globular-stage was achieved using Lloyd and McCown woody plant medium (WPM) with 2.0% sucrose, 0.3% AC, 181 μM 2,4-d and 0.8% Technical Agar Grade III. Significant effects of media pH on differentiation of early pro-embryogenic Ocotea cell aggregates were found. Low pH of media (ca. 3–4) appeared to prevent differentiation of proembryogenic cell aggregates whereas higher pH levels (ca. 5–5.5) favoured the formation of globular structures. Once globular structures formed, they developed further to form cotyledonary somatic embryos, under the same set of culture conditions. Successful conversion of these somatic embryos to plantlets was achieved after culture on a medium composed of 1/2-strength WPM (minerals only) with 2% sucrose, 0.3% AC, 0.8% Technical Agar Grade III and 90.5 μM 2,4-d, followed by transfer to a medium composed of 1/2-strength WPM (minerals only) with 2% sucrose, 0.8% Technical Agar Grade III and 0.905 μM 2,4-d and 1.4 μM gibberellic acid, in a 16-h photoperiod regime.

In vitro conservation of Cedrela fissilis Vellozo (Meliaceae),a native tree of the Brazilian Atlantic Forest

Two in vitro conservation methods have beendeveloped for the ex situ conservation of germplasm fromCedrela fissilis, an economically important tree of theBrazilian Atlantic Forest. The first method involves the medium-term storage, at 25°C, of artificial seeds comprising alginate encapsulatedvegetative propagules (shoot tips, cotyledonary and epicotyl nodal segments).Maximum post-storage (3 months) viabilities of 96–100% wereachieved for encapsulated shoot tips and cotyledonary nodal segments stored onwater-solidified agar (at 0.4–0.7% w/v). Encapsulated shoot tips storedfor 6 months on 0.4% (w/v) agar showed the highest survival rates(44%). Seeds of C. fissilis were successfully cryopreserved(100%) after direct immersion in liquid nitrogen. Ex situstorage procedures are now available for the medium- to long-term conservationof C. fissilis. These approaches offer new opportunitiesfor the conservation, sustainable management and utilization of this valuablefast growing timber tree.

Micropropagation, callus and root culture of Phyllanthus urinaria (Euphorbiaceae)

Efficient micropropagation, callus culture and root culture protocols were developed for the medicinal plant Phyllanthus urinaria(Euphorbiaceae) using single node explants. Maximum multiplication (16–20 shoots per explant) was achieved on Murashige and Skoog media supplemented with 5.0 μM kinetin. Murashige and Skoog and Anderson Rhododendron media promoted significant shoot culture growth in terms of numbers of shoots and nodes produced per explant. Rooting was achieved with 93–100% of the microshoots on Murashige and Skoog medium without growth regulators, although 1.25–5.0 μM α-naphthaleneacetic acid significantly increased the number of roots per explant. Regenerated plants were successfully acclimatized and 91% of plantlets survived under ex vitro conditions. Flowering was observed on micropropagated plants after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when single node explants were inoculated in the horizontal position on Murashige and Skoog medium supplemented with 5.0 μM indole-3-butyric acid. Other auxins such as 2,4-dichlorophenoxyacetic acid and α-naphthaleneacetic acid promoted moderate callus fresh weight increase, when used separately. Root cultures were successfully established on Murashige and Skoog medium containing 1.1 μM α-naphthaleneacetic acid. The optimized micropropagation, callus culture and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies.

In vitro and cryogenic preservation of plant biodiversity in Brazil

The Brazilian biomes (Amazon, Atlantic Forest, Cerrado, Caatinga, Pantanal, and Pampa) comprise one of the highest levels of plant diversity in the world; however, non-sustainable practices, deforestation, and land use have resulted in significant losses and fragmentation of the native forests. These ecosystems are now threatened and protection of their native plants through ex situ conservation is an urgent necessity. Cryopreservation and in vitro conservation are complementary options for securing and protecting Brazil’s native plant species because their potential economic value is critically important to develop strategies that will (1) support their sustainable utilization, (2) protect against the over-exploitation of species growing in natural habitats, and (3) conserve the genetic diversity of germplasm from species of different provenances. Biotechnological approaches will help to address future economic and environmental demands placed upon already at-risk species. Conserving seed germplasm ex situ provides an additional safeguard against the risks (e.g., loss due to disease, climate change) of field conservation. Moreover, seed banks and cryobanks permit the long-term conservation of a wider genetic base; this offsets the labor and space intensive costs of conserving in the active growing state. This paper is a compilation of the current status of strategies applied for conserving Brazilian native plant species.

In vitro culture of Phyllanthus caroliniensis (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus caroliniensis (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication (21–23 shoots per explant) was achieved on MS or AR media supplemented with either 5.0 μM BA, 1.25–5.0 μM kinetin or 2.5–5.0 μM 2iP. Rooting was achieved with 80–100% of the microshoots on MS medium without growth regulators, although 1.25 μM NAA and 1.25–5.0 μM IAA promoted significant increases in the number of roots per explant. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed on in vitro grown plantlets and after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated in the vertical position on MS medium supplemented with 5.0 μM 2,4-D. Root cultures were successfully established on MS medium containing 1.1 μM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies.

Repetitive Somatic Embryogenesis of Ocotea catharinensis Mez. (Lauraceae): Effect of Somatic Embryo Developmental Stage and Dehydration

Repetitive embryogenesis of Ocotea catharinensis from globular/early cotyledonary somatic embryos was successfully supported by WPM supplemented with 22.7 g l−1 sorbitol, 20 g l−1 sucrose, 400 mg l−1 glutamine and 2 g l−1 Phytagel. The best medium to induce repetitive embryogenesis in cotyledonary somatic embryos was half strength WPM supplemented with 20 g l−1 sucrose, 400 mg l−1 glutamine, 1.5 g l−1 activated charcoal and 2 g l−1 Phytagel. The mature somatic embryos gradually air dehydrated showed repetitive embryogenesis after subculture on half strength B5 medium supplemented with 20 g l− sucrose, 20 g l−1 Phytagel, 1.5 g l−1 activated charcoal, 115.6 µM gibberellic acid and 214.8 µM naphthaleneacetic acid. The early cotyledonary, cotyledonary and mature somatic embryos tolerated respectively 95, 86 and 54% fresh weight losses without losing their repetitive embryogenesis potential. Cotyledonary and mature somatic embryos gradually air dehydrated in sealed Petri dishes showed 40–41% repetitive embryogenesis respectively after 20 days and 12 weeks desiccation storage. Repetitive embryogenesis in cotyledonary somatic embryos was significantly stimulated by chemical dehydration with 0.5 M sorbitol and 56% repetitive embryogenesis was achieved even after exposure to 2 M sorbitol for 24 h. The cotyledonary somatic embryos when alginate-encapsulated showed 47% repetitive embryogenesis even after chemical dehydration in 1.5 M sorbitol for 4 days followed by 1 h air dehydration, but failed to survive to the same dehydration conditions without encapsulation. The optimized repetitive embryogenesis and desiccation protocols offer the possibility to use in vitro techniques for continuous reliable somatic embryo production and short term germplasm storage.