Photoperiod modulates growth and pigments and 20-hydroxyecdysone accumulation in Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen] grown in vitro

Plant Cell, Tissue and Organ Culture (PCTOC) - Photoperiod regulates different morphophysiological processes in plants, directly impacting photosynthetic performance and, consequently, primary and...     

Karyotype,genome size,and in vitro chromosome doubling of Pfaffia glomerata (Spreng.) Pedersen

Pfaffia glomerata (Spreng.) Pedersen, known worldwide as Brazilian ginseng, has an important commercial value due to its pharmaceutical properties. In addition to the newly described karyological traits and the first estimation of DNA content, this study reports a protocol for the successful induction of tetraploidy. Natural diploid individuals (2n = 34) showed a symmetric karyotype, centromeric DAPI⁺ bands, one chromosome pair with a CMA⁺ band and 45S rDNA site and another with one 5S rDNA site. To induce chromosome duplication, small nodal buds were cultured in semi-solid MS-based medium with 2.22 μM BA, 2.69 μM NAA, and colchicine or oryzalin at 10, 15, 20, 25, and 30 μM for 1 or 2 weeks before being transferred to MS basal medium. The results showed that colchicine induced tetraploid plants, mainly after 1 week of exposure, whereas oryzalin treatment induced only mixoploid plants. The tetraploid plants exhibited twice the chromosome number and DNA content and twice the number of chromosome markers observed for the diploids. Chromosome duplication reduced the dry mass of the stems and roots of the polyploid plants compared to the diploids, and the stomatal density was also reduced on the abaxial and adaxial leaf surfaces of the polyploids. Additionally, the production of β-ecdysone was 50 % higher in the tetraploids than in the diploids. Thus, chromosome doubling showed that is possible to increase the content of β-ecdysone, highlighting the considerable potential of this technique to produce new cultivars with high commercial value.     

Photoautotrophic propagation of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]

Pfaffia glomerata (Spreng.) Pedersen is a medicinal species of great interest because it produces the phytoecdysteroid 20-hydroxyecdysone (20E). Generally, because of atypical growing conditions, in vitro propagated plants function less efficiently as autotrophs and have poorly developed morphological structures. This study analyzed the autotrophic potential of P. glomerata propagated in vitro and evaluated the influence that this has on 20E biosynthesis. Physiological and structural parameters of plants subjected to heterotrophic, photomixotrophic and photoautotrophic growth conditions were evaluated. Levels of 20E were measured by HPLC. Plants were acclimatized in a mixture of soil, sand and substrate, in a greenhouse. Conditions that provided higher carbon input led to an increase in plant growth, and the presence of sucrose was critical, in closure systems without a gas permeable membrane, for normal anatomical development of the micropropagated plants. The absence of sucrose increased photosynthesis and conditions that enhanced photoautotrophy induced greater levels of 20E. The increase of 20E levels by the photoautotrophic system offers new prospects for increasing the commercial production of this species, and for studies that could elucidate the biosynthetic pathway of phytoecdysteroids in plants.     

A CO2-enriched atmosphere improves in vitro growth of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]

The aim of the present study was to evaluate the effects of forced ventilation and CO₂ enrichment (360 or 720 μmol mol^?1 CO₂) on the in vitro growth and development of Pfaffia glomerata, an endangered medicinal species, under photomixotrophic or photoautotrophic conditions. P. glomerata nodal segments showed substantial differences in growth, relative water content and water loss from leaves, photosynthetic pigments, stomatal density, and leaf anatomical characteristics under these different treatments. CO₂ enrichment led to increased photosynthetic pigments and reduced stomatal density of in vitro cultivated P. glomerata. A lack of sucrose in the culture medium increased 20-hydroxyecdysone levels, but the increase in CO₂ levels did not further elevate the accumulation of 20-hydroxyecdysone. All growth increased in a CO₂-enriched atmosphere. In addition, CO₂ enrichment, with or without sucrose, gave a lower relative water loss from leaves. This finding indicates that either a photoautotrophic or photomixotrophic system in a CO₂-enriched atmosphere may be suitable for large-scale propagation of this species.     

Morphophysiological in vitro performance of Brazilian ginseng (Pfaffia glomerata (Spreng.) Pedersen) based on culture medium formulations

Pfaffia glomerata has potential pharmacological and medicinal properties due to the production of a secondary metabolite known as the phytoecdysteroid 20-hydroxyecdysone (20E). There have been increasing efforts for massive in vitro propagation of Pfaffia plants due to high extractivism and overharvesting of this species. Research on the species has shown that photoautotrophic cultivation can improve the production of 20E. In addition, other abiotic factors such as the formulations of culture media can influence the morphophysiological behavior of the plants in vitro. Therefore, the objective of this study was to analyze the morphological and physiological performances of P. glomerata plants in different formulations of culture media, under photoautotrophic and photomixotrophic propagation conditions. Six medium formulations, the Driver and Kuniyuki medium (DKW), Correia et al. medium (JADS), Murashige and Skoog medium (MS), Quoirin and Lepoivre medium (QL), Rugini medium (OM), and Woody Plant medium (WPM), all supplemented with DKW vitamins, 100 mg L⁻¹ myo-inositol, 6.5 g L⁻¹ agar, and with or without 3% (w/v) sucrose, were evaluated. Cultures were maintained at 25 ± 2°C, with a 16 h-photoperiod under 60 μmol m⁻² s⁻¹ of irradiance under a fluorescent lamp for 50 d. Results showed that the presence or absence of sucrose, and the different nutritional formulations influenced growth, photosynthetic pigment content, endogenous levels of sugars, leaf morphology, levels of 20E, and transport of water and minerals in P. glomerata. Notably, OM, DKW, QL, and WPM media promoted higher production of 20E under photomixotrophic growth conditions.

     

CO2-enriched atmosphere and supporting material impact the growth,morphophysiology and ultrastructure of in vitro Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] plantlets

This study aimed to evaluate under photoautotrophic conditions the effect of CO₂-enriched atmosphere (360 or 1,000 μmol CO₂ mol^?1 air) combined with two substrate types (agar or Florialite^®) in vitro on plants of Pfaffia glomerata, an endangered medicinal species with promising applications in phytotherapy and phytomedicine. The effects of the treatments on the growth, stomatal density, Rubisco activity, carbon isotopic discrimination, metabolite accumulation, photosynthetic pigments and ultrastructural characteristics were investigated. After a 35-day cultivation period, the in vitro-growth of P. glomerata nodal segments under the different treatments resulted in plants with substantial differences in relation to their growth, photosynthetic pigments, stomatal density and leaf ultrastructural characteristics. The enrichment with CO₂ coupled with a porous substrate increased the growth of P. glomerata. The stomatal density in the abaxial epidermis more than doubled in response to the high CO₂ supply in both supporting types, whereas the Rubisco activity and activation state were both unresponsive to the treatments. Regardless of the CO₂ supply, the plants grown in agar displayed higher carbon isotope discrimination than their counterparts grown in Florialite^®. We propose that the long-term photosynthetic performance was improved using Florialite^® as a growth support in combination with a high CO₂ supply. No apparent signs of photosynthetic down-regulation could be found under elevated CO₂ conditions. The enrichment of in vitro atmospheres with CO₂ coupled with a porous substrate offers new possibilities for improving the growth and production on a commercial scale of high morphological and physiological quality Pfaffia plants.     

Irradiance-driven 20-hydroxyecdysone production and morphophysiological changes in Pfaffia glomerata plants grown in vitro

Pfaffia glomerata possesses potential pharmacological and medicinal properties, mainly owing to the secondary metabolite 20-hydroxyecdysone (20E). Increasing production of biomass and 20E is important for industrial purposes. This study aimed to evaluate the influence of irradiance on plant morphology and production of 20E in P. glomerata grown in vitro. Nodal segments of accessions 22 and 43 (Ac22 and Ac43) were inoculated in culture medium containing MS salts and vitamins. Cultures were maintained at 25 ± 2 °C under a 16-h photoperiod and subjected to irradiance treatments of 65, 130, and 200 μmol m⁻² s⁻¹ by fluorescent lamps. After 30 days, growth parameters, pigment content, stomatal density, in vitro photosynthesis, metabolites content, and morphoanatomy were assessed. Notably, Ac22 plants exhibited 10-fold higher 20E production when cultivated at 200 μmol m⁻² s⁻¹ than at 65 μmol m⁻² s⁻¹, evidencing the importance of light quantity for the accumulation of this metabolite. 20E production was twice as high in Ac22 as in Ac43 plants although both accessions responded positively to higher irradiance. Growth under 200 μmol m⁻² s⁻¹ stimulated photosynthesis and consequent biomass accumulation, but lowered carotenoids and anthocyanins. Furthermore, increasing irradiance enhanced the number of palisade and spongy parenchyma cells, enhancing the overall growth of P. glomerata.

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A low-cost alternative membrane system that promotes growth in nodal cultures of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]

In vitro propagated plants under conditions of low gas exchange generally show morphological and physiological anomalies that lead to high mortality rates during ex vitro acclimatization. The use of gas-permeable membranes increases natural ventilation in culture vessels, photosynthesis and growth rates. However, commercial membranes are expensive, which limits their application. In this study, low-cost, simple to manufacture, alternative membranes were developed to promote gas exchange in jars used for in vitro plant tissue culture. The membranes were developed using polytetrafluoroethylene film and two or three layers of microporous tape (Missner & Missner^?), and were designed to increase the growth of nodal cultures of Pfaffia glomerata (Brazilian ginseng). Conditions that provided higher gas exchange led to an increase in plant growth and content of photosynthetic pigments compared to a closed system without a gas-permeable membrane. The alternative membranes showed similar results for water vapor loss rate and photosynthetic pigments when compared to a commercial membrane. The alternative membranes were also an efficient barrier against contamination and remained intact after being autoclaved multiple times. Among the membranes tested, the traits of the P. glomerata in vitro-derived plants were similar when propagated using the alternative membrane with three layers of microporous tape or the commercial membrane. However, the alternative membrane has a unit cost that is ten times lower than the commercial membrane.     

Accessions of Brazilian ginseng (Pfaffia glomerata) with contrasting anthocyanin content behave differently in growth,antioxidative defense,and 20-hydroxyecdysone levels under UV-B radiation

Protoplasma - Ultraviolet-B (UV-B) radiation is an elicitor of secondary metabolites in plant tissue culture, but the effects on 20-hydroxyecdysone (20E) are still unclear. The 20E may show...     

Involvement of nitric oxide in the gastroprotective effects of an aqueous extract of Pfaffia glomerata (Spreng) Pedersen, Amaranthaceae, in rats

The plants belonging to Pfaffia genus are used in folk medicine to treat gastric disturbances. This study examined the effects of an aqueous extract of Pfaffia glomerata (Spreng) Pedersen (AEP) on the gastrointestinal tract. Wistar rats were pretreated orally (p.o.) with the AEP (125, 250, 500 and 1000 mg.kg(-1)) before induction of ulcers by hypothermic restraint stress (HRS, 3 h restraint stress at 4 degrees C), ethanol (ET, 70%; 0.5 ml/animal; p.o.) or indomethacin (IND, 20 mg.kg(-1); s.c.). Control animals received water (C) or ranitidine (60 mg.kg(-1)) p.o. The AEP protected rats against HRS and ET-induced ulcers, but was not able to protect the gastric mucosa against IND-induced ulcers. When injected into the duodenal lumen, the AEP reduced total acidity and both basal and histamine-stimulated acid secretion in pylorus-ligated rats. In addition, gastric secretion from AEP-treated animals exhibited increased concentrations of nitrite and nitrate. Treatment of animals with L-NAME (120 mg.kg(-1), p.o.) prevented both the reduction of total acidity and the increase in NOx levels promoted by AEP treatment. In conclusion, AEP effectively protected the gastric mucosa and inhibited gastric acid secretion in rats, probably by involving the histaminergic pathway and an enhanced production of nitric oxide in the stomach.     

Zinc alleviates mercury-induced oxidative stress in <Emphasis Type="Italic">Pfaffia glomerata</Emphasis> (Spreng.) Pedersen

The possible role of zinc (Zn) to reverse the oxidative stress caused by mercury (Hg) was investigated in Pfaffia glomerata plantlets. Thirty-day-old acclimatized plantlets of P. glomerata were exposed to four treatments: control, 50 μM Zn, 50 μM Hg and 50 μM Zn + 50 μM Hg for 9 days. In Zn + Hg treatment, shoot and root Hg concentrations were 59 and 24% smaller than that plants exposed to 50 μM Hg added alone. An increase in the Zn concentration in the shoot of plants exposed to Zn + Hg occurred, although in the roots Zn concentration was not altered, when compared to the control. Fresh and dry weights, as well as the activity of δ-aminolevulinic acid dehydratase (δ-ALA-D) in Hg-treated plants were significantly reduced. Percentage survival, fresh and dry weights and δ-ALA-D activity of plants treated by 50 μM Zn + 50 μM Hg were greater than of that treated by Hg alone. Moreover, Zn treatment reduced the lipid peroxidation caused by Hg, being this effect related to increased root superoxide dismutase activity, and shoot catalase and ascorbate peroxidase activities. In conclusion, the presence of Zn in the substrate caused a significant reduction in the oxidative stress induced by Hg.     

Physiological,epigenetic, and proteomic responses in Pfaffia glomerata growth in vitro under salt stress and 5-azacytidine

Protoplasma - Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the...     

Enhanced growth and quality of St. John's wort (Hypericum perforatum l.) under photoautotrophic in vitro conditions

Summary Photomixotrophic (Pm) micropropagation systems (ones that use a sugar-containing medium) have been used by many rescarchers for transplant production of St. John's wort. However, these methods have not yet been adopted for commercial applications, probably due to the low percentage of regeneration in vitro, and a low growth rate after transplanting ex vitro. In contrast, it is well known that the use of a photoautotrophic (Pa) micropropagation system (one that uses sugar-free medium) can promote the growth and improve the quality of plantlets in vitro, and enhance the growth during acclimatization for many plant species. In the current study, leafy nodal cuttings were cultured under Pa conditions and the growth and quality were compared with those cultured under Pm conditions. After 21d of culture, Pa conditions enhanced the growth and quality of St. John's wort plantlets in vitro, and these plantlets showed faster growth after transplantaing ex vitro compared with those cultured under Pm conditions.     

Evaluation of Spinacia oleracea (L.) for phytodesalination and augmented production of bioactive metabolite, 20-hydroxyecdysone

In this study, adaptive features of Spinacia oleracea to different levels of salinity, its use in desalination and production of 20-Hydroxyecdysone were studied. Plants showed survival up to EC 12 dS/m with reduced growth as compared with control. Net photosynthesis rate, transpiration, stomatal conductance, and water use efficiency of salt treated plants declines with increasing salinity stress. Higher antioxidant enzyme activities and compatible solutes accumulation were observed in salt treated plants as function of osmotic adjustment. Significant Na⁺ sequestration and Na/K ratio were noted with increase in salt stress in comparison to the control. Since the plant accumulates a bioactive, secondary metabolite 20-Hydroxyecdysone (20E), we observed significant 20E content in plants grown at EC 4–12 dS/m in comparison to control. Furthermore, a preliminary field experiment, showed significant reduction in the soil electrical conductivity by 1.8 ds/m after 90 days of plant growth with Na⁺ sequestration in plant biomass. Subsequent to this growth period, the phytodesalinized soil supported the significant growth of a glycophyte (rice). Our results suggest that S. oleracea can adapt to saline conditions with antioxidant defense and osmotic adjustment. The plant can be used as a potential candidate for desalination and also for enhanced production of 20-Hydroxyecdysone.     

Effects of the steroid hormone, 20-hydroxyecdysone, on the growth of neurites by identified insect motoneurons in vitro.

During metamorphosis in the hawkmoth, Manduca sexta, identified larval leg motoneurons survive the degeneration of their larval targets to innervate new muscles of the adult legs. The dendrites and axon terminals of these motoneurons regress at the end of the larval stage and then regrow during adult development. Previous studies have implicated the insect steroid, 20-hydroxyecdysone (20-HE), in similar examples of dendritic reorganization during metamorphosis. The present studies were undertaken to test whether 20-HE acts directly on the leg motoneurons to regulate dendritic growth. Larval leg motoneurons were labeled with a fluorescent dye to permit their identification in culture following the dissociation of thoracic ganglia at later stages of development. Leg motoneurons isolated from early pupal stage animals (just before the normal onset of dendritic regrowth) survived in vitro and grew processes regardless of whether 20-HE was added to the culture medium. The extent of process outgrowth, however, as measured by the total length of all processes and the number of branches, was significantly greater for motoneurons maintained in the presence of 20-HE. The enhancement could be blocked by the addition of a juvenile hormone analog. By contrast, larval leg motoneurons that were isolated just before the normal period of dendritic regression did not show enhanced growth of neurites in the presence of 20-HE. The results suggest that 20-HE acts directly on the leg motoneurons to regulate the growth of processes during metamorphosis.     

Effects of ionizing radiation on the growth and allyl isothiocyanate accumulation of Wasabia japonica in vitro and ex vitro

Mutations were induced in tissue-cultured wasabi (Wasabia japonica Matsumura) by treating in vitro-derived shoot tips with either γ-rays or X-rays at 0, 10, 20, 40 or 80 Gy. Doses of up to 40 Gy of either γ- or X-ray treatments resulted in a survival rate of more than 60% in culture after 3 mo. The use of γ- or X-rays at doses between 10 Gy and 40 Gy to induce mutation in W. japonica resulted in an alteration of the growth and allyl isothiocyanate (AITC) content of multiple shoots after 3 mo. in culture on Murashige and Skoog medium containing 5 μM N⁶-benzyladenine (BA). Putative mutants from the 40 Gy treatments of either γ- or X-rays exhibited a reduction in shoot weight, number, and height, whereas treatments of either γ-rays or X-rays at 10 Gy and 20 Gy doses showed no significant differences in shoot growth. All shoots treated with 80 Gy were either necrotic or irregenerable, while those treated with 40 Gy produced deformed leaves, from both types of ionizing radiation. Concentrations of AITC were measured by the use of gas chromatography-mass spectrometry (GC-MS). The accumulation of AITC was shown to decrease when doses increased in both γ- and X-ray treatments, compared with the controls. Positive responses were solely occurred at 18 mo. after transfer of in vitro rooted shoots to the shade house. The survival rate, rhizome weight and AITC content of plants derived from shoots treated with 20 Gy or 40 Gy of either γ-rays or X-rays were significantly greater than those of the controls.     

Expression of ZmGA20ox cDNA alters plant morphology and increases biomass production of switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is considered a model herbaceous energy crop for the USA, for its adaptation to marginal land, low rainfall and nutrient‐deficient soils; however, its low biomass yield is one of several constraints, and this might be rectified by modulating plant growth regulator levels. In this study, we have determined whether the expression of the Zea mays gibberellin 20‐oxidase (ZmGA20ox) cDNA in switchgrass will improve biomass production. The ZmGA20ox gene was placed under the control of constitutive CaMV35S promoter with a strong TMV omega enhancer, and introduced into switchgrass via Agrobacterium‐mediated transformation. The transgene integration and expression levels of ZmGA20ox in T0 plants were analysed using Southern blot and qRT‐PCR. Under glasshouse conditions, selected transgenic plants exhibited longer leaves, internodes and tillers, which resulted in twofold increased biomass. These phenotypic alterations correlated with the levels of transgene expression and the particular gibberellin content. Expression of ZmGA20ox also affected the expression of genes coding for key enzymes in lignin biosynthesis. Our results suggest that the employment of ectopic ZmGA20ox and selection for natural variants with high level expression of endogenous GA20ox are appropriate approaches to increase biomass production of switchgrass and other monocot biofuel crops.