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.     

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.     

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.

     

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.     

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.

Graphical abstract

     

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.     

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.     

CO2 enrichment and supporting material impact the primary metabolism and 20-hydroxyecdysone levels in Brazilian ginseng grown under photoautotrophy

Plant cell and organ cultures via the implementation of effective elicitation strategies can offer attractive biotechnological platforms for the enhanced production of phytochemicals of pharmaceutical interest. For the first time, the elicitation of exogenous signal molecules was conducted to enhance the production of pharmacologically active alkaloids and flavonoids in Isatis tinctoria L. hairy root cultures (ITHRCs). ITHRCs III and V correspondingly possessing high alkaloid and flavonoid productivity were adopted for elicitation treatments. The maximum accumulation of alkaloids in ITHRCs III elicited by 142.61 µM salicylic acid for 28.18 h and flavonoids in ITHRCs V elicited by 179.54 µM methyl jasmonate for 41.87 h increased 5.89- and 11.21-folds as compared with controls, respectively. Moreover, expressions of 11 genes involved in alkaloid and flavonoid biosynthetic pathways were significantly up-regulated following elicitation, among which YUCCA, CHI and F3′H genes might play a crucial role in the target phytochemical augmentation. Overall, two effective elicitation protocols were provided here to improve the yields of bioactive alkaloids and flavonoids in ITHRCs, which was useful for the scale-up production of these valuable compounds to meet the demands for natural bioactive ingredients by pharmaceutical industries.

     

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...     

Photoperiod modulates growth,morphoanatomy, and linalool content in Lippia alba L. (Verbenaceae) cultured in vitro

Biotechnology provides valuable tools to support conservation of plant species, especially in case of threatened taxa or when dealing with seed unavailability, low viability or sterility. However, plant cell culture methods have often to face problems associated with tissue recalcitrance to in vitro systems. Recalcitrance can be related to a variety of triggering factors, involving many efforts and manipulations within one or more of the micropropagation stages before obtaining successful results. An in vitro propagation protocol was developed for Zelkova sicula, a very rare and endangered relict tree, endemic to Sicily (Southern Italy). The species revealed extremely recalcitrant to in vitro culture approaches, but after many trials throughout a number of years an effective micropropagation protocol was completed. The rooting rate was about 84% of the treated explants, 8% of which were successfully acclimatized outdoor and reintroduced in the wild within a comprehensive conservation project. The technique allowed to overcome the problems of sexual sterility of this species, hence contributing concretely to contrast the problems connected with its conservation. However, additional efforts need to be carried out in order to refine the acclimatization step and further improve the whole process effectiveness.

     

Effects of Temperature and Photoperiod on Flowering in Soya bean [Glycine max (L.) Merrill]: a Quantitative Model

Factorial combinations of five photoperiods (8 h 20 min, 10h, 11 h 40 min, 13 h 20 min and 15 h) and three night temperatures(14, 19 and 24 C) combined with a single day temperature (30C) were imposed on nodulated plants of nine soya bean genotypes[Glycine max (L.) Merrill] grown in pots in growth cabinets.The times to first appearance of open flowers were recorded.For a photoperiod-insensitive cultivar, and for the remainingeight photoperiod-sensitive genotypes in photoperiods shorterthan the critical daylength, the rates of progress towards flowering(the reciprocals of the times taken to flower) were linear functionsof mean diurnal temperature. For all photoperiod-sensitive genotypes,times to flowering in photoperiods longer than the criticaldaylength increased as inverse functions of both increasingphotoperiod and decreasing temperature. A consequence of thesetwo relations is that the critical daylength becomes longerwith higher mean temperatures. In the five photoperiod-sensitivegenotypes which flowered in all environments before the experimentwas terminated (after 150 d) the delays in flowering due tolow temperatures or long photoperiods were limited by a maximumperiod to flowering specific for each genotype. These resultsare discussed in relation to the development of a simple techniquefor the large-scale screening of soya bean germplasm to determinephoto-thermal response surfaces for flowering. Glycine max (L.) Merrill, soya bean, flowering, photoperiod, temperature, screening, germplasm     

Modulation of root growth and organogenesis in thidiazuron-treated ginseng (Panax quinquefolium L.)

The morphoregulatory effect of thidiazuron (TDZ) applications to ginseng (Panax quinquefolium L.) seedlings and 3-year-old plants was determined. Applications of TDZ (0.22 and 2.20 ppm), either as foliar sprays or soil drench to greenhouse-grown seedlings over 18 weeks (2 weeks after sowing to 20 weeks when plants were harvested) induced similar responses, in particular, increased stem length and diameter, and shoot and root weight (economic yield). Single foliar applications of TDZ at 62.5 and 125 ppm to 3-year-old field-grown ginseng plants 3 months prior to harvest increased root biomass (economic yield) by 19 to 23%. Roots of TDZ-treated seedlings and 3-year-old field-grown plants developed thickened secondary roots on the upper part of the taproot. The root-like structure of these secondary roots was confirmed by histology. In addition, TDZ treatments induced adventitious buds on the shoulder of 3-year-old roots. These buds developed into shoots to give multi-stem plants following a period of dormancy, which was overcome with GA₃ (gibberellic acid) treatment prior to planting.Abbreviations TDZ = thidiazuron - GA₃ = gibberellic acid - BA = benzyladenine     

In vitro regeneration and transformation of pigeonpea [Cajanus cajan (L.) Millsp]

A requirement for generating transgenic pigeonpea [Cajanuscajan (L.) Millsp] plants is the development of a highly efficientin vitro regeneration procedure. This goal was achieved byusing germinated seedlings grown on B5 medium supplemented with 10 mgl^–1 6-benzylaminopurine, which induced differentiatingcallus formation in the cotyledonary node region. The calli were transferred onB5 medium with 0.2 mg l^–1 6-benzylaminopurine toobtain shoot induction. Elongated shoots were then further cultured on a B5hormone-free medium for rooting. Using this regeneration system transgenicpigeonpea plants were obtained both by particle bombardment andAgrobacterium tumefaciens-mediated gene transfer. Thepresence of the transgenes in the pigeonpea genome was confirmed by GUS assays,PCR and Southern hybridisation. The transgenic rooted plants were successfullytransferred to soil in the greenhouse. GUS and PCR assays of T1 progeniesconfirmed that the transgenes were stably transmitted to the next generation.This is the first report of successful use ofAgrobacteriumas well as particle bombardment for production of transgenic pigeonpea plants.     

Water deficit modulates growth,morphology, and the essential oil profile in Lippia alba L. (Verbenaceae) grown in vitro

Lippia alba (Miller) N.E. Brown is an aromatic plant species of great economic importance due to the medicinal properties of its essential oils, which provide stress relief, respiratory and gastrointestinal disease control, and anti-inflammatory and natural sedative effects. The plant is also effective in biological control against various pathogens and in food preservation. Water deficit is the most critical abiotic factor limiting plant growth and morpho-physiological development, as well as production of secondary metabolism compounds. The objective of this work was to evaluate the effect of water deficit on growth, photosynthesis, essential oil profile, and the expression of genes related to the biosynthesis of these compounds in L. alba grown in vitro. Nodal segments were cultured on medium supplemented with 0, 1, 2, and 3% (w/v) polyethylene glycol for 45 days. Water stress had a negative effect on primary metabolism indicators, such as growth, leaf area, and photosynthetic rate; but a positive effect on amino acid and total protein content. Similarly, secondary metabolism exhibited an increase in linalool but a reduction in germacrene levels under water deficit. These findings provide a deeper understanding of how water deficit affects primary and secondary metabolism in L. alba, showing the potential of this medicinal species to adapt to soils with low water availability, while still being able to grow and synthesize essential oils.

     

Effect of Photoperiod on Metabolism of [H] Gibberellins A(1), 3-epi-A(1), and A(20) in Agrostemma githago L

To determine whether daylength influences the rate of metabolism of gibberellins (GAs) in the long-day (LD) rosette plant Agrostemma githago L., [³H]GA₂₀ and [³H]GA₁ were applied under short day (SD) and LD. Both were metabolized faster under LD than under SD. [³H]GA₂₀ was metabolized to a compound chromatographically identical to 3-epi-GA₁. [³H]GA₁ was metabolized to two acidic compounds, the major metabolite having chromatographic properties similar to, but not identical with GA₈. [³H]3-epi-GA₁ applied to plants under LD was metabolized much more slowly than was [³H]GA₁, and formed a very polar metabolite which did not partition into ethyl acetate at pH 2.5. Very polar metabolites were also formed after the feeds of [³H]GA₂₀ and [³H]GA₁. It was not possible to characterize these very polar compounds further because of their apparent instability. The results obtained suggest that in Agrostemma GA₂₀ is the precursor of 3-epi-GA₁, but there is at present no evidence indicating the precursor of GA₁.